Thesis Title PDF

Session Number: 24
Session Type: Poster Talk

Session Number: 24
Session Title: Novel Diagnostics for Urinary Tract Infections
Session Start Date Time: 6/8/2018 11:00:00 AM
Session End Date Time: 6/8/2018 12:00:00 PM
Session Primary Track: Clinical and Public Health Microbiology
Abstract Control Number: 8903
Poster Board Number:
Abstract Title:
Rapid Antimicrobial Susceptibility Testing (Ast) Via Laser Light Scattering Technology for Gram Negative
Organisms from Screen Positive Urine
Primary Author Block:
K. M. Riederer, R. Witherell, J. T. Fishbain; St. John Hosp. and Med. Ctr., Grosse Pointe Woods, MI
Abstract Body:
Background: Rapid screening for urinary tract infection and organism identification is currently available
using narrow angle forward laser scattering technology followed by matrix assisted laser desorption
ionization-time of flight mass spectrometry (MALDI-TOF MS). Rapid AST (RAST) could add critical
information for treatment. We evaluated a new RAST method using 4 antibiotics for Gram negative
pathogens. Methods: Adult inpatient urine submitted for culture was screened for presence of >104
CFU/ml by monitoring growth kinetics (216 Dx, BacterioScan). Organisms were identified by MALDI-TOF
MS (Biotyper, Bruker) via direct smear from positive turbid cuvettes. Gram negatives were diluted in
Mueller Hinton broth and inoculated to antibiotic cuvettes for RAST (216 Dx). CLSI breakpoint
concentrations of Ciprofloxacin (CIP), Ceftriaxone (CRO), Cefazolin (KZ) and Meropenem (MEM) for
sensitive (S) and intermediate (I) interpretation were used. RAST growth kinetics along the no drug
control curve were considered resistant (R). RAST and parallel disk diffusion were interpreted in blinded
fashion then compared to MIC reports (Vitek 2, bioMerieux) from urine culture isolates (gold standard).
Categorical agreement was calculated and discordance classified as minor (I versus S or R), major (false
R) or very major (false S) errors. QC for E. coli (pan S), E. cloacae (pan R) and K. pneumoniae CIP, KZ (R)
CRO, MEM (S) was included. Time required for RAST results was assessed. Results: 1015 urines were
screened and 112 AST performed on Gram negative organisms. 90 AST were evaluated; 22 were
polymicrobic and excluded (15 with ≥ 3 organisms, 7 with 2 organisms on culture). RAST concordance
with MIC interpretation for all 4 drugs was 94.4%. Five errors from separate samples were noted: 1 CIP
minor (K. pneumoniae), 2 CRO minor (K. pneumoniae, E. coli), 1 MEM major (A. baumannii) and 1 KZ
very major (E. coli). RAST categorical agreement with MIC interpretation for individual drugs (CIP, CRO,
MEM, KZ) was 98.9, 97.8, 97.7 and 98.8% respectively. RAST results were available within 4 hours.
Conclusions: Rapid AST via laser light scattering technology provides early phenotypic evidence of
antimicrobial susceptibility and resistance. RAST at CLSI breakpoint concentrations correlates well with
standard MIC interpretation. Urine screening, Gram negative organism identification and susceptibility
information can be available within 8 hours to guide treatment. Further testing is needed to determine
RAST reliability with additional organisms and antimicrobial drugs.
Session Number: 24
Session Number: 24
Abstract Title:
Evaluation of Uriswab™ For the Collection, Transport and Preservation of Urine Specimens According to
the Clsi M40-A2 Guideline
A. Giambra, E. Orio, S. Castriciano; Copan Italia Spa, Brescia, Italy
Abstract Body:
Background: Accurate bacteriuria in urine is essential for the treatment of urinary tract infections. Urine
should be refrigerated or preserved during transport to prevent organisms overgrowth and support
pathogens viability. Copan developed the UriSwab™ (US), a collection, transport and preservation device
for urine specimen’s compatible with manual or WASP automated culturing methods. US has a leak-
proof screw-cap-tube with 2 or 3 sponges on a plastic stick attached to the cap. The sponges contain
preservatives salts to stabilize the original organisms load and are highly hydrophilic to absorb and
retain urine during transport and. The objective of this study was to evaluate the performance of the
UriSwab™ as per CLSIM40-A2 guidelines. Methods: ATCC strains of C. albicans (ATCC®24433), E. coli
(ATCC®25922), E. faecalis (ATCC®29212), P. aeruginosa (ATCC®27853), P. mirabilis (ATCC®7002), S.
saprophyticus (ATCC®15305), C. freundii (ATCC®8090), C. glabrata (ATCC®MYA-2950), E. cloacae
(ATCC®13047), M. morganii (ATCC®25829), S. agalactiae (ATCC®13813) were used for this study. From
each fresh sub-culture strain, a 0.5 McF suspension and six 10-fold serial dilutions were prepared in
sterile filtered synthetic urine to obtain a final count at time-zero of 25 to 250 organisms per plate.
Three lots of US were inoculated by submerging the sponges in 1.5x104, 1.5x103 and 1.5x102 CFU/ml
suspensions for 5 seconds and then returned into the tube. For each strain 15 US (3 at time zero, 6 at
24hs and 6 at 48hs at both 20-24°C and 2-6°C) were inoculated per lot. At each time-point, the US tubes
were centrifuged to release the urine and 100µl each were plated in triplicate onto appropriate agar.
After plates incubation, CFUs were counted to verify that each strains concentration in US at different
conditions remained within 1 log compared to time-zero as per guidelines. Results: Data obtained at all
conditions/times were within the acceptance criteria. Log reductions at 48hs for 2-6°C and 20-24°C were
-0.27 and -0.37 for C. albicans, -0.28 and -0.71 for E. Coli, -0.09 and -0.31 for E. faecalis, -0.15 and-0.66
for P. aeruginosa, -0.13 and -0.47 for P. mirabilis, -0.28 and 0.28 for S. saprophyticus, -0.13 and -0.19 for
C. freundii., -0.39 and -0.67 for C. glabrata, -0.29 and 0.15 for E. cloacae, -0.22and -0.21 for M. morganii,
-0.28 and -0.31 for S. agalactiae Conclusion: UriSwab™ device is fully compliant to the CLSI M40-A2
standard for all the strains tested. UriSwab™ is operator’s safe since absorbs spontaneously the urine
sample eliminating the use of a needle.
Session Number: 24
Session Number: 24
Abstract Title:
Comparison of Culture, Sequencing, and Isothermal Rna Amplification Assay for Detection of
Ureaplasma From First Void Urine of Patients with Urogenital Infections
Y. Liu; Peking Union Med. Coll. Hosp., Beijing, China
Abstract Body:
Background: In order to compare different assays of Culture, Sequencing, and Isothermal RNA
Amplification Assay for Detection of Ureaplasma from First Void Urine of Patients with Urogenital
Infections. Methods: Parallel aliquots of each urine specimen were tested for Ureaplasma by culture
only, culture and sequencing, and Isothermal RNA Amplification Assay (IRAA) (targeting 16S rRNA).
Results: From 2015 to 2016, a total of 103 urine specimens were collected from patients with urogenital
infections. Culture only, culture and sequencing, and IRAA, detected Ureaplasma spp in 17.48%
(18/103), 23.3% (24/103), and 25.24% (26/103) of the 103 specimens, respectively. Based on the gold
standard (culture and sequencing), the sensitivity and specificity of IRAA method were 91.67% (22/24)
and 94.94% (75/79), respectively, compared to 75.00% (18/24) and 100% (75/75), respectively, for
culture only method (p>0.05). Of the 24 Ureaplasma positive specimens, 70.83% (17/24) had U. parvum
only, 29.17% (7/24) U. urealyticum only, and none (0/24) contained both. The antibiotic susceptibility
rates of the 18 Ureaplasma spp isolates ranged from 11.1 % (ciprofloxacin) to 100 % (pristinamycin).
Ciprofloxacin and ofloxacin showed the lowest activity, with only 11.11% and 22.22% susceptibility,
respectively. Conclusions: Our findings suggest that IRAA demonstrate high sensitivity and specificity for
the detection of Ureaplasma, time-saving and high throughput while culture showed much higher
specificity, cheaper and also give antimicrobial susceptibility testing results.
Session Number: 24
Session Number: 24
Abstract Title:
Semi-Quantitative Molecular Detection of 5 Bacterial Pathogens and 47 Antibiotic Resistance Genes in
Urine Specimens and Culture Isolates
G. Walker1, B. Dews1, K. Pitzer1, J. Quan1, S. G. Higgins1, N. Toraskar1, N. Whitfield1, B. K. Lopansri2, S.
Riedel3; 1OpGen Inc., Gaithersburg, MD, 2Intermountain Hlth.care, Murray, UT, 3Beth Israel Deaconess
Med. Ctr., Boston, MA
Abstract Body:
Background: Molecular tests rapidly detect bacteria in patient specimens but do not comprehensively
predict antibiotic resistance compared with identification and antibiotic susceptibility testing (ID/AST).
Materials/methods: We developed the Acuitas® AMR Gene Panel u5.47 for detection of Escherichia coli,
Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis and Enterococcus faecalis plus 47
antibiotic resistance genes. The molecular test can be used with statistical algorithms from the Acuitas
Lighthouse® Knowledgebase to predict phenotypic resistance to 17 antibiotics. The test uses automated
DNA extraction and real-time multiplex PCR to provide results in 2.5 hours from urine specimens or
bacterial isolates. We used the test to evaluate 50 remnant clinical urine specimens and 60 bacterial
isolates from the FDA-CDC Antimicrobial Resistance Isolate Bank. Results: E. coli, E. faecalis, K.
pneumoniae, P. mirabilis and P. aeruginosa were detected with decreasing prevalence in the urine
specimens with 30% exhibiting mixed infections. The molecular test identified urine pathogens with 96%
semi-quantitative consistency compared with urine culture. The test detected two resistance genes on
average per urine specimen including genes for aminoglycosides, cephalosporins, sulfonamides,
fluoroquinolones and trimethoprim/sulfamethoxazole. The Acuitas Lighthouse® Knowledgebase
accurately predicted phenotypic resistance for molecular results from urine (e.g., 95% accuracy for
ciprofloxacin resistance). Several resistance genes were detected per FDA/CDC isolate on average: E. coli
(8), K. pneumoniae (8), P. aeruginosa (4) and P. mirabilis (3) with decreasing prevalence of genes for
aminoglycosides, cephalosporins, sulfonamides, carbapenems, fluoroquinolones and
trimethoprim/sulfamethoxazole. The Acuitas Lighthouse® Knowledgebase predicted phenotypic
resistance with average accuracy of E. coli (94%), K. pneumoniae (86%) and P. aeruginosa (82%) across
gentamicin, tobramycin, ciprofloxacin, levofloxacin, trimethoprim/sulfamethoxazole, imipenem,
cefazolin, cefepime, cefotaxime, ceftazidime, ceftriaxone, ampicillin and aztreonam. Conclusions:
Acuitas AMR Gene Panel u5.47 rapidly detects 5 pathogens and 47 resistance genes in urine specimens
and culture isolates. The Acuitas Lighthouse® Knowledgebase predicts phenotypic resistance. Rapid and
accurate detection of antibiotic resistance can improve antibiotic therapy and outcomes in patients with
bacterial infections.
Session Number: 24
Session Number: 24
Abstract Title:
Utilization of Laser Light Scattering for Rapid Detection and Subsequent Antimicrobial Susceptibility
Testing Directly from Urine Specimens
S. Collier1, A. Tomaras2, A. Skinner1, F. Albarillo1, A. Harrington1; 1Loyola Univ. Med. Ctr., Maywood,
IL, 2BacterioScan, Inc, St Louis, MO
Abstract Body:
Background: The high volumes and lengthy turnaround times required to process urine specimens
present a workflow challenge and prompt the use of empiric therapy, including to patients whose
specimens may be uninfected. Methods that return diagnostic information more rapidly would improve
patient care and facilitate antibiotic stewardship efforts. The BacterioScan 216Dx is a laser light
scattering device that detects urinary tract infections (UTIs) more rapidly than current standard of care
(SOC) methods. Our lab evaluated the 216Dx’s ability to provide rapid antimicrobial susceptibility testing
(AST) data using samples deemed positive during the UTI screening protocol. Methods: Ninety-five urine
specimens were analyzed using the 216Dx UTI detection protocol according to the manufacturer’s
instructions. Cuvette contents from positively flagged specimens were spread onto pre-warmed blood
and MacConkey agar plates and incubated at 37°C for 4 hours. MALDI-TOF MS (Bruker) identification
was performed on the resulting agar surface film. Direct-from-positive specimen AST was also evaluated
on 22 samples containing Gram negative pathogens using a panel of 4 antibiotics tested at clinical
breakpoint concentrations. Cuvette contents were diluted to ~5x105 CFU/ml into cation-adjusted
Mueller Hinton Broth prior to dispensing into antibiotic-containing cuvettes. AST runs in the 216Dx were
conducted at 35°C for 16 hours, after which optical profiles were compared to a corresponding no drug
control. Results: Fifty-four specimens possessed ≥10,000 CFU/ml of a UTI pathogen, 51 of which were
accurately detected by the 216Dx after 190 minutes. Despite contaminant presence in many of the
specimens, MALDI-TOF MS analysis of positively-flagged cuvette material showed ~75% agreement with
SOC data, with the majority of failed samples harboring lower density infections, Gram positive
pathogens, or possessing >1 UTI pathogen. The SOC AST method (MicroScan) indicated non-
susceptibility for 36.3, 36.3, 4.5, and 0% of isolates to Ciprofloxacin (CIP), Levofloxacin (LVX), Cefepime
(FEP), and Meropenem (MER), respectively. After 3-4 hours of analysis, the 216Dx demonstrated
categorical agreement at 100% for CIP, FEP, and MER, and 95.4% for LVX. Conclusion: Based on these
data, the BacterioScan 216Dx has the potential to offer accurate UTI detection and, when coupled with
MALDI-TOF MS, robust pathogen ID/AST for at least 75% of Gram negative infected urine specimens,
within as little as 8 hours post-collection. Further validation is needed and ongoing.
Session Number: 40
Session Type: Late-Breaker Poster Presentations
Session Number: 40
Session Title: FRIDAY - CPHM Late-breakers
Poster Board Number: FRIDAY - CPHM LB1
Abstract Title:
Evaluating the Trend of Bloodstream Infections: A Four Year Experience at a Newly Establishing Tertiary
Care Hospital in Western India
A. Sharma1, A. K. Maurya2, V. Hada3, V. Nag1; 1All India Inst. of Med. Sci., Jodhpur, Jodhpur, India, 2All
India Inst. of Med. Sci., Bhopal, Bhopal, India, 3ESI Hosp. and Med. Coll., Hyderabad, Hyderabad, India
Abstract Body:
Background: Increasing availability of invasive procedures at tertiary care has also increased the burden
of Bloodstream infections (BSIs) in India. Lack of effective antibiotics and associated mortality are major
public health threat. The aim of study was to evaluate the trend of pathogen profile and their
antimicrobial resistance pattern in BSIs over a period of four years at a newly establishing tertiary care
hospital in Western India. Material & Methods: We conducted a 4-year retrospective analysis of blood
cultures from all patients at All India Institute of Medical Sciences Jodhpur, Rajasthan, India from
February 2014 through February 2018. Blood was collected aseptically and BACTEC fx automated system
was used. Microscan and conventional methods were used for identification of pathogens. The
antimicrobial sensitivity testing was done as per CLSI standards. Internal and external quality controls
were established during the study period. Results: Out of 2459 blood culture samples received in our
laboratory a total of 441 isolates were obtained. Nearly 150 isolates were contaminants. Klebsiella
pneumoniae (16% isolates) and Candida spp. (16% isolates) were major isolates as well as leading cause
of mortality. Second leading cause of mortality was Escherichia coli. There was no difference in mortality
between carbapenem resistant and sensitive isolates. A notable rise in carbapenem resistance (33% in
2015-16 to 44% in 2017-18) was seen in these members of the family Enterobacteriaceae. Modified
Hodge test was positive in only one third of these isolates. Occasional Pan-resistance was seen among
the isolates of Acinetobacter baumanii though an overall decline in carbapenem resistant isolates
(CRAB) was seen. On the basis of AST Staphylococcus aureus (12.5% isolates) was CA-MRSA in two third
of its isolates. There was clustering of cases with MDROs in ICUs during winters and peak monsoon
season. The other significant isolates were Salmonella Typhi (n=12) and Salmonella Paratyphi A (n=8)
with fluoroquinolone resistance in 80% isolates; Streptococcus pneumoniae (n=11) with six PRSP;
Brucella spp. (n=7); Nocardia spp. (n=3) and NTM (n=1). A total of 50 carbapenem resistant gram
negative bacilli were responsible for BSIs in this period with their distribution in NICU (n=18), AICU (n=9),
PICU (n=3) and the rest were from the wards. Conclusion: We have tried to implement good practices
from the beginning and this analysis will help us in managing and restoring the quality work and in
establishing antimicrobial stewardship.
Session Number: 40
Session Number: 40
Abstract Title:
Point-of-care PCR Detection of Influenza A&B and Respiratory Syncytial Viruses in the Finnish Team
During the 2018 Olympic Winter Games
M. Valtonen1, O. Ruuskanen2, M. Waris3; 1Res. Inst. for Olympic Sports, Jyvaskyla, Finland, 2Turku
Univ. Hosp., Turku, Finland, 3Univ. of Turku, Turku, Finland
Abstract Body:
Elite athletes have increased susceptibility to acute respiratory infections due to high physical training
load and increased psychological stress especially before major competitions. In addition, long-distance
flights and living in households during games are well known risk factors. Furthermore, winter Olympic
Games coincidence with the season of influenza and other respiratory virus epidemics. The occurrence
and etiology of respiratory virus infections during winter Olympic Games are, however, poorly studied.
Before and during 2018 PyeongChang Olympic Winter Games influenza and respiratory syncytial virus
(RSV) epidemics were occurring in Finland and influenza epidemic in South-Korea. Therefore, the Finnish
Olympic team was prepared with a special focus on sensitive point-of-care PCR testing for influenza A
and B viruses and RSV (Xpert Xpress Flu/RSV, Cepheid). The surveillance was provided to all members of
the Finnish team in the PyeongChang Olympic Village. Flocked Coban nose swabs were taken by the
team physician from all team members with acute signs and symptoms of respiratory infection. An
informed oral consent was obtained. The Finnish team in the PyeongChang Olympic Village consisted of
46 athletes and 67 staff members. They stayed in the Olympic village for an average of 2-3 weeks per
person between February 2nd and 26th. A household contained 2-3 rooms with 4-6 team members.
Symptoms of respiratory infection were developed in 44 (39%) persons (22 athletes and 22 supporting
members). Point-of-care testing detected 11 infections; 5 persons (4 athletes and 1 supporting member)
with RSV, 5 with influenza B virus (1 and 4), and 1 athlete with influenza A/H3 virus. Positive cases were
isolated and cohorted. Persons with influenza were treated with oseltamivir (N=6) and their room mates
received prophylaxis (N=32). None of the latter individuals developed symptoms and were negative for
influenza virus in control tests. In only one case, RSV infection prevented the athlete to compete.
Respiratory infections were more common in the Olympic team than previously reported. Detection of 3
major viruses revealed etiology of the infection in 25% of the cases. Immediate isolation of the patients,
immediate treatment of influenza with oseltamivir and prophylactic oseltamivir treatment of contacts
prevented of spread of infections among athletes. Point-of-care PCR test proved a valuable tool in the
healthcare of the Finnish Olympic team.
Session Number: 40
Session Number: 40
Abstract Title:
T2Bacteria Panel: A Rapid, Culture Independent Assay for Identification of Bacteremia Causative Agents
K. M. Roberts, H. S. Lapp, G. E. Barringer, III, T. R. Higa, B. E. Jacobson, K. M. Candiloro, R. J. Griggs, C.
Hogan, J. Marturano, T. J. Lowery, R. P. Shivers, J. Townsend; T2 Biosystems, Lexington, MA
Abstract Body:
There are ~1.35M diagnosed cases of sepsis a year in the U.S., with an economic cost of $20B and a 30%
mortality rate. Blood culture, the standard for diagnosis, requires an incubation period that delays
diagnosis and proper treatment; a factor shown to increase patient mortality. To develop a culture
independent assay requires sensitivity at clinically relevant titers, as low as 1 CFU/mL. For this assay to
have clinical value requires a high analytical sensitivity while maintaining specificity and assay
performance in the presence of potential interferents. T2 Biosystems developed the T2Bacteria Panel to
identify the most common causes of bacteremia and provide physicians with rapid, actionable data that
could impact treatment of patients. Here we discuss the data demonstrating analytical sensitivity and
specificity, the effect of potential interferents on assay performance, competitive inhibition, and
performance across a range of clinically relevant titer levels. For these studies, K2EDTA-treated whole
blood from healthy donors was spiked with target bacterial species at clinically relevant concentrations.
To determine analytical sensitivity, a minimum of 80 total samples were tested across multiple days,
sample preps, and reagent lots with a criteria of ≥95% positivity rate across each set of N=20 replicates.
Specificity for the panel was determined using an initial in silico screen of potentially interacting species,
followed by testing of clinically relevant species at high titer. Interfering substances, both endogenous
and exogenous, were selected based on their potential presence in patient specimens and tested at high
physiological concentrations. Contrived sensitivity was determined across 50 strains for each species
spiked into individual donor blood samples at clinically relevant titer concentrations. Our studies show
that the T2Bacteria Panel is capable of providing species identification direct from blood at clinically
relevant concentrations. The combination of target specific primers and probes maintains the specificity
to prevent cross-reactivity with non-target species. Contrived sample data demonstrates a sensitivity of
>89% for targets at or above the limit of detection, and a specificity of >95% for each species. In total,
these data demonstrate that the T2Bacteria Panel provides a rapid and sensitive diagnostic for clinicians
to provide effective antimicrobial therapy to patients.
Session Number: 40
Session Number: 40
Abstract Title:
Product Contamination of Blood Culture Bottles with Proteus sp DNA Leads to a 45% False Positive Rate
for Rapid Molecular Testing
S. Duong, S. Condon, R. Khare, S. Juretschko; Northwell Hlth., Lake Success, NY
Abstract Body:
Introduction: Northwell Health Laboratories (NWHL) provides services to 23 hospitals within the greater
NYC area, including a molecular blood culture identification (BCID) panel for rapid sepsis diagnosis. Here
we report the product contamination of blood culture bottles of with Proteus sp. DNA and its
subsequent false detection with BCID. M&M: NWHL utilizes the BD BACTEC™ FX blood culture system,
with BD BACTEC™ Plus Aerobic, Plus Anaerobic and Peds Plus™ blood culture media, and BCID 2.0
FILMARRAY®, BioFire Diagnostics. The false-positive Proteus sp. results were observed in blood cultures
collected in 12 hospitals, 4 long-term facilities, and 1 off-campus emergency department. Results: On
February 5th, 2018, NWHL detected an abrupt, system wide increase of Proteus sp. positivity by BCID.
Prior, the weekly BCID Proteus sp. detection rate averaged 2.4%(0.6% to 4.9%) of all positive blood
cultures, but increased to 5.3%, 15.6%, 26.7%, 35.2% and subsequently 44.6%. Valid quality control
results ruled out contamination of Proteus sp. within the Microbiology laboratory. During this period of
5 weeks (Feb 4th to Mar 11th, 2018), a grant total of 232 of false results were discovered as determined
by the lack of culture and/or Gram stain confirmation. At least 21 different lots of blood culture bottles
reagents were involved (168 aerobic, 60 anaerobic and 4 pediatric). 93.5% of all Proteus sp. BCID results
(232/248) did not correlate with cultures and 70.6% of all cases had no Gram-negative rods present. Due
to the low positive predictive value (PPV 6.5%) and increased potential for patient harm, the decision
was made to stop reporting of Proteus sp. results by BCID and to add the following statement to all
reports to alert clinicians: “Due to technical problems, Proteus sp. will NOT be reported as part of the
BCID panel until further notice “. Conclusion: These false results, including 91 organisms otherwise
interpreted as skin contaminants, could have led to hundreds of cumulative days of unnecessary
antibiotic treatments for patients and hospital length of stay. Monitoring of the false-positive cases
continued on a daily basis and new cases continue to appear as of this writing. No official statements
have been issued by either BioFire Diagnostics or BD as of this writing. This report highlights the
necessity for a more rigorous validation process by the FDA when two methodologies (i.e. nucleic acid
amplification tests and culture) intertwine.
Session Number: 40
Session Number: 40
Abstract Title:
Development and Validation of a High-Throughput Testing Cascade for PCR-Based Diagnosis of Bacterial
Vaginosis and Vaginal Candidiasis Using the cobas® 6800/8800 System
A. B. HARRIS, A. J. PHERSON, C. P. CARTWRIGHT; Lab. Corp. of America, Burlington, NC
Abstract Body:
The cobas 6800/8800 systems (c68/8800; Roche Molecular Diagnostics, Indianapolis, IN) are fully
automated, high throughput, instruments designed to simplify and streamline the performance of
molecular diagnostic assays. Although primarily intended to be used for performing FDA-approved or
cleared assays, these systems incorporate a feature (cobas omni Utility Channel) that allows end-users
to develop, optimize, and then perform laboratory developed tests (LDTs) on the platform. We have
previously described an assay construct for the diagnosis of bacterial vaginosis (BV) that employs semi-
quantitative PCR-based detection of 3 sentinel markers for this condition; this assay is presently run
concurrently with a test for detecting the principal etiologic agents of vaginal candidiasis (CAN), Candida
albicans (CA) and Candida glabrata (CG). We report here on the development of modified versions of
those assays designed to be performed on the c68/8800 system. A multiplexed primary assay (BV/CAN)
is performed initially; completing the BV component of testing and identifying samples containing
Candida spp. A secondary assay (CA/CG) is then performed on any Candida spp. positive samples to
establish the presence or absence of CA and CG. The BV component of the BV/CAN assay demonstrated
96.6% (1537/1591) concordance with results obtained using the previously developed BV-PCR assay.
The combination of BV/CAN and CA/CG tests demonstrated 96.8% (1459/1507) and 99.8% (1505/1507)
concordance for CA and CG, respectively, versus the reference PCR method. Modeling of the workflow
impact of converting to the newly developed assay construct on the c8800 system indicated that a 2.5x
increase in throughput (960 versus 360 reportable results per 8h shift) could be achieved whilst
replacing 4 independent instruments with one integrated system and eliminating 77% (41/53) of the
process steps and 60% (4/7) of the risk points. Introduction of the c68/8800 systems provide a
significant opportunity for clinical laboratories to automate molecular LDTs.
Session Number: 40
Session Number: 40
Abstract Title:
Evaluation of Rapid Polymyxin NP Test for Early Detection of Colistin Nonsusceptibility in Resource
limited setting
P. Gupta, S. Sengupta, A. Kaur; Medanta, The Medicity, Gurugram, India
Abstract Body:
Background<br />India has 2nd highest prevalence (57%) of carbapenem resistance among Klebsiella
pneumoniae, leading to increased use of colistin. This has led to raised colistin resistance and pan drug
resistant strains in healthcare. Challenges in polymyxin susceptibility reporting persist as CLSI guidelines
recommend broth microdilution method (BMD) as standard. This is laborious and time consuming for
routine clinical practice. In clinical laboratory, the methods usually adopted are agar diffusion (E test,
disc diffusion, and automated systems) which are unreliable. In this study, we aimed to evaluate rapid
polymyxin Nordman Poriel test (RPNPT) as a reliable, rapid method, comparing it with the routine
modalities of colistin susceptibility testing.<br />Method<br />160 non-duplicate clinical isolates of K.
pneumoniae with known colistin susceptibility (colistin susceptible = 100, colistin resistant =60)
recovered from blood cultures were tested by BMD, rapid polymyxin test (RPNPT), gradient agar
diffusion method (E strip), and automated method (Vitek2, bioMeriuex France). RPNPT method was
followed as described by Nordman et al. The isolates were incubated aerobically in test reagent
containing cation adjusted Muller Hinton broth, red phenol indicator, 10% (D+) - glucose and 5μg/ml
colistin sulfate for 4 hours. Colistin non-susceptibility was identified by color change from orange to
yellow.<br />Results <br />Considering BMD as the standard reference method, E test and Vitek2 failed
to demonstrate colistin non-susceptibility in 15 and 7 isolates respectively, whereas RPNPT failed to
demonstrate non-susceptibility in 3 isolates. The non-susceptibility was detected by RPNPT in as early as
2 hours whereas in other methods results were available in 12 to 24 hours.<br />Conclusion <br
/>Resource limited nations like India where colistin resistant organisms are acquiring the status of
endemicity in healthcare; implementation of RPNPT routinely can be a simple and practical solution. The
rapidity of the test result can contribute in preserving polymyxin as the “last resort” antibiotic. Clinicians
can be informed early, for institution of appropriate antimicrobial therapy contributing to stewardship
measures. The results also help in implementation of contact isolation measures by infection control
team, preventing the dissemination of these multi drug resistant bugs within the hospital settings.
Session Number: 40
Session Number: 40
Abstract Title:
A Novel Phenotypic Method to Screen for Plasmid-Mediated Colistin Resistance
D. T. Bell, Y. Bergman, P. D. Tamma, P. J. Simner; Johns Hopkins Univ., Baltimore, MD
Abstract Body:
Background: Plasmid-mediated colistin resistance (PMCR), a consequence of the mcr genes, is a
significant public health concern given its potential to easily spread among clinical pathogens. Recently,
it was discovered that MCR enzymes require zinc for activity. The purpose of this study was to further
develop the colistin broth-disk elution test (CBDE) to screen for plasmid-mediated colistin resistance
genes based on the reduction of colistin MIC in the presence of EDTA (divalent cation chelator).
Methods: Broth microdilution (BMD) and CBDE were used to determine colistin MICs in 72
Enterobacteriaceae isolates. Isolates tested included, 7 MCR-1 producing Escherichia coli and 65 clinical
carbapenem-resistant Enterobacteriaceae lacking mcr genes (7 with colistin MICs ≥4 µg/mL). CBDE is
performed with 4, 10 ml cation-adjusted Muller Hinton broth (Remel) tubes per isolate, to which 0, 1, 2,
and 4 colistin disks (10 µg; BD) are added, generating a final concentration of 0 (growth control), 1, 2
and 4 µg/mL. Tubes are incubated at room temperature for 30min, after which a 50 µL aliquot of a 0.5
McFarland inoculum suspension is added. Colistin MIC values are read visually, after 18-20 h incubation
at 35C. To detect PMCR, a second set of tubes were setup in parallel with a set concentration of EDTA
(Sigma) added to each tube to observe a reduction of colistin MIC. Any reduction was considered
positive. Nine isolates (two of which were mcr-1 positive) were initially evaluated with and without
EDTA at varying concentrations (1, 2, 5mM). Results: Initial optimization of EDTA concentration revealed
that 1mM EDTA was sufficient to reduce the MIC of colistin from 2-4 µg/mL to ≤1 µg/mL in mcr-1
bearing isolates, while having little effect on isolates lacking mcr genes with varying colistin MICs.<br
/>Table 1: CBDE ± 1 mM EDTA Results for 72 Enterobacteriaceae with Varying Colistin MICs<br /><table
class="AbstractTable" id="{69E67ECF-1266-4405-A659-B772E82AB025}"><caption
class="AbstractTableCaption"></caption><tr><td rowspan="1" colspan="1"></td><td rowspan="1"
colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td
rowspan="1" colspan="1"></td></tr><tr><td rowspan="2" colspan="1">Isolates <br />(BMD colistin
results)</td><td rowspan="2" colspan="1">N</td><td rowspan="2" colspan="1">CBDE colistin MIC
(µg/mL) without EDTA</td><td rowspan="1" colspan="2">CBDE with 1 mM EDTA</td></tr><tr><td
rowspan="1" colspan="1"># of isolates with a reduction of colistin MIC </td><td rowspan="1"
colspan="1">MIC Reduction</td></tr><tr><td rowspan="1" colspan="1">mcr-1 positive strains <br />(2-
8 µg/mL)</td><td rowspan="1" colspan="1">7</td><td rowspan="1" colspan="1">2 (N=2)<br />4
(N=5)</td><td rowspan="1" colspan="1">7 (100%)</td><td rowspan="1" colspan="1">≥1-2-
fold</td></tr><tr><td rowspan="1" colspan="1">CRE <br />(MIC ≤ 2 µg/mL)</td><td rowspan="1"
colspan="1">58</td><td rowspan="1" colspan="1">≤1 (N=53)<br />2 (N=3)<br />>4 (N=2)a</td><td
rowspan="1" colspan="1">3 (5%)</td><td rowspan="1" colspan="1">≥ 1 fold</td></tr><tr><td
rowspan="1" colspan="1">CRE<br />(MIC ≥4 µg/mL)</td><td rowspan="1" colspan="1">7</td><td
rowspan="1" colspan="1">>4</td><td rowspan="1" colspan="1">1 (14%)</td><td rowspan="1"
colspan="1">≥ 1-3-fold</td></tr></table><br />a 2 Enterobacter cloacae isolates had MICs of >4 ug/ml
by CBDE and ≤0.25 ug/ml by BMD<br />The sensitivity and specificity of the CBDE with EDTA method to
detect PMCR was 100% and 94%, receptively.<br />Conclusions: Here we have developed a user-friendly
screen for PMCR. Our findings suggest that isolates with a reduction of colistin MIC with EDTA should be
further evaluated for the presence of mcr genes.
Session Number: 40
Session Number: 40
Abstract Title:
Rapid Antimicrobial Susceptibility Testing of Positive Blood Cultures using Serum Separator Tubes and
Vitek2
M. Brandt1, K. McCullor2, D. Harris1, Z. Ratzlaff1, E. Thompson1; 1Norman Regional Hlth.System,
Norman, OK, 2Univ. Of Oklahoma Hlth.Sci. Ctr., Oklahoma City, OK
Abstract Body:
Background: Rapid identification and antimicrobial susceptibility testing (AST) of blood cultures is
essential in improving patient outcome. New diagnostic platforms provide more rapid identification and
susceptibility options but remain cost prohibitive. The aim of our study was to evaluate a protocol
utilizing serum separator tubes to facilitate a faster, cost effective direct method for rapid sensitivity
testing of positive blood cultures using Vitek-2. Literature assessing this method has predominantly
been European and has not included molecular testing for identification within the testing protocol.
Methods: Blood culture bottles (BD BactecTM Aerobic plus and Anaerobic/F) were collected using
standard methods and monitored on the BactecTM FX system. Positives were gram stained to
determine the appropriate molecular ID testing, Cepheid Xpert® MRSA/SA BC (gram positive cocci) or
Biofire FilmArray® Blood culture ID panel (gram negative rods and gram positive cocci in chains/pairs).
Coagulase-negative staphylococci and mixed cultures (by gram stain) were excluded from the study. To a
BD Vacutainer® SST, 3 mL of blood was aseptically added and spun (3000 RPM for 15 minutes), and
supernatant decanted. A 0.5 McFarland suspension was used for AST. To represent rare occurring
organisms, 0.1 mL of a 0.5 McFarland suspension was added to blood culture bottles containing 3-5mL
of sterile blood. A blood-only control was included to ensure sterility. Results: Of the 155 isolates tested
(91 gram negatives and 64 gram positives), we observed a 99% essential agreement with 0.5% minor (5
isolates), 0.1% major (1), and 0.3% very major (3) error rates for the 930 antimicrobial-organism
combinations tested. The majority of errors occurred with gram negative isolates and Bactrim (SXT).
Excluding MRSA, all gram positive isolates tested were in agreement with the gold standard
susceptibility values. The majority of MRSA isolates tested (10/14) were positive by cefoxitin screen but
determined oxacillin susceptible by Vitek. Conclusion: Excluding SXT testing for gram negatives, the
serum separator tube method for direct AST demonstrated a reliable, more rapid methodology. MRSA
isolates did have discordant results (cefoxitin screen positive but oxacllin susceptible) however, cefoxitin
is given priority for interpretation and the Cepheid Xpert® panel denotes the presence of mecA. Utilizing
these methods, we observed no misidentification of MRSA as MSSA. The turnaround time was
calculated to be 24-48 hours faster than the current standard subculture method.
Session Number: 40
Session Number: 40
Abstract Title:
Successful Isolation of a Treponema pallidum Strain from a Penile Ulcer using the Rabbit Model
L. E. Pereira1, S. S. Katz1, Y. Sun1, P. Mills1, P. Atkins2, K-H. Chi1, D. Danavall1, E. Kersh1, Y. Fakile1, C. Y.
Chen1, S. Philip3, A. Pillay1; 1CDC, Atlanta, GA, 2Charles River Lab., Wilmington, MA, 3San Francisco
Dept. of Publ. Hlth., San Franscisco,
Abstract Body:
Increasing syphilis rates in the US have prompted a CDC call to action. Limited understanding of
molecular epidemiology of syphilis and predominance of certain T. pallidum strains in the US raise a
need for better molecular and pathogenic strain characterization. Freshly isolated T. pallidum from
clinical specimens will aid development of molecular genetic tools. T. pallidum can only be grown in the
rabbit model. We report the first successful rabbit propagation of T. pallidum from a cryopreserved
ulcer specimen as part of an advanced molecular detection funded study. A patient presented with a
primary lesion on the coronal sulcus that was positive by dark-field microscopy (DF) and reactive by
Venereal Disease Research Laboratory test (titer 1:4). The ulcer was cleaned with sterile saline and a
Dacron swab was gently rolled along the base of the ulcer, immediately agitated in a vial of 1ml (50%
v/v) glycerol and normal rabbit serum (NRS) and discarded. The vial was snap-frozen in liquid nitrogen
(LN), stored at -800C, shipped to CDC on dry ice, then stored in LN until use. The vial was thawed by
mixing with 0.5ml of pre-warmed (370C) NRS. The specimen was injected intravenously and in the left
testicle of an adult male rabbit aged 8 months with 0.6ml inoculum/site. Residual inoculum yielded 1.1 x
104 T. pallidum/ml by qPCR but was negative by DF. Blood was drawn once a week for PCR and syphilis
serology, with orchitis monitoring twice a week. The animal was euthanized on week 7 based on
increased antibody titers and testicular edema. Left testicle tissue was processed and 1.5ml of extract
was injected into the left testicle of a second age-matched rabbit followed by similar monitoring until
euthanasia on week 3. The first rabbit was seroreactive from week 6 (Treponemal pallidum Particle
Agglutination [TPPA] titer 1:160, Rapid Plasma Reagin [RPR] minimally reactive) and PCR+ from week 5.
Orchitis was apparent on week 6 with increasing edema. Antibody titers increased to 1:2560 (TPPA) and
1:32 (RPR) by week 7. Left testis extract was PCR+, containing 1.7 x 107/ml treponemes as determined
by DF. The second passage rabbit yielded 2.6 x 106/ml treponemes and was seroreactive from week 1
(TPPA titer 1:80, RPR-) while T. pallidum detection in blood by PCR and orchitis were evident from week
2. Our results show that immediate cryopreservation of syphilitic ulcer exudate can maintain viability of
T. pallidum for subsequent rabbit propagation. Performing serology and PCR in real time with orchitis
monitoring can also guide propagation timelines for uncharacterized clinical strains.
Session Number: 40
Session Number: 40
Abstract Title:
Clinical Performance of T2Bacteria among Patients with Bloodstream Infections Due to Five Common
Bacterial Species
M. Nguyen1, A. Pasculle1, P. Pappas2, G. Alagadin3, G. Pankey4, B. Schmitt5, M. Weinstein6, R. Widen7,
D. Hernandez8, D. Wolk9, T. Walsh10, J. Perfect11, C. CLANCY1, E. Mylonakis12; 1Univ. of Pittsburgh,
Pittsburgh, PA, 2Univ. of Alabama, Birmingham, AL,
Abstract Body:
Background: Blood cultures (BC) may require >24-48 hours to detect bacteremia. We evaluated a rapid,
culture-independent diagnostic system, based on T2 Magnetic Resonance, for direct detection of
bacteria in whole blood. Materials/Methods: In a prospective study at 11 U.S. hospitals, we compared
the T2Bacteria Panel with BC for diagnosing bacteremia. T2Bacteria identifies six targeted bacteria.
Blood for culture and T2Bacteria was obtained concurrently, and processed per standard procedures
and manufacturer’s instructions, respectively. Results: T2Bacteria lower limit of detection (CFU/mL) was
Acinetobacter baumannii (Ab),3; Escherichia coli (Eci), 11; Enterococcus faecium (Efm), 5; Klebsiella
pneumoniae (Kp), 2; Pseudomonas aeruginosa (Pa), 5; Staphylococcus aureus (Sa), 2. 1,427 blood
samples were tested. BC grew 85 bacterial species, 46% (39) of which were T2Bacteria-targeted: 16 Sa,
11 Eci, 6 Kp, 5 Pa, 1 Efm. T2Bacteria sensitivity and specificity based on BC results were 90% (35/39) and
88% (1220/1388), respectively. Sensitivity was 100% Efm, Kp and Pa, 91% Eci, and 81% Sa. There was no
BC-confirmed Ab bacteremia. Specificity was ≥98% Efm, Kp, Pa and Sa, and 95% Eci. 176 samples from
168 patients had T2+/BC- results and 4 had T2-/BC+. 24% (42) of T2+/BC- were in samples from patients
who had received an active antibiotic against the identified bacteria in the previous 21 days. 23% (41) of
T2+/BC- were from patients with positive blood cultures for the same species within the 21 preceding
days. 16% (28) of T2+/BC- were from patients with positive non-blood site cultures in the preceding 14
days. 19% (34) were obtained from patients with presumed infection due to compatible clinical scenerio
but either BC were not drawn or negative. Including these samples as true infection, recalculated
specificty was 95% (1315/1388). An explanation for T2+ results was not apparent for the remaining 73
T2+/BC- samples, which were considered false positive (30Eci, 11Ab, 19Pa, 7Kp, 3Efm, 3Sa). 89% (60/69)
of these samples were negative upon retesting. Conclusions: The T2Bacteria Panel demonstrated
excellent performance in detecting bacteremia. Advantages of the panel over BC are time to test
positivity and potential for diagnosing deep-seated infection. A majority of T2+/BC- results were
obtained in patients receiving antibiotics, or who had the same bacteria recovered from prior blood or
non-blood site cultures. Ongoing analysis of outcomes of patients with discordant results may provide
insights into the clinical significance of these findings.
Session Number: 40
Session Number: 40
Abstract Title:
Lipopolysaccharide of Marinobacter litoralis inhibits swarming motility and biofilm formation in
Pseudomonas aeruginosa PA01.
R. Sardar, Male; M L T Coll., saharsa, India
Abstract Body:
Introductions: Lipopolysaccharide is the integral component of cell-wall of all Gram-negative bacteria. It
is an amphiphilic macromolecule consists of lipid A, core oligosaccharide, and O-specific polysaccharide.
O-specific antigen determines bacterial O-serotype, and defines as fingerprint of bacteria. The LPS from
marine bacteria repeatedly show unusual chemical features most likely due to their external
environment. The number, nature and distribution of the fatty acid chains vary according to the genus,
and are responsible for bioactivity of LPS. Marine LPS show low virulence potential and compete with
the non-toxic LPS. Bacterial biofilms are inherently resistant to antimicrobial agents and detergents.
Polysaccharides portion in LPS enable to inhibit rhl system which is necessary in biofilm formation in P.
aeruginosa. Methodology: The marine bacteria were identified based on molecular and bio-chemical
methods. Identified bacteria were harvested for extraction of LPSs following the hot phenol method.
The isolated LPSs were purified and fatty acids and sugar composition were characterized by GCMS. The
isolated LPSs were tested for pyrogenicity, antibiofilm activity, swarming and pyocynin reduction activity
and also compared. Result: The marine bacterium identified as M.litoralis was used for LPS study. LPS
was constituted of (C12:0 3OH) (49%), (C12:0) (24%) and (C10:0) (19%) as major fatty acids, and the
polysaccharide constituents were fucose (53.79%), xylose (28.04%) and mannose (18.15%). The LPS did
not show any LAL gelation activity. The isolated LPS showed maximum 50 % inhibition in biofilm
formation only in P. aeruginosa PA01. Strong inhibition in swarming motility and reduction in pyocynin
production were observed by treatment of 100 μgmL-1 isolated LPS of M.litoralis. Variation in structural
composition and bioactivity of LPS of M. litoralis was noticed when compared with LPS isolated from
marine bacterium Idiomarina fontislapidosi. Conclusions: The presence of shorter fatty acid chains in the
LPS and its negative LAL test make it a potential candidate for development of drugs to prevent septic
shock. The LPS inhibited biofilm formation in P.aeruginosa that may be due to inhibition of swarming
motility and production of the virulence factor pyocyanin. The finding of this study suggests that the LPS
of M. litoralis differ in composition and bioactivity with I. fontislapidosi may help in eradicating the
biofilm-associated secondary infections in immunocompromised patient.
Session Number: 40
Session Number: 40
Abstract Title:
Whole Genome Sequencing in outbreak detection of Salmonella outbreaks: one size does not fit all
M. Chattaway, T. Dallman, S. Nair, H. Hartman, M. Day, A. Painset, L. Larkin, J. McCormick, N. Chandra,
P. Crook, P. Crook, K. Grant; Publ. Hlth.England, London, United Kingdom
Abstract Body:
Background: Whole genome sequencing (WGS) is having a revolutionary effect on reference
microbiology and provides access to a wealth of data to inform strain virulence, antimicrobial resistance
and clarify phylogenetic relationships. In April 2015, the PHE Gastrointestinal Bacterial Reference Unit
(GBRU) was the first reference laboratory internationally to introduce WGS and single nucleotide
polymorphism (SNP) analysis as a routine method for typing Salmonella. The laboratory receives
approximately 9,000 Salmonella isolates each year, principally from cases of human illness but also
from, the environment, foods and animals. The real time availability and use of this enhanced
information is having a ground breaking impact on our ability to monitor and investigate infectious
disease and will lead ultimately to better control and preventative action. Methods: The use of single
nucleotide polymorphism (SNP) typing has highlighted the complexity of microbial populations in
different outbreaks of Salmonella and typically SNP typing analysis is used as part of the case definition.
In August 2017, Public Health England was alerted of a cluster of cases of a rare serovar, Salmonella
Adjame, in North-West London; cgMLST and SNP typing was undertaken to further characterize the
strains. Results: SNP analysis showed three sub-clusters and considerable genetic variation (up to 692
SNPs between clusters) across human samples. Despite the genetic heterogeneity, epidemiological
signals indicated cases were likely to be part of single outbreak; because of this, the case definition was
based on serovar rather than fine microbiological typing. Cases were mainly older and of South Asian
descent, and no international travel was reported. Although no food vehicles were identified and the
complexity of supply chains made it difficult to determine the potential source of contamination, it was
suspected to be a fresh product bought from Indian grocers and most likely an imported product.
Conclusions:This is the first outbreak in the UK where SNP analysis was not used as part of the case
definition in a Salmonella outbreak since the introduction of routine WGS and SNP analysis due to the
heterogeneity of the strains. This study highlighted the complexity of interpretation of WGS in case
definitions, therefore standard thresholds for consideration of genetic similarity in outbreak settings
should be used with caution and in light of the context of the epidemiology of the outbreak.
Session Number: 40
Session Number: 40
Abstract Title:
Detection of blaOXA-23 and characterization of MDR Acinetobacter isolates in New York State
S. Morris, K. Mitchell, C. Marwali, E. Snavely, J. Bodnar, C. Wagner, D. Baker, L. Thompson, P. LaPierre, N.
Dumas, E. Nazarian, K. Musser; New York State Dept. of Hlth., Wadsworth Ctr., Albany, NY
Abstract Body:
In August 2016, state and local public health laboratories were funded by the Center for Disease
Control’s Epidemiology and Laboratory Capacity Cooperative agreement to collect, confirm, and
characterize carbapenem-resistant Enterobacteriaceae (CRE) and Pseudomonas aeruginosa (CRPA)
isolates. The goal is to provide characterization of the CRE and CRPA isolates to continue to inform the
understanding of carbapanem resistance. The Wadsworth Center (WC), as the Northeast Regional
Antimicrobial Resistance Laboratory Network laboratory is also funded to perform CRE colonization
testing from rectal swab specimens for the detection of epidemiologically important CRE and recruit for
a targeted surveillance isolate collection. This surveillance project will allow regional laboratories to to
contribute to and understand emerging or changing antimicrobial resistant threats. The emergence of
multi-drug resistant (MDR) Acinetobacter baumannii is currently a focus of this surveillance particularly
with respect to carbapenem resistance. Acinetobacter species, predominantly, A. baumannii, are
emerging pathogens worldwide, and commonly involved in healthcare associated infections. A.
baumannii exhibit drug resistance to carbapenems mainly from acquisition of OXA-type carbapenemase
genes, particularly blaOXA-23, blaOXA-40, and blaOXA-58. There are few therapeutic options for the
treatment of infections caused by these MDR A. baumannii and carbapenems are one of the remaining
options and considered a last resort drug for treatment. To improve detection of these pathogens, WC
developed and validated a TaqMan® real-time PCR assay for the detection of blaOXA-23 DNA in clinical
MDR A. baumannii isolates. This assay was determined to be highly sensitive, specific, and reproducible.
In addition, a blinded validation study of 40 samples was performed, with 100% concordance. Testing
was implemented in October 2017, and to date 6 positive Acinetobacter isolates have been identified.
All positive samples are confirmed with WGS, and additional carbapenem resistance mechanisms have
been identified. Furthermore, pulsed-field gel electrophoresis and SNP analysis has been performed on
MDR Acineteobacter outbreaks identified in NYS in 2016 and 2017. Finally, a retrospective analysis of 57
archived Acinetobacter isolates from 2011 to 2016 was performed. Future work will include assay
validation on rectal swabs which will prove useful in understanding the transmission of Acinetobacter in
healthcare facilties.
Session Number: 41
Session Type: Poster
Session Number: 41
Session Title: CPHM02 - Antimicrobial Susceptibility Testing: ESBL and Carbapenemase Testing
Poster Board Number: FRIDAY - 205
Abstract Title:
A Hosp. and Community-Based Study to Promote Antimicrobial Stewardship in Nepal: Molecular Esbl
and Carbapenemase Identification of Acinetobacter, E. coli, And K. pneumoniae Isolates from
Kathmandu Model Hospital
E. Olson1, M. Zervos2, L. Kaljee2, P. Kilgore1, R. Tibbetts2, E. Herc2, B. Shrestha3, D. Bajracharya4, D.
Joshi3, R. Joshi3, National Public Health Laboratory, Kathmandu, Public Health Concern Trust (Phect),
Kathmandu, Group for Technical Assistance, Kath
Abstract Body:
Background: In Kathmandu Model Hospital (KMH), as part of an ongoing project on antimicrobial
stewardship program, this study evaluated the prevalence of extended spectrum beta lactamase (ESBL)
in isolates of Klebsiella pneumoniae, and Escherichia coli and of carbapenem resistance in gram negative
bacterial isolates including Acinetobacter calcoacetricus baumanii complex (ACBC), E. coli, and K.
pneumoniae isolates from inpatients and outpatients. Methods: KMH is a 125-bed private hospital
located in the Kathmandu Valley, Nepal. Surgical, general medicine, and obstetrics/gynecology
departments at KMH were included in the study. Patient cultures were collected from May through
August 2017. Cultures were processed by the KMH clinical laboratory using conventional microbial
identification methods and Kirby-Bauer disc diffusion. Genomic and plasmid DNA extraction with
DNeasy blood and tissue and Plasmid kits (Qiagen, Tokyo, Japan) was performed on cultures identified
as ACBC, E. coli or K. pneumoniae with resistance patterns indicating ESBL producers or carbapenem
resistance. PCR amplification of CTX, SHV, and TEM genes from both genomic and plasmid DNA was
performed to verify the presence or absence of resistance genes. Results: Genomic DNA was evaluated
from 77 isolates and plasmid DNA from 50 isolates. These isolates (obtained from 52 inpatients and 25
outpatients) included 41 E. coli, 21 ACBC and 15 K. pneumoniae isolates. Isolate origin included 47 from
urine, 22 from sputum, seven from pus, one from body fluid, one from tissue, and one of unspecified.
Forty-six genomic isolates and 31 plasmid isolates were found to have CTX resistance genes. Thirty-one
genomic and eighteen plasmid isolates were found to have TEM resistance genes. Twenty-one genomic
and thirteen plasmid isolates were found to have SHV resistance genes. Conclusion: This study shows
CTX, TEM, SHV genes in both community and health care associated isolates of multi antibiotic resistant
gram-negative bacteria from patients attending an urban hospital in Nepal. These findings underscore
the urgent need for community-and hospital-based programs that improve the use of a wide array of
important antimicrobial agents.
Session Number: 41
Session Number: 41
Abstract Title:
Impact of Removing Esbl Designation from Culture Reports on the Selection of Antibiotics for the
Treatment of Infections Associated with Esbl-Producing Organisms
M. Lesher, C. Hale, D. S. S. Wijetunge, M. England, D. Myers, D. Craft; Penn State Hlth., Hershey, PA
Abstract Body:
Introduction: Implementation of revised breakpoints (RB) for cephalosporins and eliminating ESBL
confirmatory results may confuse clinicians when selecting antibiotic therapy for ESBL producers. In May
2017, our institute implemented new breakpoints for Gram negative organisms to comply with CLSI MIC
RB. Prior to this it was common to prescribe carbapenems for the treatment of infections due to ESBLs,
regardless of beta-lactam/beta-lactamase inhibitor (BL/BLI) susceptibility test results. This practice
evolved due to concerns for impaired efficacy on the basis of ESBL production. The removal of the ESBL
designation from reports in the Electronic Medical Record (EMR) was therefore hypothesized to reduce
carbapenem and increase BL/BLI prescribing for treatment of infections due to ESBLs. The objective of
this study was to evaluate the impact on antibiotic selection by removing ESBL designation from
microbiology results in the EMR. Methods: This was a retrospective chart review of patients with
positive cultures for ESBLs during the 6 months before (Pre-RB) and after (Post-RB) the implementation
of RB. ESBL production of all isolates used in the study was confirmed according to CLSI guidelines. EMRs
of patients with ESBLs were reviewed to retrieve the antibiotic therapy prescribed on the basis of
culture and susceptibility results. Data were further analyzed by treatment setting with a focus on
inpatients. This study was approved by IRB. Results: 253 patient charts were reviewed (Pre-RB, n=99
patients and Post-RB, n=154 patients). A total of 292 ESBL producing bacteria were identified in these
patients. Out of these, 58 were cultured from inpatients in the Pre-RB and 63 from inpatients in the
Post-RB. The most common organism from inpatients was E. coli (51.7% Pre-RB and 77.8% Post-RB). In
the Pre-RB period, 91.1% of inpatient patients with an ESBL isolate were treated compared to 78.3% in
the Post-RB period. Inpatient antibiotics utilized after cultures results were reported in the EMR
illustrated an increase in BL/BLI utilization (19.5% Pre-RB and 40.4% Post-RB) and a reduction in
carbapenem utilization (43.9% Pre-RB and 28.0% Post-RB). Conclusions: By implementing RB and
eliminating ESBL comments in the EMR, our data suggest a change in inpatient antibiotic prescribing. We
observed a reduction in carbapenem use and an increase in BL/BLI use. Further study of this change in
antibiotic usage for ESBL producing organisms and its impact on antibiotic treatment success and patient
outcomes is warranted.
Session Number: 41
Session Number: 41
Abstract Title:
Rapid Identification of Carbapenemase Producing Bacteria from Positive Blood Culture Broth
M. D. Patel, E. McElvania, K. Hambourger, A. Horwitz, S. Das, R. B. Thomson, Jr; NorthShore Univ.
Hlth.System, Evanston, IL
Abstract Body:
Background: Rapid identification and susceptibility testing of blood culture pathogens is of utmost
importance for the clinical microbiology laboratory. Conventional testing requires 18-48 hr. Most blood
stream pathogens can be identified by MALDI-TOF from agar subculture in 6 hours. We describe rapid
resistance screening to identify MDRO Enterobacteriaceae, Acinetobacter baumannii and Pseudomonas
aeruginosa using direct disk diffusion. Methods: 63 Gram-negative bacterial isolates from the FDA-CDC
isolate bank [A. baumanii (n=11) Enterobacteriaceae (n=48); P. aeruginosa (n=4);] containing a variety of
carbapenemase enzymes [NDM (n=12); KPC (n=9); OXA (n=13); IMP (n=4); VIM (n=5)] and non-
carbapenemase producing controls (n=20) were seeded into blood culture bottles in duplicate. Once the
seeded bottles signaled positive, broth was removed from the bottle, and bacteria were washed and
resuspended to 1.0 McFarland concentration. Disk diffusion antimicrobial susceptibility testing was
performed using meropenem, ertapenem, cefepime, ceftriaxone, and ceftazidime. Kiestra Total
Laboratory Automation (TLA) was used for incubation and imaging. Agar plates were imaged at 4, 5, 6, 7,
and 8 hours of incubation to determine the earliest time point at which carbapenemase production
could be reliably determined, using 18 hr incubation as the gold standard. Results: Our resistance
screening assay detect carbapenemase producing Enterobacteriaceae and A. baumanii starting at 4 hr
incubation, with 97% isolates correctly identified by 6 hr (57/59) and 100% correctly identified by 7 hr
(Table 1). Slow growth of P. aeruginosa on agar plates hindered resistance detection until time points of
≥ 8 hr. Conclusions: Antimicrobial resistance screening using disk diffusion with TLA provides a rapid and
cost-effective method for the detection of carbapenemase production by Gram-negative bloodstream
isolates. Combined with MALDI-TOF, the use of antimicrobial resistance screening allows clinicians to
transition patients to effective antimicrobial therapy more rapidly. Table 1: Hours of incubation
necessary to detect carbapenemase production.<table class="AbstractTable" id="{A040F7C2-1E5E-4756-
AD80-9D36D68A571C}"><caption class="AbstractTableCaption"></caption><tr><td rowspan="1"
rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1"
colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td
rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1"> </td><td rowspan="1"
colspan="6">Hours of Incubation</td></tr><tr><td rowspan="1" colspan="1">Organism</td><td
rowspan="1" colspan="1">n=</td><td rowspan="1" colspan="1">4 h</td><td rowspan="1"
colspan="1">5 h</td><td rowspan="1" colspan="1">6 h</td><td rowspan="1" colspan="1">7 h</td><td
rowspan="1" colspan="1">8 h</td><td rowspan="1" colspan="1">18 h</td></tr><tr><td rowspan="1"
colspan="1">Acinetobacter baumanii</td><td rowspan="1" colspan="1">11</td><td rowspan="1"
colspan="1">0 (0%)</td><td rowspan="1" colspan="1">3 (27%)</td><td rowspan="1" colspan="1">11
(100%)</td><td rowspan="1" colspan="1">11 (100%)</td><td rowspan="1" colspan="1">11
(100%)</td><td rowspan="1" colspan="1">11 (100%)</td></tr><tr><td rowspan="1"
colspan="1">Enterobacteriaceae</td><td rowspan="1" colspan="1">48</td><td rowspan="1"
(96%)</td><td rowspan="1" colspan="1">48 (100%)</td><td rowspan="1" colspan="1">48
(100%)</td><td rowspan="1" colspan="1">48 (100%)</td></tr><tr><td rowspan="1"
colspan="1">Pseudomonas aeruginosa</td><td rowspan="1" colspan="1">4</td><td rowspan="1"
(0%)</td><td rowspan="1" colspan="1">0 (0%)</td><td rowspan="1" colspan="1">2 (50%)</td><td
rowspan="1" colspan="1">4 (100%)</td></tr></table>
Session Number: 41
Session Number: 41
Abstract Title:
Evaluation of Modified Carbapenem Inactivation Method (Mcim) for Phenotypic Detection of
Carbapenemase Producing Enterobacteriaceae
D. S. S. Wijetunge, D. Myers, D. Craft; Penn State Hlth., Hershey, PA
Abstract Body:
Introduction: Carbapenemase Producing Carbapenem Resistant Enterobacteriaceae (CP-CRE) pose a
major challenge to healthcare due to limited treatment options as well as spread in health care facilities.
Accurate identification and reporting of CP-CRE is a strategy for infection control and prevention of CRE
dissemination. The mCIM is a phenotypic test recently recommended by CLSI to detect CP-CRE. The
objective of this study is to evaluate the performance of mCIM for detection of CP-CRE in our facility.
Methods: 161 CRE isolates were used in the study, including 84 isolates obtained from the CDC
Antimicrobial Resistant (CDCAR) isolate bank and 77 clinical isolates from our facility from 2015-2017.
mCIM was performed according to CLSI guidelines (M100S27E). Briefly, a suspension of each isolate was
incubated for 4 hours at 37°C with a 30µg meropenem disk. The disk was then removed and placed on a
lawn of meropenem susceptible E. coli ATCC 25922 strain and incubated overnight. Carbapenemase
production was evaluated using the inhibitory zone diameter interpretive criteria established by CLSI.
Typing of carbapenemases in patient isolates was performed using multiplex PCR targeting blaKPC,
blaNDM and blaOXA48 genes as previously described. CDCAR isolates were characterized by whole
genome sequencing (WGS) at CDC. Results of mCIM test were compared to carbapenemase encoding
genes detected by WGS or targeted PCR. Results: CDCAR isolates included 58 CP-CREs that were positive
for seven different carbapenemases (blaKPC=23, blaNDM=20, blaSME=6, blaIMI=2, blaVIM=2,
blaOXA181=4 and blaOXA48=1) and 26 non-CP CREs. The only carbapenemase detected among our
patient isolates was blaKPC (n=71). mCIM sensitivity and specificity was 96.6 % and 96.2% for CDCAR
isolates and 95.7% and 83.3 % for clinical isolates. For clinical isolates, E. cloacae bearing blaKPC showed
indeterminate mCIM test result. In both populations, false negative mCIM results were associated with
blaKPC positive isolates of K. pneumoniae (n=3), M. morganii (n=1) and P. mirabilis (n=1). False positive
mCIM results were observed with two isolates, E. aerogenes (CDCAR) and Providencia spp. (clinical).
Conclusions: The mCIM test showed a high level of sensitivity for CP-CRE in both CDCAR and clinical
isolates indicating that mCIM is a sensitive method to detect CP-CRE. However, mCIM specificity was
lower for clinical isolates than CDCAR isolates. Additional isolates of non CP-CRE should be tested to
further assess the specificity of mCIM for patient isolates in a health care facility.
Session Number: 41
Session Number: 41
Abstract Title:
Old Breakpoint, New Breakpoint: Reliance on Historical Breakpoints for Enterobacteriaceae Creates
Discrepancies in Interpretation of Susceptibility Testing for Carbapenems and Gaps in Detection of Cre
M. L. Yarbrough1, M. A. Wallace1, R. F. Potter1, A. W. D'Souza1, S. Andleeb2, G. Dantas1, C-A. D.
Burnham1; 1Washington Univ. Sch. of Med., Saint Louis, MO, 2Atta-ur-Rahman Sch. of Applied BioSci.s,
Natl. Univ. of Sci. and Technology, Islamabad, Pakistan
Abstract Body:
Background: Carbapenem-resistant Enterobacteriaceae (CRE) are an urgent threat to public health. CRE
are typically detected by antimicrobial susceptibility testing (AST) results upon recovery in clinical
cultures. Although AST interpretive standards for Enterobacteriaceae were updated in 2010 for
carbapenems and 2014 for cefepime, many laboratories have yet to adopt new breakpoints (BPs). The
objective of our study was to measure the impact of BP changes on AST interpretation for CRE.
Methods: Clinical and environmental CRE isolates (n = 358) with meropenem zone sizes of ≤ 23 mm
were analyzed. Zone sizes from disk diffusion testing were recorded for cefepime and carbapenems and
interpreted according to historical (CLSI M100-S19) and current (CLSI M100-S28) BPs. Errors were
calculated according to FDA definitions. Results: In total, 180 (50%) Klebsiella pneumoniae, 35 (10%)
Escherichia coli, and 143 (40%) other Enterobacteriaceae species were analyzed (Table 1). Using M100-
S28 breakpoints as the reference method, 137 (38%) minor errors (mE) and 19 (5%) very major errors
(VME) occurred for categorical interpretation of cefepime using historical BPs. For meropenem, 110
(31%) mE and 87 (24%) VME occurred. The overall categorical agreement for CRE between historical and
current BPs was 56%, 45%, 42%, and 84% for cefepime, meropenem, imipenem, and ertapenem,
respectively. For 149 isolates that harbored a confirmed blaKPC gene, there were 70 (47%) mE and 10
(7%) VME for cefepime and 54 (36%) mE and 42 (28%) VME for meropenem. The overall categorical
agreement for KPC+ CRE between historical and current BPs was 46%, 36%, 23%, and 80% for cefepime,
meropenem, imipenem, and ertapenem, respectively. Conclusions: Failure to adopt new BPs may lead
to reports of false susceptibility for antimicrobials commonly used to treat Gram-negative infections and
preclude recognition of CRE. Such errors may negatively impact patient care and hamper infection
control efforts.<p><a
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Session Number: 41
Session Number: 41
Abstract Title:
Rapid Detection of Oxa β-Lactamases by Multiplex Real-Time Pcr
S. Cossette, M. Torres, J. Lechner, C. Connelly; Streck, LaVista, NE
Abstract Body:
Background: The OXA β-lactamases have evolved to metabolize cephalosporins and carbapenems, in
addition to the penicillins, making them a growing problem when selecting effective antibiotic therapies.
These enzymes are often associated with Acinetobacter spp., but, due to the mobility of these genes,
other organisms have acquired resistance to this class of enzymes facilitating the spread of this type of
antibiotic resistance. As such, assays that identify such resistance mechanisms are needed for faster
detection of a resistance-associated genes. This data can be used to supplement phenotypic test results
and promote improved antimicrobial stewardship and surveillance. In this study, we describe a multiplex
real-time PCR assay that successfully discriminates six genetically similar OXA β-lactamase gene families.
Methods: The NCBI GenBank database was used to identify sequences for each OXA gene family.
Sequences for each of the six groups were aligned to identify genetic variation between each OXA
group, which guided primer and probe design to improve discrimination of each OXA family within the
multiplex PCR reaction. A custom 2X qPCR MasterMix was used to optimize thermal cycling conditions
on the ABI QuantStudio 7 FLEX Real-Time PCR System for the multiplex and an internal control (IC), that
targets a conserved region in Gram-negative bacteria, was included to reduce false negative results.
Results: Together, the six oligo sets in this assay will amplify a total of 224 OXA-like variants without
cross reactivity between the OXA subgroups. Positive samples were identified within the first 30 cycles
of PCR. Sensitivity and Specificity for the control DNA tested in this assay was greater than or equal to
95% in each case. Conclusions: β-lactamases are a major mechanism of antibiotic resistance, in Gram-
negative bacteria, that continues to threaten health care facilities by reducing the available treatment
options. Additionally, there are many genes associated with antibiotic resistance and, as such, it is
critical that tests such as these are developed to comprehensively detect these mechanisms. The assays
described here provide a rapid detection strategy for genotypic monitoring of oxacillinase-based
antibiotic resistance in Gram-negative bacteria. More rapid identification of these genes provides an
added tool to improve antibiotic stewardship practices and active surveillance of resistance
mechanisms.
Session Number: 41
Session Number: 41
Abstract Title:
A Multicenter Comparative Evaluation of Four Phenotypic Tests for Detection of Carbapenemase-
Producing Enterobacteriaceae
M. Zhou1, Q. Yang2, Y. Xu2; 1Peking Union Med. Coll. Hosp., Bei, China, 2Peking Union Med. Coll. Hosp.,
Beijing, China
Abstract Body:
Background: With the worldwide increase in carbapenem resistance and transferable carbapenemases,
early identification of carbapenemase-producing Enterobacteriaceae (CPE) is of paramount importance
to prevent their dissemination within health care settings. Methods: We performed a comprehensive
method comparison study to assess the accuracy of these four screening methods, the modified Hodge
test (MHT), Carba NP test, meropenem hydrolysis assay (MHA) with 1/2 h incubation and modified
carbapenem inactivation method (mCIM) with meropenem (MEM), imipenem (IMP), and
ertapenem(ETP), in carbapenemases detection on a total of 342 carbapenem-resistant
Enterobacteriaceae (CRE) including 244 CPE and 98 non-CPE using genotypic assay as a gold standard.
Results: The 2 h-incubation MHA had an overall sensitivity, specificity, positive predicted value and
negative predicted value of 100%, the highest observed among all assays, followed by Carba NP with a
sensitivity of 99.6% while the 1 h-incubation MHA performed much worse with an overall sensitivity of
71.3%, mainly due to failure in KPC detection. Comparison between different carbapenem disks using
mCIM showed that MEM performed best among the three. The MHT performed worst among all assays
which was mainly manifested in specificity (88.8%). Comparison between specific carbapenemase
classes showed that all assays, except for the 1 h-incubation MHA, had a >98% sensitivity in detecting
the 172 KPCs, which failed to recognize 68 KPC-2 carbapenemase. Likewise, all assays demonstrated a
>95% sensitivity in detecting the 70 Class B carbapenemases except for MHT (82.9%) due to invalid
results for one IMP and 11 VIM producers. As for different species, almost all the assays had sensitivities
and specificities over 90% for the ten species with the notable exceptions in sensitivities for Klebsiella
pneumoniae (56.2%) and Serratia marcescens (75.0%) by 1 h-incubation MHA. Remarkably, the 2 h-
incubation MHA significantly improved the accuracy in CPE detection compared to 1 h-incubation MHA
and performed best in our study in all the species and classes of carbapenemases. Conclusions: To the
best of our knowledge, we are the first to report on the comparative performance of the mCIM with
MHA, along with MHT and Carba NP. Our findings suggested that the MHA was the most practical assay
to select for carbapenemase detection. For those who can’t afford the equipment at the moment, both
Carba NP and mCIM were good alternatives according to practical requirements of speed and cost.
Session Number: 41
Session Number: 41
Abstract Title:
Evaluation of Hardychrom™ Cre As A Detection Method of Carbapenem Non-Susceptible
Enterobacteriaceae: A Multi-Centric Study
E. Palavecino1, S. Whittier2, J. Turner1, M. Blevins1, M. L. Faron3, B. Buchan3, J. Connolly3, R. Myslymi2,
N. A. Ledeboer3; 1Wake Forest Univ. Baptist Med. Ctr., Winston-Salem, NC, 2NewYork-
Presbyterian/Columbia Univ. Med. Ctr., New York, NY, 3Med. Coll
Abstract Body:
Background: HardyCHROM™ CRE is a selective and differential chromogenic medium designed to screen
for carbapenem-resistant Enterobacteriaceae (CRE) from fecal specimens. Based on the colony color,
this medium differentiates Escherichia coli and Citrobacter spp. (pink) from members of the KES group,
namely Klebsiella spp., Enterobacter spp., and Serratia spp., (blue). Methods: HardyCHROMTM CRE was
compared to a traditional culture method utilizing a TSB enrichment at three different hospital
laboratories across the United States. Stool samples were inoculated onto HardyCHROMTM CRE plates
and incubated for 18 hours. Simultaneously, as the reference method, the samples were also inoculated
into 10 ml TSB with 1 µg/mL meropenem and 3 µg/mL vancomycin and incubated overnight followed by
subculture to MacConkey. The organisms growing on the CRE medium and on the MacConkey plates
were identified to the species level and tested for antimicrobial susceptibility using an FDA-cleared
system. CLSI breakpoints were used for interpretation of results. Results: A total of 1,638 fecal
specimens were tested on HardyCHROMTM CRE in parallel with the TSB enrichment. There were 96
target CRE organisms recovered by traditional culture. All of these were also recovered on
HardyCHROM™ CRE after 18 hours of incubation. HardyCHROM™ CRE was 91.4% sensitive for the
screening of carbapenem non-susceptible E. coli, Klebsiella spp., Enterobacter spp., Serratia spp,
Citrobacter spp. and 96.9% specific. There were 47 instances where HardyCHROM™ CRE recovered
target organisms while the reference method did not. Of the strains recovered by HardyCHROM™ CRE
but not the reference method, 7 were carbapenem NS E. coli strains, two were carbapenem NS
Citrobacter strains, and 38 were carbapenem NS KES strains. Conclusions: Overall, the data illustrates
the reliability of HardyCHROMTM CRE for the selective screening of carbapenem non-susceptible
microorganisms.
Session Number: 41
Session Number: 41
Abstract Title:
Stability Study of Beta-Lactamase Detection from Gram-Negative Bacilli Directly from Positive Blood
Cultures Using Two Commercially Available Pcr Kits
D. Hernandez1, A. Styer1, M. Updike1, N. Hanson2, D. Wolk1; 1Geisinger, Danville, PA, 2Creighton
Univ., Omaha, NE
Abstract Body:
Multi-drug resistant organisms (MDROs) are one of the greatest threats to public health. To prevent
morbidity and mortality due to sepsis and to target appropriate therapy, early detection of antibiotic
resistance is of extreme importance. Combined, the ARM-D® PCR kits for β-lactamase and ampC,
(Streck, Omaha, NE) detect over 450 variants including carbapenemase genes and are the only
commercially available molecular kits to detect plasmid-mediated ampC. The kits are based on real-time
PCR and use a Cq (quantification cycle) of 10 to 26 to qualify a positive reaction. Processing time is 50
min and run time is 45 min. This work represents the first time the ARM-D® kits are used in clinical blood
culture samples, the stability of blood culture samples after a positive flag were also assessed. To test
the feasibility of their use for β-lactamase gene detection from microbes in positive blood cultures, 8
isolates, characterized as extended spectrum β -lactamase (ESBL) or AmpC-producing MDROs by
phenotypic methods, were spiked in triplicate, into healthy-donor blood, and incubated until positive.
DNA was extracted using the EZ1 Tissue Kit (Qiagen, Germantown, MD) at time 0, and after 2, 4, 6, and 8
h of storage at room temperature. 6 frozen positive blood culture samples known to contain ESBL
producers were also tested. Of the 8 spiked isolates tested, 2 isolates were positive for CMY-2, 3 for
CTX-M-15, and 3 could not be confirmed by the ARM-D® kits. No difference in confirmed bacterial
viability was detected during the course of the experiments. No observable difference in Cq values was
detected at any of the time points for ampC or ESBL-spiked positive blood cultures. Six positive blood
cultures, frozen for 2 years, were thawed and tested; 4 were positive for CTX-M-15, one for CTX-M-14,
and 1 was negative for all assay markers. The ARM-D® kits are accurate for detection in positive blood
cultures of claimed ESBL- or AmpC-gene targets found in gram-negative bacteria stored at room
temperature for up to 8 h or frozen for 2 years. Increasing the number of isolates tested will validate the
results presented. Once validated these assays can be used to investigate discrepant results obtained by
phenotypic methods and detect antibiotic resistant genes prior to susceptibility results. Results obtained
using these kits on positive blood cultures can provide guidance for targeted antimicrobial therapy for
patients with bacteremia caused by gram-negative bacteria and provide epidemiologic information
during potential outbreaks of gram-negative MDROs.
Session Number: 41
Session Number: 41
Abstract Title:
Impact of Incubation Volume on Zone Size for the Carbapenem Inactivation Method (Cim) and Modified
Cim (Mcim)
K. Starr, H. Berger, L. Bourassa, A. Bryan; Univ. of Washington Med. Ctr., Seattle, WA
Abstract Body:
The carbapenem inactivation method (CIM) and the modified CIM (mCIM) have been described as
effective phenotypic methods for fast, simple, and reliable identification of carbapenemase-production,
but limited data directly compares the two assays and their optimization. First, to determine method-
dependent cut-off zone sizes for the CIM interpretation and acceptable QC ranges, we evaluated the
precision of the assay independent of carbapenemase production with a negative control strain. Overall
mean zone size and standard deviation from 3 independent meropenem lots was 26.1 +/- 1.2mm
(n=29), with individual lots of disks yielding 25.8 +/- 1.1mm, 25.3 +/- 0.9mm, and 27.4 +/- 0.5mm. Based
on these data, CIM zone interpretations were designated as follows: <= 19mm positive, indeterminate
20-23mm, and negative >= 24mm. Using these cut-offs, indeterminate values are >1.7 SDs and positive
zone sizes are >5 SDs from the mean negative QC result. To compare the CIM method of van der Zwaluw
et al. 2015 using the above interpretations to the CLSI mCIM, we compared the methods using 43
carbapenem-resistant Enterobacteriales isolates molecularly determined to have a carbapenemase and
20 carbapenem-resistant, carbapenemase-negative isolates. Both assays were 100% sensitive and
specific for Enterobacteriales. However, we noted larger zone sizes for carbapenemase negative isolates
using the CIM compared to the mCIM: 28.95mm vs 22.95mm, respectively (p <0.0001), suggesting a
larger zone cut-off could be used to improve the sensitivity of the CIM compared to the mCIM without
compromising specificity. We hypothesized that this difference was due to dilution of the meropenem
during the incubation period. To test this, we performed the CIM, but varied the incubation volume. As
expected, mean zone size decreased when volume was increased. Using small incubation volumes and
adjusting zone-size cut-offs has the potential to maximize sensitivity while allowing shorter incubation
times more conducive to clinical workflows (2 vs. 4 hrs for the CIM vs. mCIM). Similarly, smaller volumes
may facilitate detection of low-level carbapenemase production in Pseudomonas and Acinetobacter spp.
Sensitive phenotypic detection of low-level carbapenemase production has the potential to facilitate
infection control interventions and antimicrobial stewardship while detecting resistant determinants not
present on common molecular panels, but further work is needed to optimize incubation conditions,
particularly for Pseudomonas and Acinetobacter spp.
Session Number: 42
Session Number: 42
Session Title: CPHM02 - Antimicrobial Susceptibility Testing: Gram-Negative Bacteria
Abstract Title:
Determination of Oral Antimicrobial Agents with the Lowest Resistance Rates among Community
Urinary Isolates
S. E. Farhat1, I. Coelho1, G. Lim1, W. P. Shih1, B. Shea1, M. Villasis1, S. Alcantara1, B. Premraj1, N.
Azim1, I. Niazi1, A. Obesi1, A. L. Joaquin1, I. Dapiosen1, A. E. Simor2; 1Alpha Lab, Toronto, ON, Canada,
2Sunnybrook Hlth. Sci. Ctr., Toronto, ON, Ca
Abstract Body:
Background: Urinary tract infections (UTIs) are commonly encountered worldwide. Oral antimicrobial
agents are the mainstay of treatment in the community. Identifying oral agents with low resistance (R)
can be advantageous to informing empirical therapy. Two studies from our lab have identified
amoxicillin-clavulanate (AMC) or fosfomycin (FOS) as the oral agent with the lowest R rate among
urinary isolates from non-hospitalized pregnant women or community patients, respectively. As neither
study included both agents and in light of current antimicrobial resistance, we sought to test urinary
isolates for their R against AMC, FOS, and five other oral agents commonly used in the treatment of
UTIs. Methods: Consecutive isolates were identified by conventional methods from urine cultures
processed over a 2 month period ending December 2017. Isolates were tested by disk diffusion or Vitek-
2 system (bioMérieux), in accordance with CLSI guidelines, against AMC, FOS, ampicillin (AM), cefazolin
(KZ), ciprofloxacin (CIP), nitrofurantoin (FM), and trimethoprim/sulfamethoxazole (SXT). Due to lack of
FOS interpretive criteria for all organisms, CLSI Escherichia coli and Enterococcus faecalis breakpoints
were applied to Gram-negative and -positive organisms, respectively, similar to recently published
investigations. Screening was performed for carbapenem-resistant Enterobacteriaceae (CRE), extended-
spectrum beta-lactamases (ESBLs) in E. coli and Klebsiella spp, methicillin-resistant Staphylococcus
aureus (MRSA), and vancomycin-resistant enterococci (VRE), in accordance with CLSI guidelines. Results:
Of 15,949 urine specimens processed, a total of 3,313 non-duplicate isolates were tested, including E.
coli (n =2,025), Streptococcus agalactiae (283), Klebsiella spp (272), E. faecalis (232), E. faecium (1),
Proteus mirabilis (160), Staphylococcus spp including S. aureus (131), Pseudomonas aeruginosa (22),
Citrobacter (84), Enterobacter (62), Morganella (32), Serratia (7), Providencia (1), and Acinetobacter (1)
species. R rates for AMC, FOS, AM, KZ, CIP, FM, and SXT were 9.8%, 3.2%, 43.5%, 20.1%, 12.1%, 12.7%,
and 31.8%, respectively. 528 isolates were R to ≥ 3 antimicrobial classes, including 157 ESBL-producing
(150 E. coli, 7 Klebsiella), 4 MRSA and 1 CRE isolates. Conclusions: Of the oral agents reported in this
study, FOS followed by AMC, had the lowest R profile among urinary isolates. These results provide
support for FOS and AMC as the most likely useful oral agents for current empirical therapy of UTIs in
the community.
Session Number: 42
Session Number: 42
Abstract Title:
Emerging Opportunistic Nosocomial Pathogen Ochrobactrum Anthropi: Characterization of
Carbapenem-Resistant (Cr) Strains Involved in An Outbreak
M. Almuzara1, J. S. Fernandez2, M. Barenboim1, S. Montaña3, M. Hernandez2, M. Carulla1, M. S.
Ramirez2; 1Hosp. Interzonal de Agudos Eva Perón, Laboratorio de Bacteriología, San Martin, Argentina,
2California State Univ., Fullerton, Fullerton, CA, 3Inst.
Abstract Body:
Background: In the last years the use of technologies in the microbiology diagnostic laboratory have
contributed to the increase in identification of uncommon pathogens. Ochrobactrum anthropi is an
emerging pathogen that was lately recognized as an opportunistic nosocomial pathogen. In 2016 an
outbreak caused by O. anthropi occurred in a hospital. Surprisingly the four isolates involved in the
outbreak were CR. The aim of this project was to characterize the strains involved in the outbreak and
identify the resistant determinants that can explain the multidrug-resistance (MDR) phenotype
observed. Methods: Four isolates were recovered from the blood sample of four patients. The isolates
were identified as O. anthropi by 16s rDNA sequencing. Susceptibility tests were performed and PCR
reactions for various carbapenemases were carried. OD-PCR was used to determine their genetic
relationship. In addition, the draft genome sequence of one of the strains (OA 107383) was obtained
with Illumina MiSeq-I and Nextera XT DNA library. De novo assembly was performed with SPADES and
RAST was used to predict the open reading frames (ORF). Further genomic analysis was carried out using
ARG-ANNOT, ISFinder, and PHAST. Results: All isolates were MDR and EDTA synergies exhibited positive
results, suggesting the presence of a MBL in each isolate. However, PCR reaction for blaIMP, blaVIM,
blaKPC and blaNDM were negative. The same pattern of bands was obtained by OD-PCR suggesting an
epidemiological relationship. The whole genome sequencing of OA 107383 was performed, and its draft
genome consists of 4,792,719-bp sequences. By RAST server, 4554 protein-coding genes were predicted.
Sequence analysis of the genome identified the presence of multiple resistance genes including various
class C β-lactamases, and most importantly, a metal-depended hydrolase, phnP. Moreover, genes
coding for efflux pumps and two intact phages were also found. Conclusion: To our knowledge, this is
the first description of an outbreak cause by carbapenem-resistant O. anthropi. We have identified
different β-lactamases genes in the genome of O. anthropi as well as various efflux pumps. The presence
of these determinants could explain the observed resistance phenotype. We also highlight the
importance of the isolation of uncommon MDR pathogens from clinical specimens since they can serve
as a reservoir of resistance determinants.
Session Number: 42
Session Number: 42
Abstract Title:
Performance of Microscan Colistin Well in Detecting Colistin Resistance in A Clin. Microbiology
Laboratory
A. Kim1, J. D. Lutgring2, M. Karlsson2, D. Campbell2, A. C. Brown2, E. M. Burd3; 1CFD Res. Corp.,
Huntsville, AL, 2CDC, Atlanta, GA, 3Emory Univ. Dept. of Pathology and Lab. Med., Atlanta, GA
Abstract Body:
Background: The prevalence of antimicrobial resistance is increasing, which has led to a renewed
interest in using colistin as a therapeutic option. The emergence of the plasmid-mediated mcr gene
threatens the utility of colistin. Clinical microbiology laboratories need to be able to detect colistin
resistance but options are limited. This study evaluated whether growth in the MicroScan colistin well
was an accurate indicator of colistin resistance compared with either reference broth microdilution
(BMD) or gradient diffusion strips (ETEST®). Methods: At a single institution, from May 1, 2017 to
October 25, 2017, all Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii with
growth in the MicroScan colistin well were included in the study. Each isolate was retested at the CDC
using BMD, ETEST®, and examination of the colistin well (4 µg/ml) on a MicroScan Neg Breakpoint
Combo 44 Panel. All isolates displaying a colistin minimum inhibitory concentration (MIC) of ≥4 µg/ml by
BMD were considered resistant and evaluated for the presence of mcr-1 and -2 by real-time PCR.
Results: Of the 63 isolates that grew in the colistin well, we excluded 16 samples (25.4%) that were
polymicrobial (including an organism of interest and an organism intrinsically resistant to colistin) and 3
isolates (4.8%) that were misidentified (organism was intrinsically resistant to colistin), leaving 44
isolates for further evaluation. Reference BMD confirmed 41 of the 44 isolates (93.1%) to be colistin-
resistant, indicating that MicroScan overestimated colistin resistance in 3 instances (6.8%). Of the 41
isolates confirmed to be colistin-resistant by BMD, ETEST® detected resistance in 27 isolates (65.9%). All
41 isolates tested negative for the mcr gene by PCR. Conclusions: The MicroScan colistin well showed a
higher rate of categorical agreement than gradient diffusion strips compared to reference BMD.
Laboratories lacking capacity to perform colistin susceptibility testing by reference BMD may consider
MicroScan as an alternative tool to detect colistin-resistant bacteria.
Session Number: 42
Session Number: 42
Abstract Title:
Evaluation of Polymyxin B and Colistin Antibiotic Susceptibility Testing Methods for Gram-Negative
Bacteria
A. E. Clark1, C. M. Crooks2, R. A. Weingarten1, J. P. Dekker1, K. M. Frank1; 1NIH, Bethesda, MD, 2Univ.
of Wisconsin-Madison, Madison, WI
Abstract Body:
Background: Antimicrobial susceptibility testing (AST) of polymyxin B and colistin is challenging due to
the physical properties of these compounds that interfere with testing. Accurate AST is critical, as
polymyxins are often last-line options for the treatment of patients with multidrug-resistant Gram-
negative infections. We evaluated the performance of the Sensititre® system (ThermoFisher) for routine
colistin and polymyxin B AST of Enterobacteriaceae and non-Enterobacteriaceae isolates. Methods: We
compared the performance of Sensititre® Gram-negative panels against agar dilution performed both in-
house and at a reference laboratory for colistin and polymyxin B AST. A collection of Gram-negative
clinical isolates with polymyxin minimum inhibitory concentrations (MICs) ranging from ≤0.25 to >4
µg/mL was used: A. baumannii (n=7), Achromobacter sp. (n=15), C. freundii complex (n=1), Enterobacter
sp. (n=14), E. coli (n=18), K. pneumoniae (n=27), Pseudomonas sp. (n=22), Pantoea sp. (n=1), R.
ornithinolytica (n=1), and S. maltophilia (n=2). Samples with discrepant MIC results were repeated, and
panels were manually evaluated for bacterial growth. Categorical agreement (CA) was calculated using a
breakpoint of S ≤2 and R >2 µg/mL. Agar dilution was performed in accordance with CLSI guidelines.
Results: A total of 108 Gram-negative isolates were tested. Of these isolates, 20% and 31% were called
resistant to polymyxin B and colistin respectively by the Sensititre® system. The Sensititre® CA was 85%
compared to polymyxin B agar dilution and 90% CA compared with colistin agar dilution results. Repeat
testing and manual evaluation of discrepant Sensititre® panels revealed the presence of skipped wells or
failure of the instrument to accurately detect bacterial growth. Conclusions: Polymyxin AST is
challenging, and although agar dilution appears to be the most reliable method, it remains too laborious
for most routine laboratories. Laboratories choosing to use the Sensititre® system for polymyxin AST
should consider manual evaluation of microdilution results due to the presence of skipped wells or
failure of the instrument to detect growth of some species.
Session Number: 42
Session Number: 42
Abstract Title:
In Pursuit of the Holy Grail: the Colistin Np Test As A Rapid, Cost Effective and Reliable Screen for Colistin
Resistance
C. Kingsburgh, M. M. Kock, B. Mitton, N. M. Mbelle, K-A. Strydom; Natl. Hlth.Lab. Services, Univ. of
Pretoria, Pretoria, South Africa
Abstract Body:
Background: Colistin minimum inhibitory concentration (MIC) determination is associated with several
issues. Broth microdilution (BMD) is regarded as the gold standard, but requires expertise, is labour
intensive and cannot routinely be implemented in all settings. A need for accurate, rapid and user-
friendly methods of detecting colistin resistance exists, esp. in light of the emergence of plasmid
mediated colistin resistance mechanisms. In this study an in-house prepared colistin NP test was
compared against colistin BMD and Etest as a possible rapid screen for colistin resistance. Methods: A
total of 32 stored Enterobacteriaceae isolates were evaluated using colistin BMD, Etest (bioMèrieux)
and an in-house prepared colistin NP test. These isolates comprised of 25 E. coli (of which 17 confirmed
mcr-1 positive) isolates and 7 K. pneumoniae. The BMD was performed according to CLSI M07-A10 and
regarded as the gold standard. The in-house colistin NP test was prepared as previously described with
the modification of only adding colistin sulphate powder (Abtek Biological Ltd, UK). The Etest and BMD
results were interpreted according to EUCAST clinical breakpoints v.7.1. Results: Twenty-five (78%) of
the isolates tested resistant using BMD, with MICs ranging from 4 µg/mL to 64 µg/mL (average MIC 11
µg/mL). Seven of the isolates tested sensitive using BMD, with an average MIC of 0.25 µg/mL. The
essential and categorical agreement between Etest and BMD were both 94%. A 6% very major error rate
was detected with the Etest. Thirty-one (97%) of the isolates showed a categorical agreement between
BMD and the colistin NP. The colistin NP had one false negative result (3%), which was obtained from a
K. pneumoniae isolate with an unknown mechanism of resistance and a MIC of 8 µg/mL. The sensitivity,
specificity, positive, and negative predictive values of the Etest was 92%, 100%, 100%, and 78%, whilst it
was 96%, 100%, 100% and 88% for the colistin NP test. Conclusions: The colistin NP test is a rapid, user-
friendly, inexpensive test that shows potential as a screening assay for colistin resistance, esp. in
resource limited settings where access to BMD is often limited.
Session Number: 42
Session Number: 42
Abstract Title:
Evaluation of Microelution Methods to Determine the Susceptibility of Enterobacteriaceae To
Polymyxins
T. V. Dalmolin1, H. Avila1, L. P. de Castro1, D. Lima-Morales2, A. L. Barth1; 1Univ.e Federal do Rio
Grande do Sul, Porto Alegre, Brazil, 2Hosp. de Clínicas de Porto Alegre, Porto Alegre, Brazil
Abstract Body:
Background: Polymyxins (polymyxin B and colistin) are considered the last resort for the treatment of
serious infections caused by multidrug resistant Enterobacteriaceae. The standard technique to
determine the susceptibility of polymyxins is the broth microdilution which requires material which may
present high costs. Recently, the Colistin Broth Microelution was described as a technique which
requires materials of low cost and easily obtained in the routine microbiology laboratory (antibiotics disk
as a source of antibiotic). The aim of this study was to evaluate the microelution method with
polymyxins (Polymyxins Broth Microelution - PBM) as well to propose a modified protocol using
diminished volumes of polymyxins (microelution-plates test - MPT). Methods: The tests were performed
with 26 isolates of Enterobacteriaceae. To perform the test with colistin, 3 tubes with 10mL of Cation
Adjusted Mueller Hinton Broth (CAMHB) were prepared and colistin disks (10µg) were added as follow:
one disk in tube 1 (1µg/mL), two in tube 2 (2µg/mL) and four in tube 3 (4µg/ml). Polymyxin B disks
(300UI=30µg) were also tested as follow: one disk in 30mL of CAMHB (1µg/mL)–Tube 1, one disk in
15mL of CAMHB (2µg/mL)–Tube 2 and two disks in 15mL of CAMHB (4µg/mL)–Tube 3. One tube without
antibiotic was used as the growth control. Polymyxins were allowed to elute from the disks for 60
minutes. The solutions were fractionated in tubes (1mL) for the PBM tests and in microtiter plates
(200µL) for the MPT tests. For the PBM test a volume of 5µl of the bacterium inoculum (108UFC/mL)
was added to each tube containing 1, 2, 4µg/mL of the antibiotics and the control. For the MPT test a
volume of 2µL of the inoculum (108UFC/mL) was added in each well containing 1, 2, 4µg/mL of the
antibiotics and the control. The tubes and plates were incubated for 16-20 hours and the test results
were interpreted according to the MIC value as susceptible (≤2µg/mL) or resistant (>2µg/mL). Results:
Among the 26 isolates, 12 were susceptible (MIC90=0.5µg/mL) and 14 were resistant (MIC90=64µg/mL)
to polymyxins according to standard technique. All 26 isolates presented categorical agreement with the
PBM and MPT for either antibiotics. Considering this classification, the sensitivity and specificity of the
tests were 100%. Conclusion: The PBT and MPT exhibited excellent discrimination between polymyxins-
resistant and polymyxins-susceptible isolates. Moreover, the microelution tests (PBT and MPT) proved
to be easy to perform and require materials commonly found in a routine microbiology laboratory.
Session Number: 42
Session Number: 42
Abstract Title:
Novel Media for Identification of Colistin Heteroresistant Enterobacter
D. Hufnagel, D. Weiss; Emory Univ., Atlanta, GA
Abstract Body:
Current trends in bacterial antibiotic resistance threaten multitudes of modern medical procedures from
transplants, to in-dwelling medical devices, to control of bacterial infections. The increasing frequency of
multi-drug resistant bacterial isolates has caused physicians and researchers to revisit the use of the
antibiotic colistin. Colistin is a cationic antimicrobial that binds and disrupts the negatively charged
membrane of Gram-negative bacteria. An issue with colistin is the inaccuracy and numerous problems
involved with susceptibility testing of isolates. Etest strips contain a gradient of antibiotic and are placed
on top of bacteria growing on an agar plate creating a halo if bacterial growth is inhibited. Etests are
currently not recommended for colistin susceptibility testing due to inaccurate results. A common
colistin resistance mechanism is to decorate the bacterial lipid A with positively charged residues
through the PhoPQ and PmrAB two component systems to repel the positively charged drug. The Weiss
lab has found that certain bacteria have a sub-population of resistant cells that can actively grow in the
presence of antibiotic, instead of the entire culture being recalcitrant to treatment, as occurs in classic
antibiotic resistance. This phenomenon, termed heteroresistance, is quite common in carbapenem-
resistant Enterobacteriaceae. I have designed and detailed a novel media termed DIAG that can not only
detect colistin resistant and susceptible strains, but also low frequency colistin heteroresistant
Enterobacter cloacae. Heteroresistance can cause antibiotic treatment failure in a mouse model of
sepsis, and certain heteroresistant isolates are undetected by susceptibility testing, showing the
importance for accurate identification of colistin heteroresistant isolates. DIAG contains decreased
cations and doesn’t inhibit the resistant sub-population of heteroresistant isolates. Detection of
heteroresistant isolates on DIAG is dependent on PhoPQ. Heteroresistant isolates that normally appear
as susceptible by Etest are able to grow alongside the Etest strip to high concentrations of antibiotic on
DIAG plates, accurately showing the underlying highly drug resistant subpopulation. DIAG holds great
promise for unearthing antibiotic resistant subpopulations and properly advising physicians on the
resistance profiles of bacteria.
Session Number: 42
Session Number: 42
Abstract Title:
Colistin Susceptibility Testing for Enterobacteriaceae by Broth Microdilution and Agar Dilution Vs. Rapid
Polymyxin Np
A. Bryson, P. Ramanan, P. Kohner, N. Cole, J. Uhl, R. Patel, A. Schuetz; Mayo Clinic, Rochester, MN
Abstract Body:
Background: Polymyxins, like colistin, serve as last resort antibiotics against multidrug resistant Gram-
negative bacteria; however, resistance to polymyxins among Enterobacteriaceae has been increasing
worldwide. Colistin resistance can arise though chromosomal mutations or plasmid-mediated
acquisition of mcr-1, mcr-2, or mcr-3. The Clinical and Laboratory Standards Institute (CLSI) established
epidemiological cutoff values (ECVs) for Enterobacteriaceae and colistin, but additional and faster
methods would be helpful to identify polymyxin resistance. Here we use 117 clinical Enterobacteriaceae
isolates to evaluate three methods of colistin testing: 1) broth microdilution (BMD), 2) agar dilution
(AD), and 3) rapid polymyxin NP (RPNP). We also used a laboratory developed test (LDT) PCR to detect
mcr-1 and mcr-2. Methods: We compared colistin MICs by AD and BMD for 117 Enterobacteriaceae
isolates from a variety of clinical sources. We included 24 different species with the most abundant
being Enterobacter cloacae complex (29.1% of all isolates), Escherichia coli (19.7%) and Klebsiella
pneumoniae complex (11.1%). Organisms with intrinsic resistance (e.g., Proteus mirabilis) made up 12%
of all isolates. BMD served as the reference method. MIC ≤2 µg/mL was wildtype (WT); MIC ≥4 µg/mL
was non-wild-type (NWT) as per CLSI ECVs. AD and BMD results were compared to RPNP with results
read at 2 hours. Molecular detection of mcr-1 and mcr-2 was performed by a LDT PCR. Results:
Categorical and essential agreement between AD and BMD was 98.0% and 67.3%, respectively. There
were no very major errors and major errors were 3.8% (2/53). Sensitivity and specificity of RPNP were
90.7% and 96.2%, respectively, as compared to BMD. One E. coli isolate, which had an MIC of 4 µg/mL
(NWT) for both AD and BMD was positive for mcr-1. No isolates were positive for mcr-2. Excluding
organisms with intrinsic resistance, BMD categorized 39.8% of isolates as WT, 51.5% as NWT, and 8.7%
as indeterminate (due to skipped wells). E. cloacae complex isolates accounted for 89% of skipped wells
for BMD and 100% for AD. Conclusions: AD demonstrated an acceptable categorical agreement with
BMD of 98.0%, with no very major errors. The essential agreement was low (67.3%), and the major error
rate was 3.8% (2/53). The RPNP assay demonstrated adequate sensitivity and specificity, with same-day
results. Consistent with previously published literature, the majority of skipped wells occurred with E.
cloacae complex isolates. Only one isolate was mcr-1 positive.
Session Number: 42
Session Number: 42
Abstract Title:
Cefepime Susceptibility Testing for Enterobacteriaceae Using Current Clsi Interpretive Criteria is
Inconsistent between Multiple Testing Platforms
V. E. Stegner1, D. L. Oblack2, M. V. Powers-Fletcher3; 1UC Hlth., Cincinnati, OH, 2VA Med. Ctr.,
Cincinnati, OH, 3Univ. of Cincinnati, Cincinnati, OH
Abstract Body:
Background: In 2010, the Clinical Laboratory and Standards Institute (CLSI) revised the clinical
interpretive breakpoints (CBPs) for cephalosporins tested against Enterobacteriaceae. Updates to the
FDA-approved antimicrobial susceptibility testing (AST) platforms were delayed, leaving many
laboratories faced with validating the modified CBPs. While completing this validation, we identified a
unique subset of isolates for which AST on an FDA-approved platform using lowered CBPs was not
reliable. The purpose of this study was to determine the extent of this inaccuracy in hopes of identifying
a method that could be used to reliably guide patient care. Methods: The initial validation study
included Enterobacteriaceae from the CLSI Breakpoint Implementation Tool (BIT), all of which had
published susceptibility profiles. Testing was performed using the bioMérieux Vitek® 2 AST-GN73 panel
(software version 7.01), along with the CLSI-approved disk diffusion method as a second comparator. An
additional set of Enterobacteriaceae isolates, cultured from clinical specimens as part of routine patient
care, was also tested using the Vitek® 2 and disk diffusion methods. A subset of isolates was then tested
using a third method, the BD Phoenix™ NMIC/ID-304 panel (software version 7.00A/6.21A). Results: A
total of 31 isolates were tested in the initial study. There was 77.4% essential agreement between the
expected cefepime AST results and that of the Vitek® 2. All errors occurred in isolates with known
resistance genes. A total of 207 additional Enterobacteriaceae were then tested, all with profiles
suggesting the presence of a resistance gene. The rate of minor, major, and very major errors for
cefepime AST was 45%, 0% and 3%, respectively. Discrepant cefepime results were found most
frequently in the species more commonly associated with extended-spectrum β-lactamase production.
Ten isolates were also tested using the Phoenix™; essential agreement occurred for only 50% of these
isolates between the two FDA-approved platforms and there was a minor and major error rate of 50%
and 10%, respectively, when comparing the Phoenix™ to disk diffusion. Conclusions: This data highlights
significant discrepancy between cefepime AST using different methodologies and underscores the
importance of “wet lab” testing when validating modified CBPs. If not resolved, such inconsistencies
could have a negative impact on clinical care decisions, especially when considering the use of cefepime
in Enterobacteriaceae that are resistant to other cephalosporins.
Session Number: 42
Session Number: 42
Abstract Title:
A Comparison of Piperacillin-Tazobactam and Colistin Sensitest Mic to Clsi Broth Microdilution Mic for
Gram Negative Challenge Strains
L. Koeth, J. DiFranco-Fisher; Lab. Specialists, Inc., Westlake, OH
Abstract Body:
Background: The Sensitest was recently developed by Liofilchem to provide a manual broth
microdilution option for MIC testing of single antimicrobial agents using a 32-well dried panel. The
piperacillin-tazobactam (P-T) Sensitest includes a wide range of concentrations (0.008-128 ug/mL),
which provides for testing of 2 isolates/panel. The colistin Sensitest is configured to test 4 isolates/panel
(4 rows each containing 0.25-16 ug/mL). There have been challenges in testing piperacillin-tazobactam
by some automated and gradient strip methods. There have also been difficulties in testing colistin by
gradient strip methods and an overall lack of testing methods available for colistin. This study was
performed as an initial evaluation at a single testing site using challenge organisms specific to each of
the two agents. Method: Each strain was tested once with frozen panels (CLSI reference) and by
Sensitest using the same inoculum in cation adjusted Mueller Hinton broth. Incubation conditions
(ambient at 33-37oC for 16-20 hrs) and reading of the MIC were similar for both methods. The strains
tested included A. baumanii, Enterobacteriaceae, P. aeruginosa and quality control strains. The colistin
challenge set consisted of 52 strains, which were from the CDC resistant collections and included
characterized strains with a range of colistin MIC results. The P-T challenge set consisted of 28 strains,
which were from the LSI collection and were chosen because the majority of MIC results were near the
breakpoints (between 16-≥128 µg/mL). Results: Colistin and P-T agreement rates and error rates are
shown in the table.<table class="AbstractTable" id="{A39E8003-160A-4C0F-91FB-
57848AEF1602}"><caption class="AbstractTableCaption"></caption><tr><td rowspan="1"
rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1"
colspan="1">Antimicrobial Agent</td><td rowspan="1" colspan="1">n</td><td rowspan="1"
colspan="1">EA</td><td rowspan="1" colspan="1">CA</td><td rowspan="1" colspan="1">Minor Error
Rate</td></tr><tr><td rowspan="1" colspan="1">Colistin</td><td rowspan="1"
colspan="1">52</td><td rowspan="1" colspan="1">98.1%</td><td rowspan="1"
colspan="1">100%</td><td rowspan="1" colspan="1">NA</td></tr><tr><td rowspan="1"
colspan="1">Piperacillin/tazobactam</td><td rowspan="1" colspan="1">28</td><td rowspan="1"
colspan="1">100%</td><td rowspan="1" colspan="1">75%</td><td rowspan="1"
colspan="1">25%</td></tr><tr><td rowspan="1" colspan="5">EA – essential agreement (within ± 1
dilution); CA – category agreement; NA – not applicable</td></tr></table> Although essential
agreement rate was 100% for P-T, the category agreement was 75% as a result of 7 strains with MICs
that differed by one dilution and were near the breakpoints. Conclusions: There was excellent
correlation of Sensitest and reference broth microdilution MIC results for both piperacillin-tazobactam
and colistin. Additional testing, with a larger number of isolates and testing sites, is warranted. The
Sensitest method is a simple MIC method that would provide an option, other than gradient diffusion,
for testing of a single agent as a supplement to a clinical laboratory’s automated system.
Session Number: 42
Session Number: 42
Abstract Title:
Development of Clsi Mic and Disk Diffusion Quality Control Ranges for Cefepime/VNRX-5133
L. Koeth1, S. Cusick2, L. Xerri2; 1Lab. Specialists, Inc., Westlake, OH, 2VenatoRx Pharmaceuticals,
Malvern, PA
Abstract Body:
Background: VNRX-5133 is a novel cyclic boronate Broad-Spectrum β-lactamase inhibitor under
development by VenatoRx Pharmaceuticals, Inc. The addition of VNRX-5133 to cefepime (FEP/5133)
restores the activity of cefepime against Enterobacteriaceae and P. aeruginosa that are resistant due to
the production of certain beta-lactamases, including ESBL, AmpC, OXA, KPC, NDM, and VIM. For control
of susceptibility testing methods, this study was performed to obtain CLSI quality control ranges for both
MIC broth microdilution and disk diffusion methods. Methods: Disk and MIC results were obtained in
accordance with CLSI M23-4 Tier 2 QC guidelines for six strains (shown in table) and included 10
replicates tested at 9 laboratories using 3 lots of media for both MIC and disk methods. MIC testing of
cefepime/VNRX-5133 (fixed concentration of 4 µg/mL) was performed by broth microdilution method
along with 2 comparator agents, cefepime and cefepime/tazobactam. Disk diffusion testing was
performed with cefepime/VNRX-5133 30/20 µg disks from two manufacturers and with cefepime 30 µg
and ceftolozane/tazobactam 30/10 µg disks. Results: As shown in the table, at least 98% of
cefepime/VNRX-5133 results were within a 3-4 well MIC range and a 6-8 mm disk range for all strains.
Comparator agent results were within established ranges for ≥99% of results.Conclusions: CLSI quality
ranges have been established for six strains. CLSI has recommended that either E. coli NCTC 13353 or K.
pneumoniae ATCC BAA-1705 are used as routine QC strains. The remaining strains and approved ranges
are available for supplemental testing. These quality control ranges will not appear in the CLSI M100
document until the product is named; the summarized data and CLSI recommendations are available as
the “June 2017 AST Meeting Minutes” on the CLSI website.<table class="AbstractTable" id="{05737954-
BA6F-47CF-ABF2-998A3422D5C7}"><caption class="AbstractTableCaption"></caption><tr><td
colspan="1"></td></tr><tr><td rowspan="1" colspan="1">Quality Control Strain</td><td rowspan="1"
colspan="2">Approved CLSI Range (% within Range) for FEP/5133</td></tr><tr><td rowspan="1"
colspan="1"></td><td rowspan="1" colspan="1">MIC (µg/mL)</td><td rowspan="1" colspan="1">Disk
(mm)</td></tr><tr><td rowspan="1" colspan="1">E. coli ATCC 25922</td><td rowspan="1"
colspan="1">0.03/4 – 0.12/4<br />(100%)</td><td rowspan="1" colspan="1">31-37<br
/>(99.0%)</td></tr><tr><td rowspan="1" colspan="1">E. coli ATCC 35218 <br />(TEM-1) </td><td
rowspan="1" colspan="1">0.016/4-0.06/4<br />(100%)</td><td rowspan="1" colspan="1">31-37<br
/>(98.7%)</td></tr><tr><td rowspan="1" colspan="1">E. coli NCTC 13353<br />(CTX-M-15)</td><td
rowspan="1" colspan="1">0.12/4-1/4<br />(99.6%)</td><td rowspan="1" colspan="1">24-30<br
/>(99.8%)</td></tr><tr><td rowspan="1" colspan="1">K. pneumoniae ATCC 700603<br />(SHV-
18)</td><td rowspan="1" colspan="1">0.12/4-0.5/4<br />(99.3%)</td><td rowspan="1"
colspan="1">24-31<br />(99.4%)</td></tr><tr><td rowspan="1" colspan="1">K. pneumoniae ATCC
BAA-1705<br />(KPC)</td><td rowspan="1" colspan="1">0.12/4-0.5/4<br />(98.9%)</td><td
rowspan="1" colspan="1">22-27<br />(99.0%)</td></tr><tr><td rowspan="1" colspan="1">P.
aeruginosa ATCC 27853</td><td rowspan="1" colspan="1">0.5/4-4/4<br />(100%)</td><td
rowspan="1" colspan="1">25-31<br />(99.4%)</td></tr></table>
Session Number: 42
Session Number: 42
Abstract Title:
Multicenter Evaluation of Ceftolozane/Tazobactam Mic Results for Enterobacteriaceae and
Pseudomonas Aeruginosa Using Microscan Dried Gram Negative Mic Panels
R. Brookman1, A. Harrington2, J. A. Hindler3, M. Traczewski4, S. Campeau3, S. DesJarlais2, D. M.
Beasley4, C. J. Hastey1, D. Roe-Carpenter1, A. Chipman5; 1Beckman Coulter, Inc., West Sacramento, CA,
2Loyola Univ. Med. Ctr., Maywood, IL, 3UCLA Hlth.System
Abstract Body:
Background: A multicenter study was performed to evaluate the accuracy of ceftolozane/tazobactam on
a MicroScan Dried Gram Negative MIC (MSDGN) Panel when compared to frozen CLSI broth
microdilution reference panels. Materials/Methods: For efficacy, an evaluation was conducted at three
sites by comparing MICs obtained using the MSDGN to MICs using a CLSI broth microdilution reference
panel. A total of 575 Enterobacteriaceae and Pseudomonas aeruginosa clinical isolates were tested
using the turbidity and PromptTM* methods of inoculation. For challenge, a set of 118 organisms was
tested on MSDGN panels at one site. For reproducibility, a set of 17 organisms was tested on MSDGN
panels at all three sites. MSDGN panels were incubated at 35 ± 2ºC and read on the WalkAway System,
the autoSCAN-4 instrument, and read visually. Read times for the MSDGN panels were at 16-20 hours.
Frozen reference panels, prepared according to CLSI methodology, were inoculated using the turbidity
inoculation method. All frozen reference panels were incubated at 35 ± 2ºC and read visually. Frozen
reference panels were read at 16-18 hours. FDA breakpoints (µg/ml) used for interpretation of MIC
results were: Enterobacteriaceae ≤ 2/4 S, 4/4 I, and ≥ 8/4 R and Pseudomonas aeruginosa ≤ 4/4 S, 8/4 I,
and ≥ 16/4 R. Results: When compared to frozen reference panel results, essential and categorical
agreements for all isolates tested in Efficacy and Challenge are as follows:<table class="AbstractTable"
id="{FA40F1ED-DBB0-4402-9205-C0596A253FED}"><caption
colspan="1">Read Method</td><td rowspan="1" colspan="2">Essential<br />Agreement %</td><td
rowspan="1" colspan="2">Categorical Agreement %</td><td rowspan="1" colspan="2">VMJ %</td><td
rowspan="1" colspan="2">MAJ %</td><td rowspan="1" colspan="2">MIN %</td></tr><tr><td
rowspan="1" colspan="1"></td><td rowspan="1" colspan="1">T</td><td rowspan="1"
colspan="1">P</td><td rowspan="1" colspan="1">T</td><td rowspan="1" colspan="1">P</td><td
rowspan="1" colspan="1">T</td><td rowspan="1" colspan="1">P</td><td rowspan="1"
colspan="1">T</td><td rowspan="1" colspan="1">P</td><td rowspan="1" colspan="1">T</td><td
rowspan="1" colspan="1">P</td></tr><tr><td rowspan="1" colspan="1">Visually</td><td rowspan="1"
colspan="1">96.5<br />(669/693)</td><td rowspan="1" colspan="1">95.2<br />(660/693)</td><td
rowspan="1" colspan="1">98.0<br />(679/693)</td><td rowspan="1" colspan="1">97.0<br
/>(672/693)</td><td rowspan="1" colspan="1">0.0<br />(0/63)</td><td rowspan="1"
colspan="1">1.6<br />(1/63)</td><td rowspan="1" colspan="1">1.0<br />(6/623)</td><td rowspan="1"
rowspan="1" colspan="1">2.0<br />(14/693)</td></tr><tr><td rowspan="1"
colspan="1">WalkAway</td><td rowspan="1" colspan="1">96.4<br />(668/693)</td><td rowspan="1"
/>(1/63)</td><td rowspan="1" colspan="1">3.2<br />(2/63)</td><td rowspan="1" colspan="1">0.3<br
colspan="1">1.7<br />(12/693)</td><td rowspan="1" colspan="1">1.7<br />(12/693)</td></tr><tr><td
rowspan="1" colspan="1">autoSCAN-4</td><td rowspan="1" colspan="1">95.5<br />(662/693)</td><td
/>(11/693)</td></tr><tr><td rowspan="1" colspan="11">T = Turbidity inoculation method, P = Prompt*
inoculation method</td></tr></table> Reproducibility among the three sites were greater than 95% for
all read methods for both the turbidity and Prompt* inoculation methods. Conclusion: This multicenter
study showed that ceftolozane/tazobactam MIC results for Enterobacteriaceae and Pseudomonas
aeruginosa obtained with the MSDGN panel correlate well with MICs obtained using frozen reference
panels. * PROMPT is a registered trademark of 3M. © 2018 Beckman Coulter. All rights reserved.
Beckman Coulter, the stylized logo and the Beckman Coulter product and service marks mentioned
herein are trademarks or registered trademarks of Beckman Coulter, Inc. in the United States and other
countries.
Session Number: 42
Session Number: 42
Abstract Title:
Performance of Ceftolozane-Tazobactam Hardydisk™ Compared to Reference Broth Microdilution for
Enterobacteriaceae and Pseudomonas Aeruginosa Isolates
A. Klavins1, K. Alby2, P. Edelstein2, R. Malherbe1, S. Hepler1, R. Cage1, C. Massey1, A. Hsiung1, J.
Hardy1; 1Hardy Diagnostics, Santa Maria, CA, 2Hosp. of the Univ. of Pennsylvania, Philadelphia, PA
Abstract Body:
Ceftolozane-tazobactam (Zerbaxa) is a cephalosporin that is combined with a beta-lactamase inhibitor
for complicated intra-abdominal and urinary tract infections caused by select gram-negative organisms.
This drug was introduced in 2014 by Merck, and subsequently FDA-cleared susceptibility devices were
launched in the market, firstly as susceptibility disk (HardyDisk™) for the Kirby Bauer disk diffusion
method. A recent study performed by Humphries et al. found that comparable performance between
the HardyDisk™, gradient method, and reference broth microdilution against 308 beta-lactam resistant
P. aeruginosa strains from three clinical laboratories in Los Angeles, California. The aim of the our study
was to further affirm the performance of ceftolozane-tazobactam HardyDisk™, as described by
Humphries et al., and expand the evaluation to Enterobacteriaceae. In our study, the testing was
evaluated in triplicate (3 different lots) on Hardy Diagnostics Mueller Hinton Agar in parallel with
reference broth microdilution for 49 strains of Enterobacteriaceae and 20 strains of P. aeruginosa that
exhibited a range of susceptibility profiles to ceftolozane-tazobactam. The strains were collected from
antimicrobial resistance panels from the CDC and kindly supplied by University of Pennsylvania. Each
fresh organism was prepared into a TSB suspension equivalent to a 0.5 McFarland and the same
suspension was also used to prepare serial dilutions to the appropriate inoculum for the reference broth
microdilution tray. Additionally, all dilutions were verified by aseptic spread-plate to blood agar to
confirm inoculum. Using Clinical and Laboratory Standards Institute (CLSI) breakpoints for ceftolozane-
tazobactam, a scattergram of the broth microdilution minimum inhibitory concentration (MIC) versus
the average disk zone size was created. Of the Enterobacteriaceae tested, 91.8% strains showed
categorical agreement between disk zone size and reference broth MIC. Additionally, 88.9% of the P.
aeruginosa tested showed categorical agreement. These results show that the disk diffusion method
delivers an accurate and reliable result that is comparable to reference BMD MIC values in evaluating
antimicrobial susceptibility.
Session Number: 42
Session Number: 42
Abstract Title:
Multicenter Evaluation of Ceftazidime/Avibactam Mic Results for Enterobacteriaceae and Pseudomonas
Aeruginosa Using Microscan Dried Gram Negative Mic Panels
R. K. Brookman1, A. Harrington2, J. A. Hindler3, M. Traczewski4, S. A. Campeau3, S. Desjarlais2, D. M.
Beasley4, C. J. Hastey1, D. Roe-Carpenter1, A. Chipman5; 1Beckman Coulter, Inc., West Sacramento, CA,
2Loyola Univ. Med. Ctr., Maywood, IL, 3UCLA Hlth.
Abstract Body:
Background: A multicenter study was performed to evaluate the accuracy of ceftazidime/avibactam on a
MicroScan Dried Gram Negative MIC (MSDGN) Panel when compared to frozen CLSI broth microdilution
reference panels. Materials/Methods: For efficacy, an evaluation was conducted at three sites by
comparing MICs obtained using the MSDGN panel to MICs using a CLSI broth microdilution reference
panel. A total of 618 Enterobacteriaceae and Pseudomonas aeruginosa clinical isolates were tested
using the turbidity and PromptTM* methods of inoculation. For challenge, a set of 116 organisms was
tested on MSDGN panels at one site. For reproducibility, a subset of 16 organisms was tested on MSDGN
panels at each site. MSDGN panels were incubated at 35 ± 2ºC and read on the WalkAway System, the
autoSCAN-4 instrument, and read visually. Read times for the MSDGN panels were at 16-20 hours.
reference panels were read at 16-18 hours. FDA breakpoints (µg/ml) used for interpretation of MIC
results were: Enterobacteriaceae and Pseudomonas aeruginosa ≤ 8/4 S and ≥ 16/4 R. Results: When
compared to frozen reference panel results, essential and categorical agreements for all isolates tested
in Efficacy and Challenge are as follows:<table class="AbstractTable" id="{E65812AC-CC6C-424F-AAF5-
B6292AE57478}"><caption class="AbstractTableCaption"></caption><tr><td rowspan="1"
rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1">Read Method</td><td
rowspan="1" colspan="2">Essential<br />Agreement %</td><td rowspan="1" colspan="2">Categorical
Agreement %</td><td rowspan="1" colspan="2">VMJ^ %</td><td rowspan="1" colspan="2">MAJ^
%</td></tr><tr><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1">T</td><td
rowspan="1" colspan="1">P</td><td rowspan="1" colspan="1">T</td><td rowspan="1"
rowspan="1" colspan="1">T</td><td rowspan="1" colspan="1">P</td></tr><tr><td rowspan="1"
colspan="1">Visually</td><td rowspan="1" colspan="1">98.8<br />(725/734)</td><td rowspan="1"
/>(1/703)</td><td rowspan="1" colspan="1">0.3<br />(2/703)</td></tr><tr><td rowspan="1"
colspan="1">autoSCAN-4</td><td rowspan="1" colspan="1">98.8<br />(725/734)</td><td rowspan="1"
colspan="9">T = Turbidity inoculation method, P = Prompt* inoculation method<br />^ = calculated
without 1 well dilution errors</td></tr></table> Reproducibility among the three sites were greater
than 95% for all read methods for both the turbidity and Prompt* inoculation methods. Conclusion: This
multicenter study showed that ceftazidime/avibactam MIC results for Enterobacteriaceae and
Pseudomonas aeruginosa obtained with the MSDGN panel correlate well with MICs obtained using
frozen reference panels. * PROMPT is a registered trademark of 3M. © 2018 Beckman Coulter. All rights
reserved. Beckman Coulter, the stylized logo and the Beckman Coulter product and service marks
mentioned herein are trademarks or registered trademarks of Beckman Coulter, Inc. in the United States
and other countries.
Session Number: 42
Session Number: 42
Abstract Title:
Performance of Etestceftazidime/Avibactam (Cza) (0.016-256 μG/Ml) for Antimicrobial Susceptibility
Testing of Enterobacteriaceae And Pseudomonas Aeruginosa
S. Garrett1, T. Armstrong1, D. Halimi2, R. Martelin2, H-P. Dwivedi1, C-A. Burnham3, R. Humphries4, J.
Hindler5, S. Campeau5, G. Denys6, G. Zambardi2; 1bioMerieux, Hazelwood, MO, 2bioMerieux, La Balme
les Grottes, France, 3Washington Univ. Sch. of Med., S
Abstract Body:
Background: Ceftazidime/Avibactam (CZA) is a cephalosporin and beta-lactamase inhibitor combination
drug with activity against multidrug-resistant (MDR) Pseudomonas aeruginosa and MDR
Enterobacteriaceae that produce beta-lactamases, including extended-spectrum beta-lactamases
(ESBLs) and Klebsiella pneumoniae carbapenemases (KPCs), but not metallo-beta lactamases (MBLs). In
this study, the performance of Etest® CZA, an in vitro technique for determining the antimicrobial
susceptibility of Enterobacteriaceae and P. aeruginosa, was evaluated against the Clinical Laboratory
Standards Institute (CLSI) broth microdilution reference method (BMD). Method: A total of 1239 isolates
(1087 Enterobacteriaceae and 152 P. aeruginosa) were tested by Etest® CZA method and BMD reference
procedure (CLSI M07-A10). Of the 1239 isolates tested, 699 were fresh clinical isolates collected at the
three external testing sites. The Enterobacteriaceae tested included 13 different species. The results
were analyzed for essential agreement (EA), category agreement (CA), major error (ME) and very major
error (VME) rates and compared to the FDA performance criteria using the FDA breakpoints for
Ceftazidime/Avibactam (Enterobacteriaceae and P. aeruginosa S <u><</u> 8/4 µg/mL and R ≥16/4
µg/mL). Results: Etest® CZA performance for Enterobacteriaceae and P. aeruginosa met the FDA
performance criteria for EA and CA (≥ 90%) and ME (≤ 3.0% ). Etest® CZA met the VME rate criterion (≤
1.5%) with Pseudomonas aeruginosa. Etest® CZA failed to meet the VME rate criterion with
Enterobacteriaceae with a VME rate of 4.4%. Both VMEs occurred in Morganella morganii strains which
had results of 16 µg/mL by the BMD reference test and 8 µg/mL by Etest® CZA. The performances of
these two strains were within essential agreement. Since there is no intermediate breakpoint, the
adjusted categorical VME rate based on the essential agreement is acceptable at 0.0% for
Enterobacteriaceae. Etest® CZA performance evaluated is summarized in the table below.<table
class="AbstractTable" id="{9E856FD9-6509-47F0-8CFF-34D78F89F692}"><caption
rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1">Organism</td><td rowspan="1"
colspan="1">EA</td><td rowspan="1" colspan="1">CA</td><td rowspan="1" colspan="1">CA ME
Rate</td><td rowspan="1" colspan="1">CA VME Rate</td></tr><tr><td rowspan="1"
colspan="1">Enterobacteriaceae</td><td rowspan="1" colspan="1">99.1%<br />(1077/1087)</td><td
rowspan="1" colspan="1">99.6%<br />(1083/1087)</td><td rowspan="1" colspan="1">0.2%<br
/>(2/1042)</td><td rowspan="1" colspan="1">4.4%<br />(2/45)</td></tr><tr><td rowspan="1"
colspan="1">Pseudomonas aeruginosa</td><td rowspan="1" colspan="1">99.3%<br
/>(151/152)</td><td rowspan="1" colspan="1">99.3%<br />(151/152)</td><td rowspan="1"
colspan="1">0.8%<br />(1/118)</td><td rowspan="1" colspan="1">0.0%<br />(0/34)</td></tr></table>
Conclusion: When compared to the BMD reference method, Etest® CZA proved to be a suitable test for
susceptibility testing of Enterobacteriaceae and P. aeruginosa.
Session Number: 42
Session Number: 42
Abstract Title:
Performance of Ceftazidime-Avibactam Disk Diffusion Compared with Broth Microdilution for Antibiotic
Susceptibility Testing of Pseudomonas Aeruginosa And Enterobacteriaceae
B. B. Yoo1, M. J. Machado2, R. Balbuena3, J. Ilutsik3, A. Kim4, E. Ransom5, D. Lonsway1, A. C. Brown1;
1CDC, Atlanta, GA, 2Eagle Med. Services, Atlanta, GA, 3IHRC, Atlanta, GA, 4CFD Res. Corp., Huntsville,
AL, 5Association of Publ. Hlth.Lab., Silver Spri
Abstract Body:
Background: Ceftazidime-avibactam (CZA) is a β-lactamase inhibitor combination antimicrobial with
enhanced activity against Gram negative bacteria (GNB), including Pseudomonas aeruginosa (PSA) and
Enterobacteriaceae (ENT). CZA was approved by the U.S. Food and Drug Administration (FDA) to treat
intra-abdominal and urinary tract infections. CZA currently has no intermediate FDA breakpoints for disk
diffusion (DD), thus one mm difference on DD reading could create a very major error (VME; false
susceptible) or major error (ME; false resistant) when compared to reference broth microdilution
(BMD). There is a difference in CZA disk potency between Clinical and Laboratory Standards Institute
(CLSI) (30/20µg) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) (10/4µg).
The aim of this study was to test GNB isolates by both BMD and DD in order to see if CZA DD using a CLSI
potency is a reliable method for testing. Methods: We evaluated 62 isolates of PSA and 183 isolates of
ENT, including 57 Klebsiella pneumoniae, 50 Escherichia coli, 28 Enterobacter spp., and 48 others from
the CDC-FDA Antibiotic Resistance Isolate Bank. We performed susceptibility testing to CZA on all
isolates using BMD and DD in parallel, using a single inoculum. We used FDA breakpoints to interpret
results: ≤ 8/4 S and ≥ 16/4 R for BMD (µg/ml); ≥ 18 S and ≤ 17 R for PSA DD (mm); ≥ 21 S and ≤ 20 R for
ENT DD (mm). All isolates were tested for resistance genes using real-time PCR or whole genome
sequencing. Results: Overall interpretive category agreement was 87.1 % (54/62) for PSA and 94.5%
(173/183) for ENT. Among 32 PSA isolates that were resistant to CZA by BMD, 8 isolates were
susceptible by DD, resulting in a 25% VME; none of these isolates with VME were carbapenemase
producers. Among 9 ENT isolates in the range of MICs 8-16, VME was 11% and ME was 33%; these
discrepancies occurred mainly among KPC producers. Conclusions: Because current CZA DD breakpoints
for PSA and ENT gave unacceptably high VME and ME, we suggest either introducing an intermediate
CZA breakpoint category to help minimize the discrepancy rates between DD and BMD or adjusting the
CLSI CZA disk potency to be equivalent to EUCAST for better correlation.
Session Number: 42
Session Number: 42
Abstract Title:
Performance of VITEK® 2 AST-GN Ceftazidime/Avibactam S <u><</u> 8/4 µg/mL and R ≥16/4 µg/mL) for
Antimicrobial Susceptibility Testing of Enterobacteriaceae and Pseudomonas aeruginosa
S. Franklin, H. Dwivedi, V. LaBombardi, T. Davis, R. Humphries, J. Hindler, S. Campeau, P. Deol;
bioMerieux Inc., Hazelwood, MO
Abstract Body:
Background: Ceftazidime/Avibactam (CZA) is a cephalosporin and beta-lactamase inhibitor combination
drug with activity against extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae and
multidrug-resistant (MDR) Pseudomonas aeruginosa. In this study, the performance of VITEK®2 CZA, an
in vitro technique for determining the antimicrobial susceptibility of Enterobacteriaceae and
Pseudomonas aeruginosa, was evaluated against the CLSI broth microdilution (BMD) reference method.
Method: A total of 1073 isolates (866 Enterobacteriaceae and 207 Pseudomonas aeruginosa) were
tested by VITEK®2 CZA method and BMD reference procedure (CLSI M07-A10). The results were
analyzed for essential agreement (EA), category agreement (CA), major and very major error rates
following FDA/ISO performance criteria using the FDA/EUCAST breakpoints for Ceftazidime/Avibactam
(Enterobacteriaceae and Pseudomonas aeruginosa S <u><</u> 8/4 µg/mL and R ≥16/4 µg/mL). Results:
VITEK®2 CZA overall performance for Enterobacteriaceae and Pseudomonas aeruginosa met the
performance criteria for EA and CA, major errors and very major errors. The overall categorical major
error rate for Enterobacteriaceae and Pseudomonas aeruginosa combined was 1.4% (14/998). Nine (9)
major errors were one dilution apart from the reference method and as such fall within EA. Based on
the EA, and the lack of an intermediate breakpoint for Ceftazidime/Avibactam, the adjusted FDA
categorical major error rate was 0.4% (3/829) for Enterobacteriaceae , 1.2% (2/169) for Pseudomonas
aeruginosa, and 0.5% (5/998) for all organisms combined. VITEK®2 CZA performance is summarized in
Table 1. Table 1<table class="AbstractTable" id="{5D4DBD2A-C03E-468F-83DA-
3283FF7B4F65}"><caption class="AbstractTableCaption"></caption><tr><td rowspan="1"
colspan="1">Claimed Species</td><td rowspan="1" colspan="1">EA</td><td rowspan="1"
colspan="1">CA</td><td rowspan="1" colspan="1">CA Major Error Rate</td><td rowspan="1"
colspan="1">CA Very Major Error Rate</td></tr><tr><td rowspan="1" colspan="1">Enterobacteriaceae
FDA</td><td rowspan="1" colspan="1">94.2% (816/866)</td><td rowspan="1" colspan="1">99.2%
(859/866)</td><td rowspan="1" colspan="1">0.8% (7/829)</td><td rowspan="1" colspan="1">0.0%
(0/37)</td></tr><tr><td rowspan="1" colspan="1">Pseudomonas aeruginosa-FDA</td><td rowspan="1"
colspan="1">95.7% (198/207)</td><td rowspan="1" colspan="1">96.6% (200/207)</td><td
rowspan="1" colspan="1">4.1% (7/169)</td><td rowspan="1" colspan="1">0.0%
(0/38)</td></tr><tr><td rowspan="1" colspan="1">Overall-FDA</td><td rowspan="1"
colspan="1">94.5%(1014/1073)</td><td rowspan="1" colspan="1">98.7%(1059/1073)</td><td
(0/75)</td></tr><tr><td rowspan="1" colspan="1">Overall-EUCAST</td><td rowspan="1"
colspan="1">94.5%(1014/1073)</td><td rowspan="1" colspan="1">98.9%(1059/1073)</td><td
rowspan="1" colspan="1">1.2%(12/998)*</td><td rowspan="1" colspan="1">0.0%
(0/75)</td></tr></table> *resolved data shown Conclusion: When compared to the broth microdilution
reference method, VITEK®2 CZA proved to be a suitable test for susceptibility testing of
Enterobacteriaceae and Pseudomonas aeruginosa.
Session Number: 42
Session Number: 42
Abstract Title:
Comparative Evaluation of Meropenem/Vaborbactam Mic Determination with the New Etest® Mev*
and Clsi 2017 Broth Microdilution Method
V. Sauvonnet1, O. Lomovskaya2, M. Bouvier1, V. Collin1, D. Halimi1, R. Martelin1, G. Zambardi1;
1bioMérieux, La Balme les grottes, France, 2The Medecines Company, San Diego, CA
Abstract Body:
Background: Carbapenems antibiotics are a key weapon in the fight against beta-lactam resistant Gram-
negative infections. Increased occurrence of carbapenems resistance has led to the development of new
drug and inhibitor combinations, such as meropenem/vaborbactam. Vabomere® (TheMedCo) is FDA
approved for the treatment of adults complicated urinary tract infections (cUTI) including pyelonephritis
caused by the following susceptible microorganisms : Escherichia coli, Klebsiella pneumoniae and
Enterobacter cloacae species complex. The new ETEST® MEV (meropenem/vaborbactam - MIC range
0.004/8-64/8 µg/L) has been developed and calibrated versus the broth microdilution reference method
(BMD) as described by the Clinical and Laboratory Standards Institute (CLSI). This test is intended to
determine the MIC of meropenem/vaborbactam toward the Enterobacteriaceae group. The aim of this
study was to perform a comparative evaluation of ETEST MEV with the CLSI BMD method on a panel of
225 strains. Methods: The panel consisted of 198 Enterobacteriaceae (including 23 resistant strains), 22
Pseudomonas aeruginosa, and 5 CLSI QC strains. BMD was performed using the 2017 CLSI
recommendations. ETEST MEV was evaluated using the standard ETEST MIC procedure for aerobic
strains (inoculum 0.5 McF, Mueller Hinton medium, incubation at 35°C). For each method, the MIC was
read at complete inhibition of growth. The FDA approved breakpoints for Enterobacteriaceae were
applied : S≤4µg/L - R≥16µg/L. Results: The MICs for the QC strains were in the expected ranges with
reproducible results. The essential MIC agreement [±1 dilution] was 94.5% without overestimation or
underestimation trend. The categorical agreement was 96%. 2 Very Major Errors (2 NDM-1 producing
strains), 6 minor errors and no Major Error were found. Conclusions: In this study, the new ETEST MEV is
found to be substantially equivalent to the CLSI reference method. MIC end points are easy to read.
With a 15-dilution range and simplicity of use, ETEST MEV could represent a valuable tool for MIC
determination and could be an alternative to BMD. * For Research Use Only. The performance
characteristics of this product have not been established yet.
Session Number: 42
Session Number: 42
Abstract Title:
Development of Meropenem-Vaborbactam Mic Assay for Gram Negative Bacteria on Microscan Dried
Gram Negative Mic Panels
S. Wood, R. Williams, V. Carr, D. Burtner, R. Miller; Beckman Coulter, West Sacramento, CA
Abstract Body:
Background: Development of a meropenem-vaborbactam assay was completed for the MicroScan Dried
Gram Negative MIC (MSDGN) Panel when compared to CLSI broth microdilution reference panels.
Materials/Methods: Development was conducted by comparing MICs obtained using the MSDGN panel
to MICs using a CLSI broth microdilution reference panel. A total of 197 Enterobacteriaceae, and 30
Pseudomonas aeruginosa isolates were tested (for a total of 227 isolates) using the turbidity and
Prompt®* methods of inoculation. MSDGN panels were incubated at 35 ± 2ºC and read on the
WalkAway System, the autoSCAN-4 instrument, and read visually. Read times for the MSDGN panels
were at 16-20 hours for the autoSCAN-4 instrument and visual reads, and 16-18 hours for the WalkAway
System. Each isolate was read in triplicate on the autoSCAN-4 instrument. Frozen reference panels,
prepared according to CLSI methodology, were inoculated using the turbidity inoculation method. All
frozen reference panels were incubated at 35 ± 2ºC and read visually. Frozen reference panels were
read at 16-18 hours. FDA breakpoints (μg/ml) used for interpretation of MIC results were:
Enterobacteriaceae ≤ 4/8 S, 8/8 I, and ≥ 16/8 R. Results: When compared to frozen reference panel
results, essential agreement for all isolates tested and categorical agreement for all Enterobacteriaceae
isolates tested are as follows:<table class="AbstractTable" id="{5EB6ADB2-35E0-48E9-9289-
5B873CB52708}"><caption class="AbstractTableCaption"></caption><tr><td rowspan="1"
colspan="1"></td></tr><tr><td rowspan="2" colspan="1">Read Method</td><td rowspan="1"
colspan="2">Essential<br />Agreement %</td><td rowspan="1" colspan="2">Categorical Agreement
%</td><td rowspan="1" colspan="2">VMJ %</td><td rowspan="1" colspan="2">MAJ %</td><td
rowspan="1" colspan="2">MIN %</td></tr><tr><td rowspan="1" colspan="1">T</td><td rowspan="1"
rowspan="1" colspan="1">P</td></tr><tr><td rowspan="1" colspan="1">Visual</td><td rowspan="1"
colspan="1">1.99<br />(3/151)</td><td rowspan="1" colspan="1">1.99<br />(3/151)</td></tr><tr><td
rowspan="1" colspan="1">autoSCAN-4</td><td rowspan="1" colspan="1">94.01<br
colspan="1">1.52<br />(9/591)</td></tr><tr><td rowspan="1" colspan="11">T = Turbidity inoculation
method, P = Prompt* inoculation method</td></tr></table> Conclusion: The MIC results obtained with
the MSDGN panel with an extended dilution series correlate well with MICs obtained using frozen
reference panels. The elevated Very Major Error Rate may be inflated due to the low number of
resistant isolates tested. * PROMPT is a registered trademark of 3M. © 2018 Beckman Coulter, Inc. All
rights reserved. Beckman Coulter, the stylized logo and the Beckman Coulter product and service marks
mentioned herein are trademarks or registered trademarks of Beckman Coulter, Inc. in the United States
and other countries.
Session Number: 42
Session Number: 42
Abstract Title:
Development of Eravacycline Mic Assay for Gram Negative Bacteria on Microscan Dried Gram Negative
Mic Panels
S. Wood, R. Kwong, V. Carr, R. Williams, R. Miller; Beckman Coulter, West Sacramento, CA
Abstract Body:
Background: Development of an eravacycline assay was completed for the MicroScan Dried Gram
Negative MIC (MSDGN) Panel when compared to CLSI broth microdilution reference panels.
Materials/Methods: Development was conducted by comparing MICs obtained using the MSDGN panel
to MICs using a CLSI broth microdilution reference panel. A total of 284 Enterobacteriaceae, 43
Acinetobacter baumannii and 27 Pseudomonas aeruginosa isolates were tested (for a total of 354
isolates) using the turbidity and Prompt®* methods of inoculation. MSDGN panels were incubated at 35
± 2ºC and read on the WalkAway System, the autoSCAN-4 instrument, and read visually. Read times for
the MSDGN panels were at 16-20 hours for the autoSCAN-4 instrument and visual reads, and 16-18
hours for the WalkAway System. Each isolate was read in triplicate on the autoSCAN-4 instrument.
reference panels were read at 16-18 hours. Results: When compared to frozen reference panel results,
essential agreement for all isolates and tested are as follows:<table class="AbstractTable"
id="{2055AD0B-BA61-461B-A2C0-0D4271F78533}"><caption
colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="2" colspan="1">Read
Method</td><td rowspan="1" colspan="2">Essential<br />Agreement %</td></tr><tr><td rowspan="1"
colspan="1">T</td><td rowspan="1" colspan="1">P</td></tr><tr><td rowspan="1"
colspan="1">Visual</td><td rowspan="1" colspan="1">99.72<br />(353/354)</td><td rowspan="1"
colspan="1">98.87<br />(350/354)</td></tr><tr><td rowspan="1" colspan="1">WalkAway</td><td
/>(275/285)</td></tr><tr><td rowspan="1" colspan="1">autoSCAN-4</td><td rowspan="1"
colspan="1">96.80<br />(1028/1062)</td><td rowspan="1" colspan="1">94.92<br
/>(1008/1062)</td></tr><tr><td rowspan="1" colspan="3">T = Turbidity inoculation method,<br />P =
Prompt* inoculation method</td></tr></table> Conclusion: The MIC results obtained with the MSDGN
panel with an extended dilution series correlate well with MICs obtained using frozen reference panels.
Essential agreement is >90% for all inoculation and read methods. * PROMPT is a registered trademark
of 3M. © 2018 Beckman Coulter, Inc. All rights reserved. Beckman Coulter, the stylized logo and the
Beckman Coulter product and service marks mentioned herein are trademarks or registered trademarks
of Beckman Coulter, Inc. in the United States and other countries.
Session Number: 42
Session Number: 42
Abstract Title:
A Multi-Site Study Comparing A Commercially Prepared Dried Mic Susceptibility Sys. to the Clsi/Iso Broth
Microdilution Method for Plazomicin Using Non-Fastidious Gram-Negative Organisms
S. M. Andrus1, C. C. Knapp1, N. M. Holliday1, S. B. Killian1, J. M. Lindley2, J. M. Streit2, B. J. Olson3, T. R.
Fritsche3, J-W. W. Decousser4, E. Scopes5, A. M. Leonte5, A. W. Serio6; 1Thermo Fisher Scientific,
Oakwood Village, OH, 2JMI Lab., North Libe
Abstract Body:
Background: Plazomicin (PLZ) (Achaogen, South San Francisco, CA) is a next-generation aminoglycoside
active against multidrug resistant Enterobacteriaceae spp., including carbapenem-resistant
Enterobacteriaceae (CRE). A 4-site study was performed to determine the accuracy and reproducibility
of PLZ susceptibility testing using the Thermo Scientific™ Sensititre® dried MIC susceptibility system
(Thermo Fisher Scientific, Cleveland, OH) compared with the CLSI (M07) and ISO 20776-1 (CLSI/ISO)
reference broth microdilution method (BMD). Both automated and manual reading methodologies were
employed. Methods: PLZ (0.06-128 µg/mL) was tested against 473 recent clinical isolates, 96 challenge
isolates, and 15 reproducibility isolates. These isolates consisted of E. coli (122), K. pneumoniae (111), K.
oxytoca (51), E. cloacae (49), E. aerogenes (28), C. koseri (24), C. freundii (26), P. mirabilis (48), P.
vulgaris (24), M. morganii (28), P. stuartii (19), P. rettgeri (23), and S. marcescens (29). The Sensititre MIC
susceptibility system was inoculated per manufacturer’s instructions, and the BMD method was
performed per CLSI/ ISO guidelines. Recommended quality control organisms were tested daily and
within the acceptable ranges. Results: Comparison of Enterobacteriaceae spp. MIC results on the
Sensititre system to the CLSI/ISO BMD method for automated and manual reads resulted in 99.5% and
99.5% essential agreement (EA, +/- 1 log2 dilution) for PLZ, respectively. Overall the essential
agreements for reproducibility (+/- 1 log2 dilution of the modal MIC) using automated and manual reads
were 99.4% and 99.6%. Conclusion: The Sensititre susceptibility system (both automated and manual
read methodologies) demonstrates an equivalent level of performance compared to the CLSI/ISO BMD
method when testing PLZ against Enterobacteriaceae spp. The high level of agreement obtained by the
Sensititre susceptibility system and the CLSI/ISO BMD method demonstrates that this is an acceptable
method for susceptibility testing of PLZ in the laboratory.
Session Number: 42
Session Number: 42
Abstract Title:
Multicenter Evaluation of Ciprofloxacin Mic Results for Gram Negative Bacteria Using Microscan Dried
Gram Negative Mic Panels
R. Brookman1, A. Harrington2, J. A. Hindler3, M. Traczewski4, F. Xu5, S. A. Campeau3, S. DesJarlais2, D.
M. Beasley4, C. J. Hastey1, D. Roe-Carpenter1, A. Chipman6; 1Beckman Coulter, Inc., West Sacramento,
CA, 2Loyola Univ. Med. Ctr., Maywood, IL, 3UCLA
Abstract Body:
Background: A multicenter study was performed to evaluate the accuracy of an extended dilution series
of ciprofloxacin on a MicroScan Dried Gram Negative MIC (MSDGN) Panel when compared to frozen CLSI
broth microdilution reference panels. Materials/Methods: For challenge, an evaluation was conducted
at three sites by comparing MICs obtained using the MSDGN panel to MICs using a CLSI broth
microdilution reference panel. A total of 74 Salmonella enterica serotype Typhi isolates were tested at
each of three sites (for a total of 222 replicates) using the turbidity and PromptTM* methods of
inoculation. For reproducibility, a subset of 11 organisms was tested on MSDGN panels at all three sites.
MSDGN panels were incubated at 35 ± 2ºC and read on the WalkAway System, the autoSCAN-4
instrument, and read visually. Read times for the MSDGN panels were at 16-20 hours. Frozen reference
panels, prepared according to CLSI methodology, were inoculated using the turbidity inoculation
method. All frozen reference panels were incubated at 35 ± 2ºC and read visually. Frozen reference
panels were read at 16-18 hours. FDA breakpoints (µg/ml) used for interpretation of MIC results were:
Salmonella enterica serotype Typhi ≤ 0.06 S, 0.12 – 0.5 I, and ≥ 1 R. Results: When compared to frozen
reference panel results, essential and categorical agreements for all isolates tested in Efficacy are as
follows:<table class="AbstractTable" id="{6086E206-1850-43E0-B054-457A97F53196}"><caption
colspan="1">Read Method</td><td rowspan="1" colspan="2">Essential<br />Agreement %</td><td
rowspan="1" colspan="2">Categorical Agreement %</td><td rowspan="1" colspan="2">VMJ %</td><td
rowspan="1" colspan="2">MAJ %</td><td rowspan="1" colspan="2">MIN %</td></tr><tr><td
rowspan="1" colspan="1"></td><td rowspan="1" colspan="1">T</td><td rowspan="1"
rowspan="1" colspan="1">P</td></tr><tr><td rowspan="1" colspan="1">Visually</td><td rowspan="1"
colspan="1">100<br />(222/222)</td><td rowspan="1" colspan="1">100<br />(222/222)</td><td
colspan="1">100<br />(222/222)</td><td rowspan="1" colspan="1">94.1<br />(209/222)</td><td
colspan="1">autoSCAN-4</td><td rowspan="1" colspan="1">100<br />(222/222)</td><td rowspan="1"
colspan="1">100<br />(222/222)</td><td rowspan="1" colspan="1">95.0<br />(211/222)</td><td
colspan="11">T = Turbidity inoculation method, P = Prompt* inoculation method</td></tr></table>
Reproducibility among the three sites were greater than 95% for all read methods for both the turbidity
and Prompt* inoculation methods for best case. Conclusion: This multicenter study showed that
ciprofloxacin MIC results for Salmonella enterica serotype Typhi obtained with the MSDGN panel with
an extended dilution series correlate well with MICs obtained using frozen reference panels. * PROMPT
is a registered trademark of 3M. © 2018 Beckman Coulter. All rights reserved. Beckman Coulter, the
stylized logo and the Beckman Coulter product and service marks mentioned herein are trademarks or
registered trademarks of Beckman Coulter, Inc. in the United States and other countries.
Session Number: 42
Session Number: 42
Abstract Title:
Microdilution Method for Fda Approved Delafloxacin (Baxdela) Using Gram-Negative Non-Fastidious
Organisms
N. M. Holliday1, C. C. Knapp1, S. M. Andrus1, S. B. Killian1, J. M. Lindley2, J. M. Streit2, B. J. Olson3, T. R.
Fritsche3, J. W. Decousser4, E. Scopes5, A. M. Leonte5, S. McCurdy6; 1Thermo Fisher Scientific,
Oakwood Village, OH, 2JMI Lab., North Liberty
Abstract Body:
Background: Delafloxacin (DLX) (BAXDELATM) (Melinta Therapeutics, New Haven, CT), is a
fluoroquinolone antibacterial that has been FDA approved for the treatment of acute bacterial skin and
skin structure infections (ABSSSI) caused by gram-negative and gram positive organisms. A 4-site
evaluation was designed to determine the accuracy and reproducibility of DLX susceptibility testing
against non-fastidious gram negative organisms using the Thermo ScientificTM SensititreTM dried MIC
susceptibility system compared with the CLSI (M07)/ ISO 20776-1, ISO 20776-2 (CLSI/ISO) reference
broth microdilution method (BMD). Materials and Methods: The Sensititre 18-24 Hour MIC or
Breakpoint Susceptibility System with DLX in the dilution range of 0.004-8μg/ml was tested against 304
recent clinical isolates, 75 challenge isolates and 11 reproducibility isolates. Microorganisms tested
included 132 E.coli, 139 K.pneumoniae, 58 E.cloacae, and 61 Ps.aeruginosa. The Sensititre dried MIC
susceptibility system was inoculated per manufacturers’ instructions. BMD was performed per CLSI/ISO
guidelines. Recommended CLSI quality control (QC) organisms were tested daily and all results were
within the published QC ranges.Results: Comparisons of the indicated non-fastidious gram-negative
organisms MIC results on the FDA cleared Sensititre system to the CLSI/ISO BMD for automated and
manual reads resulted in 98.1% and 98.9% essential agreement (EA; +/- 1 log2 dilution) and 96.2% and
96.6% categorical agreements respectively. Overall agreement for the reproducibility (+/- 1 log2 dilution
of the modal MIC) using automated and manual reads was 99.7% and 99.5%.Conclusions: The results for
DLX indicate that the Sensititre dried MIC susceptibility system for all clinical and challenge gram-
negative non fastidious organisms gave reliable results using either the automated or manual read
methods compared to the reference CLSI/ISO BMD. The FDA has determined that DLX is substantially
equivalent for the indications of E.coli, E. cloacae, K. pneumoniae and Ps. aeruginosa on the Sensititre
dried MIC susceptibility system and has been cleared for in vitro diagnostic use.
Session Number: 42
Session Number: 42
Abstract Title:
In-Vitro Activity of Delafloxacin and Meropenem-Vaborbactam against Multi-Drug Resistant Bacteria
S. Jean, M. A. Wallace, C-A. D. Burnham; Washington Univ. in St. Louis Sch. of Med., Saint Louis, MO
Abstract Body:
Background: Delafloxacin (DFX) is a novel fluoroquinolone with activity against Gram-positive and Gram-
negative organisms including methicillin-resistant Staphylococcus aureus (MRSA), Enterobacteriaceae
and Pseudomonas aeruginosa. Meropenem-vaborbactam (MEV), a new β-lactam/β-lactamase inhibitor
combination, has activity against some carbapenemase producing strains including those with Klebsiella
pneumoniae carbapenemase (KPC). Our objective was to assess the in vitro activity of DFX and MEV
against a cohort of multi-drug resistant (MDR) strains. Methods: Antimicrobial susceptibility testing was
performed on MDR isolates from the Barnes-Jewish Hospital Microbiology laboratory (St. Louis, MO).
DFX and MEV activity was measured using Kirby-Bauer disk diffusion (Hardy Diagnostics) and gradient
diffusion (Liofilchem®) according to manufacturer instructions. MEV activity was tested against
carbapenemase-producing (CP) K. pneumoniae (15) and MDR P. aeruginosa (20) while DFX activity was
tested against MRSA (20) and extended-spectrum β-lactamase (ESBL) producing Enterobacteriaceae (41)
as well as the aforementioned organisms. Susceptibility to ciprofloxacin (CIP), meropenem (MEM), and
ceftazidime-avibactam (CZA50) was also determined where appropriate using Kirby-Bauer disk diffusion
(BD BBL™) according to CLSI guidelines. Results: 85% of MRSA isolates tested were susceptible to DFX
while only 25% were susceptible to CIP or levofloxacin. DFX was less active than CIP against the MDR P.
aeruginosa, CP- K. pneumoniae and ESBL Enterobacteriaceae isolates tested with 24% susceptible to DFX
and 36% susceptible to CIP in this cohort. Though 100% of KPC- K. pneumoniae isolates were susceptible
to MEV, MEV was not active against NDM1- and OXA48-producing K. pneumoniae. Of the P. aeruginosa
evaluated, 25% were susceptible to MEM, 95% susceptible to CZA50 and while no interpretive criteria
have been established for MEV and P. aeruginosa, MEV had an MIC50 = 16 µg/mL and MIC90 >256
µg/mL for the P. aeruginosa isolates tested. Conclusions: These data indicate that DFX and MEV have
increased activity against MRSA and KPC- K. pneumoniae compared to CIP and MEM respectively, and
may be useful therapeutic alternatives against these MDR bacteria.<table class="AbstractTable"
id="{8F7F8065-7493-4BF1-B624-60FC751C0F9B}"><caption
colspan="6">Table 1. Susceptibility of MDR isolates to delafloxacin (DFX) and meropenem-vaborbactam
(MEV)</td></tr><tr><td rowspan="1" colspan="1">Organism (n)</td><td rowspan="1"
colspan="1">CIP</td><td rowspan="1" colspan="1">DFX</td><td rowspan="1"
colspan="1">MEM</td><td rowspan="1" colspan="1">CZA50</td><td rowspan="1"
colspan="1">MEV</td></tr><tr><td rowspan="1" colspan="1">MRSA (20)</td><td rowspan="1"
colspan="1">5 (25%)</td><td rowspan="1" colspan="1">17 (85%)</td><td rowspan="1"
colspan="1">nt</td><td rowspan="1" colspan="1">nt</td><td rowspan="1"
colspan="1">nt</td></tr><tr><td rowspan="1" colspan="1">ESBL Enterobacteriaceae (41)</td><td
rowspan="1" colspan="1">12 (30%)</td><td rowspan="1" colspan="1">9 (22%)</td><td rowspan="1"
colspan="1">nt</td><td rowspan="1" colspan="1">nt</td><td rowspan="1"
colspan="1">nt</td></tr><tr><td rowspan="1" colspan="1">MDR P. aeruginosa (20)</td><td
colspan="1">5 (25%)</td><td rowspan="1" colspan="1">19 (95%)</td><td rowspan="1"
colspan="1">*</td></tr><tr><td rowspan="1" colspan="1">CP K. pneumoniae (15)</td><td
(40%)</td></tr><tr><td rowspan="1" colspan="1">KPC (5)</td><td rowspan="1" colspan="1">1
(20%)</td><td rowspan="1" colspan="1">1 (20%)</td><td rowspan="1" colspan="1">0</td><td
rowspan="1" colspan="1">4(80%)</td><td rowspan="1" colspan="1">5(100%)</td></tr><tr><td
rowspan="1" colspan="1">OXA48 (3)</td><td rowspan="1" colspan="1">1 (33%)</td><td rowspan="1"
(100%)</td><td rowspan="1" colspan="1">1(33%)</td></tr><tr><td rowspan="1" colspan="1">NDM1
(7)</td><td rowspan="1" colspan="1">0</td><td rowspan="1" colspan="1">0</td><td rowspan="1"
colspan="1">0</td><td rowspan="1" colspan="1">0</td><td rowspan="1"
colspan="1">0</td></tr><tr><td rowspan="1" colspan="6">Abbreviations: nt- not tested<br
/>*Interpretive criteria for MEV and P. aeruginosa have not been established; MIC50= 16 µg/mL, MIC90
>256 µg/mL</td></tr><tr><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td
colspan="1"></td><td rowspan="1" colspan="1"></td></tr></table>
Session Number: 42
Session Number: 42
Abstract Title:
Multi-Site Evaluation of Meropenem/Vaborbactam Mic Test Strip Compared to Broth Microdilution for
Enterbacteriaceae and P. Aeruginosa
L. Koeth1, A. Windau2, D. Hardy3, J. Mortensen4, O. Lomovskaya5; 1Lab. Specialists, Inc., Westlake, OH,
2Microtech Lab., Inc., Westlake, OH, 3Univ. of Rochester Med. Ctr., Rochester, NY, 4Cincinnati Children's
Hosp. Med. Ctr., Cincinnati, OH, 5The Med.s
Abstract Body:
Background: MIC Test Strips (MTS, Liofilchem, Roseto degli Abruzzi, Italy) consists of specialized paper
impregnated with a pre-defined concentration gradient of an antimicrobial agent, which is used to
determine the minimum inhibitory concentration against bacteria as tested on agar media using
overnight incubation and manual reading procedures. Meropenem/vaborbactam (M/V) was recently
approved in the US for the treatment of patients 18 years of age and older with complicated urinary
tract infections (cUTI) including pyelonephritis caused by the following susceptible microorganisms:
Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae species complex. This study was
performed to evaluate the performance of M/V MTS compared to a broth microdilution method (BMD)
for FDA 510(k) submission. Methods: Clinical and challenge isolates were tested by M/V BMD with
frozen panels (according to CLSI M7-A10 and M100-S27) and by M/V MTS. Clinical isolates were
collected and tested at 3 sites, 10 reproducibility isolates/agent were shared and tested in triplicate on 3
days at 3 sites and challenge isolates were tested at 1 site. Challenge isolates included majority with MIC
results near or above the susceptible breakpoint. The total number of clinical isolates tested were: 390
Enterobacteriaceae and 75 Pseudomonas aeruginosa. The total number of challenge isolates tested
were: 88 Enterobacteriaceae and 25 P. aeruginosa. QC strains (K. pneumoniae ATCC BAA-1705 and P.
aeruginosa ATCC 27853) were tested a minimum of 20 times by each site. Results: As shown in the table,
M/V MTS MIC results for consolidated clinical and challenge organisms were within +/- one doubling
dilution (essential agreement) of BMD MIC results for >90% of isolates.<table class="AbstractTable"
id="{C2CD88A5-A57D-4C3A-BD7B-7719CE5963E2}"><caption
rowspan="1" colspan="1">Organism</td><td rowspan="1" colspan="1">N</td><td rowspan="1"
colspan="1">% Essential Agreement</td><td rowspan="1" colspan="1">% Category
Agreement</td></tr><tr><td rowspan="1" colspan="1">Enterobacteriaceae</td><td rowspan="1"
colspan="1">478</td><td rowspan="1" colspan="1">95.6</td><td rowspan="1"
colspan="1">97.1</td></tr><tr><td rowspan="1" colspan="1">P. aeruginosa</td><td rowspan="1"
colspan="1">100</td><td rowspan="1" colspan="1">96.0</td><td rowspan="1"
colspan="1">NA</td></tr></table> For reproducibility strains, 97% of M/VMTS results were within a
doubling dilution of BMD results. All MTS and BMD QC results were within CLSI ranges. Conclusions: The
M/V MTS against clinically indicated Enterobacteriaceae species and P. aeruginosa perform similar to
the reference broth microdilution method. The M/V MTS received clearance by FDA, Center for Devices
and Radiological Health, for testing of relevant Enterobacteriaceae species.
Session Number: 42
Session Number: 42
Abstract Title:
Validation of Meropenem-Vaborbactam in the Bd PhoenixTmAutomated Microbiology System
G. A. Denys1, V. Collazo-Velez1, S. Campeau2, M. Weinstein3; 1Indiana Univ. Sch. of Med., Indianapolis,
IN, 2UCLA Hlth.System, Los Angeles, CA, 3Rutgers Robert Wood Johnson Med. Sch., New Brunswick, NJ
Abstract Body:
Background: Meropenem-Vaborbactam* (MEV) is a carbapenem plus non-suicidal beta-lactamase
inhibitor combination that demonstrates activity against multidrug-resistant (MDR) Gram-negative
bacteria, including certain serine beta-lactamases, such as Klebsiella pneumoniae carbapenemase (KPC).
For this study, the BD PhoenixTM Automated Microbiology System (BD Life Sciences, Sparks MD) was
compared to the CLSI-recommended broth microdilution (BMD) method for performance against
Enterobacteriaceae isolates. Methods: A total of 1,141 isolates, encompassing 900 fresh clinical strains,
114 stock and 127 challenge set isolates, were tested in Phoenix and reference panels containing MEV
(range 0.125/8 - 32/8) at three geographically diverse U.S. clinical sites. The challenge set was comprised
of well-characterized beta-lactamase-producing Enterobacteriaceae, and included KPC, OXA and AmpC-
producers. All isolates were tested in parallel using the BD Phoenix System according to manufacturer’s
instructions, and CLSI reference BMD panels. The CLSI-recommended quality control (QC) isolates, were
tested daily in both panels at each site. The breakpoints for Enterobacteriaceae published in the FDA-
approved drug insert (Enterobacteriaceae S ≤4/8, R ≥16/8) were used in the analyses of the essential
(EA) and categorical agreements (CA), very major (VME) and major error (ME) rates. Results: The MEV
results for all Enterobacteriacae tested in the BD Phoenix System produced EA and CA rates of 98.9%
(n=1,128) and 99.7% (n=1,138), respectively. There were no (0%) VME out of 10 resistant strains, and no
(0%) ME with 1,126 susceptible strains. The Phoenix MEV categorically agreed with the BMD for all
isolates identified as KPC-producers. For each of the CLSI QC strains, the results fell within the expected
range ≥97.8% of the time. Conclusions: The BD Phoenix System provided accurate MICs for MEV against
a large collection of gram-negative bacteria including carbapenemase-producing isolates. The BD
Phoenix System provides the clinical laboratory with a reliable method for antimicrobial susceptibility
testing with MEV. *Product not for sale in the US, BD Phoenix™ Automated Microbiology System - GN
Meropenem-vaborbactam is pending 510(k) review. ©2018 BD, BD and BD Phoenix are trademarks of
Becton, Dickinson and Company.
Session Number: 42
Session Number: 42
Abstract Title:
Microdilution Method for Eravacycline Using Gram Positive and Gram Negative Non-Fastidious
Organisms
Fritsche3, K. Becker4, E. A. Idelevich4, J. W. Decousser5, E. Scopes6, A. M. Leonte6, C. Fyfe7; 1Thermo
Fisher Scientific, Oakwood Village, O
Abstract Body:
Background: Eravacycline (ERV) (Tetraphase Pharmaceuticals, Watertown, MA) is a novel, fully-synthetic
fluorocycline displaying broad spectrum activity against non-fastidious organisms, including multidrug-
resistant bacteria. A 4-site study was performed to determine the accuracy and reproducibility of ERV
susceptibility testing against gram positive and gram negative non-fastidious organisms using the
Thermo Scientific™ Sensititre® dried MIC susceptibility system (Thermo Fisher Scientific, Cleveland, OH)
compared with the CLSI (M07) and ISO 20776-1, (CLSI/ISO) reference broth microdilution method
(BMD). Both automated and manual reading methodologies were tested. Methods: ERV (0.001-
16µg/mL) was tested against 848 recent clinical isolates, 180 challenge isolates, and 25 reproducibility
isolates consisting of: Staphylococcus aureus (MRSA, 254), Staphylococcus aureus (MSSA, 256),
Enterococcus spp. (129), E. coli (122), Klebsiella spp. (166), Enterobacter spp. (77), Citrobacter spp. (49).
The Sensititre dried MIC susceptibility system was inoculated per manufacturers’ instructions and the
BMD method was performed per CLSI (M07) and ISO 20776-1 guidelines. CLSI quality control organisms
were tested daily and were within the published ranges. Results: Comparison of gram positive non-
fastidious MIC results on the Sensititre system to the CLSI/ISO BMD method for automated and manual
reads resulted in 99.2% and 98.9% essential agreement (EA, +/- 1 log2 dilution) for ERV, respectively.
Overall the essential agreements for reproducibility (+/- 1 log2 dilution of the modal MIC) using
automated and manual reads were 100% and 100%. Comparison of gram negative non-fastidious MIC
results on the Sensititre system to the CLSI/ISO BMD method for automated and manual reads resulted
in 100% and 100% essential agreement (EA, +/- 1 log2 dilution) for ERV, respectively. Overall the
essential agreements for reproducibility (+/- 1 log2 dilution of the modal MIC) using automated and
manual reads were 99.8% and 99.4%. Conclusion: The Sensititre susceptibility system demonstrates an
equivalent level of performance compared to the CLSI/ISO BMD method when testing ERV against gram
positive and gram negative non-fastidious organisms. The high level of agreement obtained by the
Sensititre susceptibility system and the CLSI/ISO BMD method suggests that this is an acceptable
method for susceptibility testing of ERV.
Session Number: 42
Session Number: 42
Abstract Title:
Evaluating Clin. and Lab. Standards Inst. Testing Recommendations for the Burkholderia Cepacia
Complex
H. K. Huse1, S. A. Campeau1, J. F. Hindler1, J. J. LiPuma2, O. B. Garner1, R. M. Humphries3; 1Univ. of
California, Los Angeles, Los Angeles, CA, 2Univ. of Michigan, Ann Arbor, MI, 3Accelerate Diagnostics,
Tucson, AZ
Abstract Body:
Background: The Burkholderia cepacia complex (BCC) is a group of Gram-negative bacteria that cause
lung infections in persons with cystic fibrosis (CF). CF patients may require lung transplantation due to
severe lung damage caused by BCC infections. BCC is difficult to treat because of intrinsic resistance to
many antimicrobial agents and acquired resistance to other agents. Some transplant centers consider
BCC infection a contraindication to lung transplantation owing to this resistance, which renders post-
operative treatment very challenging. While the Clinical and Laboratory Standards Institute (CLSI)
recommends antimicrobial susceptibility testing (AST) of BCC, the European Committee on Antimicrobial
Susceptibility Testing (EUCAST) does not due to observations of irreproducible results. The goal of this
study was to evaluate CLSI AST methods for BCC. Methods: Disk diffusion (DD), broth microdilution
(BMD), and Etest were performed on 44 BCC isolates following CLSI guidelines or the manufacturer’s
instructions (Etest). DD and Etest were compared to BMD to determine categorical agreement (CA), very
major errors (VME), major errors (ME), minor errors (MiE), and essential agreement (EA; when
applicable). Ceftazidime (CAZ), meropenem (MEM), minocycline (MIN), and
trimethoprim/sulfamethoxazole (TMP/SMX) were tested by DD, BMD and Etest. Levofloxacin (LVX) was
tested by Etest and BMD. Results:<table class="AbstractTable" id="{4C67807A-F4DE-4131-9B64-
38DE22ECA904}"><caption class="AbstractTableCaption"></caption><tr><td rowspan="1"
colspan="1"></td><td rowspan="1" colspan="4">DD vs. BMD</td><td rowspan="1" colspan="5">Etest
vs. BMD</td></tr><tr><td rowspan="1" colspan="1">Antimicrobial</td><td rowspan="1"
colspan="1">CA<br />(%)</td><td rowspan="1" colspan="1">VME<br />(%)</td><td rowspan="1"
colspan="1">ME<br />(%)</td><td rowspan="1" colspan="1">MiE (%)</td><td rowspan="1"
colspan="1">CA (%)</td><td rowspan="1" colspan="1">VME (%)</td><td rowspan="1" colspan="1">ME
(%)</td><td rowspan="1" colspan="1">MiE<br />(%)</td><td rowspan="1" colspan="1">EA<br
/>(%)</td></tr><tr><td rowspan="1" colspan="1">CAZ</td><td rowspan="1" colspan="1">37/44<br
/>(84)</td><td rowspan="1" colspan="1">1/11<br />(9)</td><td rowspan="1" colspan="1">0/27<br
/>(0)</td><td rowspan="1" colspan="1">8/44 (18)</td><td rowspan="1" colspan="1">32/44
(73)</td><td rowspan="1" colspan="1">1/11 (9)</td><td rowspan="1" colspan="1">0/27 (0)</td><td
rowspan="1" colspan="1">10/44 (23)</td><td rowspan="1" colspan="1">20/39<br
/>(51)</td></tr><tr><td rowspan="1" colspan="1">MEM</td><td rowspan="1" colspan="1">32/44<br
/>(73)</td><td rowspan="1" colspan="1">0/16<br />(0)</td><td rowspan="1" colspan="1">0/16<br
/>(77)</td></tr><tr><td rowspan="1" colspan="1">MIN</td><td rowspan="1" colspan="1">19/44
(43)</td><td rowspan="1" colspan="1">12/25<br />(48)</td><td rowspan="1" colspan="1">0/14<br
/>(77)</td></tr><tr><td rowspan="1" colspan="1">TMP/SMX</td><td rowspan="1"
colspan="1">35/44<br />(78)</td><td rowspan="1" colspan="1">0/25<br />(0)</td><td rowspan="1"
colspan="1">6/19 (32)</td><td rowspan="1" colspan="1">3/44 (7)</td><td rowspan="1"
colspan="1">2/19 (11)</td><td rowspan="1" colspan="1">0/44<br />(0)</td><td rowspan="1"
colspan="1">26/40<br />(65)</td></tr><tr><td rowspan="1" colspan="1">LVX</td><td rowspan="1"
colspan="1">NA</td><td rowspan="1" colspan="1">NA</td><td rowspan="1" colspan="1">NA</td><td
rowspan="1" colspan="1">NA</td><td rowspan="1" colspan="1">38/44 (86)</td><td rowspan="1"
colspan="1">24/44 (55)</td><td rowspan="1" colspan="1">36/37<br />(97)</td></tr></table> NA=not
applicable. Conclusion: For most drugs tested, CA and/or EA was low (< 90%) using either DD or Etest.
Similarly, VME and/or ME rates were unacceptably high (> 3%) for most drugs tested. Further studies
will help clarify whether clinicians should consider AST results when deciding whether BCC-infected CF
patients should undergo lung transplantation.
Session Number: 42
Session Number: 42
Abstract Title:
Antiseptic Alcohol Alters the Antibiotic Susceptibility of Proteus MirabiliS
J. J. Gacad, C. B. Lattao; Univ. of Santo Tomas, Manila, Philippines
Abstract Body:
Proteus infections are becoming a public health problem as their resistance to antiseptics and antibiotics
has increased. To investigate the change in the susceptibility of Proteus mirabilis to antibiotics after
alcohol treatment, the bacterium was isolated from uncooked street-vended chicken intestine (“isaw”)
sold fried at Padre Noval Street, Ermita, Manila. The isolate was subjected initially for susceptibility to
the antiseptics 70% Isopropyl alcohol and 70% Ethyl alcohol as well as to the antibiotics ciprofloxacin,
amoxicillin, and cefalexin. It was then tested likewise for susceptibility to lower concentrations of
antiseptics (17.5%, 8.75%, and 4.375%) and again with the different antibiotics. It was observed that P.
mirabilis is more susceptible to 70% Isopropyl alcohol than to 70% ethyl alcohol and also more
susceptible to ciprofloxacin than to the other two antibiotics. Treatments with lower concentrations of
the alcohols caused an increase in susceptibility to ciprofloxacin and cefalexin and a decrease in
susceptibility to amoxicillin. Statistical analysis showed a significant difference (p<0.05) in the
susceptibility of the bacterium to antibiotics and alcohols and in the change of its susceptibility after
treatments with lower concentrations of alcohols. The results obtained merit investigations to establish
the genetic mechanism(s) that caused the change in susceptibility to antibiotics after alcohol treatment.
Session Number: 42
Session Number: 42
Abstract Title:
In Vitro Susceptibility of Yersinia Pestis Strains to Antimicrobial Agents
M. Grdzelidze1, S. Chubinidze1, C. Das2, S. Tsanava1; 1Natl. Ctr. for Disease Control and Publ. Hlth.of
Georgia, Tbilisi, Georgia, 2Intralytix, Inc., Baltimore, MD
Abstract Body:
Background: Plague is an infectious disease caused by the bacterium Yersinia pestis. It is considered a
reemerging bacterial zoonotic disease, which can progress rapidly and can lead to death if treatment is
not timely received. The use of antimicrobial agents for the treatment of Plague started in 1938. The
purpose of our study was to determine the antibiotic susceptibility of Y. pestis strains to 11 different
antibiotics by using the disk diffusion method. Currently, 46 strains of Y. pestis isolated from rodents and
fleas are kept at the National Repository of Bacteria and Viruses of NCDC. The sources of these strains
are as follows: animal carcasses, n=15 (C. musicus n=1; M. arvalis n=11; M. cibicus (erythrourus) n=2; M.
caudata n=1), fleas n=31 (N. setosa n=1; C. caspius n=8; C. teres n=22); Methods: Susceptibility test was
performed in a BSL-3 facility at the NCDC/Lugar Center. Isolates stored at −80°C were reconstituted in
BHI Broth, kept overnight at 35°C and then cultured on MHA plates and incubated at the same
temperature. After incubation, growth was used to make a suspension adjusted to McFarland standard
1.0 (3.0 × 108 CFU/ml) that was inoculated on MHA plate and the following 11 antibiotic disks:
Streptomycin, ampicillin, cefoxitin, trimethoprim-sulfamethoxazole, rifampin, gentamicin, ceftriaxone,
tetracycline, imipenem, chloramphenicol, ceftazidime (BBL Sensi-Disc, Becton Dickinson, Sparks, MD,
USA) were applied. After overnight incubation at 35°C, plates were examined and data were recorded.
All experiments were performed in duplicate. Interpretation criteria were based upon National
Committee for Clinical Laboratory Standards guidelines for Enterobacteriaceae. Control ATCC strains
included: S. aureus 29213, P. aeruginosa 27853 and E. coli 25922. Results: All 46 Y. pestis strains were
found susceptible to the following antimicrobial agents: Streptomycin, ampicillin, cefoxitin,
trimethoprim-sulfamethoxazole, gentamicin, ceftriaxone, tetracycline, imipenem, chloramphenicol,
ceftazidime. 43 strains revealed in vitro resistance to only rifampin with the 3 remaining strains being
borderline intermediate-resistant to rifampin. All Y. pestis strains demonstrated in vitro resistance and
intermediate-resistance to Ansamicin. Strains show susceptibility to antimicrobial class of Cephems and
Carbapenem. Conclusions: All collected strains were found susceptible to antimicrobial class
traditionally recommended for the treatment of Plague, including Streptomycin, Penicillin,
Aminoglycosides, Tetracycline.
Session Number: 42
Session Number: 42
Abstract Title:
The Influence of Single Nucleotide Polymorphism (Snp) Mutations in Gyra, Gyrb, Parc and Pare on the
Acquisition of Fluoroquinolone and Cross-Resistances in Salmonella enterica Subspecies Enterica Serovar
Enteritidis
R. An; Dept. of Vet Sci., St Paul, MN
Abstract Body:
The emergence and dissemination of antimicrobial resistance, including resistance to clinically relevant
antibiotics, has become a significant public health problem worldwide. Fluoroquinolones (FQs), broad-
spectrum bactericides, are the antibiotic of choice for treatment of invasive, life-threatening infections
caused by Salmonella enterica. The consistently Increasing incidence of FQ-resistant S. enterica has
sparked intense worldwide debate aiming to find a solution to this global health problem. In this study,
we aimed to identify and characterize non-synonymous single nucleotide polymorphisms (nsSNPs)
mutations in the genes gyrA, gyrB, parC and parE, responsible for FQ-resistance in four resistant (MIC 8-
16 μg/mL) and four highly resistant (MIC > 128 μg/mL) S. enterica serovar Enteritidis strains,
respectively. Initially, we used four clinical FQ-susceptible (MIC 0.06-0.12 μg/mL) S. Enteritidis strains
(A5-S, A7-S, A21-S, and A33-S) and selected four spontaneous FQ-resistant (A5-R, A7-R, A21-R, and A33-
R) and four FQ-highly resistant (A5-HR, A7-HR, A21-HR, and A33-HR) S. Enteritidis strains via a stepwise
assay using ciprofloxacin as a selective agent. Among both, the resistant and highly resistant strains, no
nsSNP was found in the gyrB and parE. In contrast, all the resistant strains had a substitution of S (Ser)
for Y (Tyr) and F (Phe) at position 83 of the GyrA. The highly resistant strains together with A5-R strain
acquired a second substitution of D (Asp) to G (Gly) at position 87 of the GyrA. For the ParC enzyme, we
found a substitution of G for D at position 78 in the A7-HR strain and S for R (Arg) and I (Ile) at position
80 in the A5-R and A5-HR, respectively. By performing a cross-resistance assay using the resistant and
highly resistant strains, we found that exposure of S. Enteritidis to FQ selects for additional resistance to
cefoxitin, amikacin, chloramphenicol, and ceftiofur, antibiotics that do not belong to FQ class of
antibiotics. Also, exposure to FQ does not affect the susceptibility of S. Enteritidis to
amoxicillin/clavulanic acid, kanamycin, ampicillin, ceftriaxone, and gentamicin, while results for
tetracycline and streptomycin were variable. Our study showed that amino acid substitution at position
83 of the GyrA is crucial for the acquisition of FQ-resistance, while substitution at position 87 of the
same enzyme provides a high-level FQ-resistance.
Session Number: 42
Session Number: 42
Abstract Title:
The Antimicrobial Effects of Zno Nanoparticles against Psuedomonas Aeruginosa
J. C. Nixon; Northest Nazarene Univ., Nampa, ID
Abstract Body:
Background: In the USA alone, there are around 100,000 deaths annually due to healthcare associated
infections. An issue that compounds this problem is the rise in antibiotic resistant bacteria, making
treatment options more difficult to determine. Due to this need for new treatment options, metal oxide
nanoparticles, such as zinc oxide, are promising treatment options due to their antimicrobial properties.
Published studies have revealed antibacterial effects of ZnO nanoparticles on organisms such as E. coli
and Staphylococcus aureus. (1,2) Methods: For this project, the antimicrobial properties of ZnO
nanoparticles (NP) were assessed against Pseudomonas aeruginosa by analyzing growth over time in the
presence or absence of three different ZnO nanoparticles and two different concentrations. Optical
density values of bacterial broths were measured and the optical density values were analyzed using the
computer program R. Results: Data revealed that P. aeruginosa is susceptible to some but not all of the
ZnO nanoparticles tested at different concentrations. Two of the three ZnO nanoparticles tested were
bacteriostatic and signifcantly increased the lag phast of the bacterial growth. Conclusions: These data
indicate that the shape of the nanoparticle, method of synthesis, concentration or a combination of
these three factors are important features to consider when investigating ZnO nanoparticles for
antibacterial applications.
Session Number: 42
Session Number: 42
Abstract Title:
The Challenge of Multidrug- and Extensively Drug Resistant Pseudomonas Aeruginosa (Mdr- and Xdr-
Pa): Using Disc Diffusion Assays and Mechanism-Based-Susceptibility Testing (Mbst) to Guide Therapy in
A Lung Transplant Recipient
M. Yasmin1, S. Marshall2, F. Perez2, O. Martinez3, A-P. Cardona4, L. M. Abbo3, R. A. Bonomo2; 1Univ.
Hosp. Cleveland Med. Ctr., Cleveland, OH, 2Louis Stokes Cleveland Veteran's Affairs Med. Ctr.,
Cleveland, OH, 3Univ. of Miami Miller Sch. of Med., Miami,
Abstract Body:
Background Recurrent lung infections caused by MDR and XDR-Pa are a scourge in persons with cystic
fibrosis (CF) and lung transplantation. Effective treatment is challenging due to the emergence of
antibiotic resistance. When faced with XDR-Pa, clinicians often resort to combination antimicrobial
therapy. Here, we describe a novel application based on disc diffusion assays of antibiotic combinations
with different mechanisms of action. Isolates were obtained from a person with CF who underwent a
lung transplant and had persistent XDR-Pa. We aimed to explore the hypothesis that selective disc
diffusion assays can assist clinicians in choosing active antibiotic combinations. Methods Consecutive
MDR and XDR-Pa respiratory isolates were collected from April 2017 to January 2018. Isolates were
tested using single antibiotic discs to which further antibiotics were sequentially added to create double
(DDD) or triple disc diffusion assays (TDD). We incorporated combinations of anti-pseudomonal
antibiotics, β-lactamase inhibitors, and cell membrane agents to achieve MBST according to CLSI
methods. A regimen was then suggested to the treating physician. Strain relatedness was assessed by
multi-locus sequence typing (MLST). Results Nine isolates from the same patient had varying antibiotic
susceptibility patterns in single and combination susceptibility testing (Table 1). MLST identified isolates
as sequence type (ST) 2100, except for one ST463. In strains resistant to individual antibiotics, discs with
specific targeted combinations revealed active regimens. Conclusions The characterization of strains
with different susceptibility patterns reveals the dynamic nature of Pa in CF and lung transplantation.
Double or even triple disc diffusion antibiotic susceptibility testing was successfully applied to a
persistent Pa respiratory infection after lung transplantion. MBST can be a useful tool to help clinicians
identify treatment options against XDR-Pa. <p><a
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src="http://files.abstractsonline.com/CTRL/ed/1/ad1/ba4/79d/4f4/bbd/b9f/0a2/bf8/2d7/2a/g5915_2.J
PG" alt="" border="0" width="600" height="353" /></a></p>
Session Number: 43
Session Number: 43
Session Title: CPHM03 - Diagnostic Bacteriology: Let's Get Clinical
Abstract Title:
Clinical Evaluation of A Rapid Blood Culture Identification Panel for the Diagnosis of Bacteremia among
Critically Ill Patients in Uganda
M. Workneh1, H. Kajumbula2, O. Abrahim1, F. Nalubega2, O. Mbabazi3, K. C. Carroll1, Y. C. Manabe1, S.
J. Reynolds4; 1Johns Hopkins, Baltimore, MD, 2Makerere Univ., Kampala, Uganda, 3Infectious Diseases
Inst., Kampala, Uganda, 4NIH, Bethesda, MD
Abstract Body:
Background: In low-income settings like Uganda, limited availability of blood cultures and long turn-
around time limits ability to appropriately diagnose bacteremia. The Biofire™ FilmArray BCID panel is a
multiplex PCR system designed to identify 24 pathogens commonly causing bacteremia and 3 antibiotic
resistance genes. The goal of this study was to compare the FilmArray BCID to culture-based biochemical
methods for identification of organisms responsible for bacteremia. Methods: Septic patients 18 years
and older were recruited from the Intensive Care Unit at Mulago Hospital and the Uganda Cancer
Institute in Kampala, Uganda. Two sets of blood cultures collected from independent venipuncture sites
were inoculated into BD Bactec bottles and loaded into the Bactec 9120 blood culture system
immediately upon delivery to the laboratory. Positive blood cultures were identified using Gram-stain
and routine biochemical tests. In addition, an aliquot was tested on the FilmArray BCID according to
manufacturer’s instructions. Results: 73 patients were enrolled in the study between July and October
2017. 16 had positive blood cultures (22%). 13 were tested by the FilmArray BCID and all 16 samples
were tested by routine microbiologic methods. The majority of organisms were Gram-negative, 69% by
the FilmArray BCID and 64% by routine biochemical testing. Organisms identified included Acinetobacter
baumannii (n=4), Klebsiella pneumoniae (K.pneumoniae) (n=2), Escherichia coli (n=1), Pseudomonas
aeruginosa (n=1), Salmonella spp. (n=1), methicillin-resistant Staphylococcus aureus (n=1), Candida
tropicalis (n=1), Enterococcus spp. (n=1). The Salmonella isolate was identified as Enterobacteriaceae by
the FilmArray BCID. 1 of the 2 K.pneumoniae isolates was a metallo-beta-lactamase producer by
meropenem-EDTA combined disc test. All identified Enterobacteriaceae were KPC negative by the
FilmArray BCID. Polymicrobial growth was identified in 3 specimens by FilmArray BCID compared to in 1
specimen by routine methods. Conclusion: These early results suggest increased prevalence of Gram-
negative organisms in this setting in contrast to multi-center evaluations in the United States where
majority of isolates are Gram-positive organisms. Relatively common organisms in this setting, such as
Salmonella spp. are only identified to the family level of Enterobacteriaceae by the FilmArray BCID.
FilmArray BCID may be able to identify polymicrobial growth more readily but misses non-KPC
carbapenemases that are more common in this setting.
Session Number: 43
Session Number: 43
Abstract Title:
Development and Evaluation of A Metagenomic Approach for the Diagnosis of Bloodstream Infections in
Oncology Patients
E. A. Powell1, T. McMillen1, B. Fanelli2, N. A. Hasan2, M. Dadlani2, N. Babady1; 1Mem. Sloan Kettering
Cancer Ctr., New York, NY, 2CosmosID, Rockville, MD
Abstract Body:
Background: Oncology patients are at increased risk for infections, particularly during periods of
neutropenia. Diagnosis of infections in this patient population may be more challenging and the use of
multiple test methods often results in delayed and expensive work-up. Next generation sequencing
(NGS) applications including metagenomcis, microbiome and whole genome sequencing are now being
evaluated and used in diagnostic microbiology. In this pilot study, we aimed to develop and evaluate an
unbiased, metagenomics approach to diagnosis of bloodstream infection in our oncology patient
population Methods: Plasma samples from patients with bloodstream infections detected by standard
of care (SOC) testing (bacterial/fungal blood cultures, viral PCRs) were included. Healthy volunteers were
recruited as negative controls. Extraction was performed using the EasyMag system followed by library
preparation with the Nextera XT DNA Library preparation kit. Libraries were sequenced on the Illumina
MiSeq using the 150-cycle MiSeq V3 reagents. Data were uploaded to CosmosID GENIUS bioinformatic
tool (Rockville, MD) and analyzed using standard settings and filters. Results: A total of 45 samples were
included in the study: 32 positive patients’ samples (10 bacteria, 10 viruses, and 1 yeast); 10 healthy
donors’ samples, and 3 reagents controls. 8/32 positive samples had results concordant between SOC
and NGS (1/10 bacteria, 0/1 yeast, 7/21 viruses). 24/32 had no pathogens detected or were positive for
potential contaminants (Clostridium phage, Torque Teno virus). 1/10 healthy volunteers patients had no
pathogens detected while 9/10 had potential contaminating organisms. 2/3 reagent controls showed
contaminating organisms. Toxoplasma and Pseudomonas were detected in three samples without
corresponding SOC. Conclusions: We developed a metagenomics approach that was concordant with
SOC for 25% of samples and negative for pathogens in healthy volunteers. Data analysis, which is often
challenging, was easily performed using the CosmosID application, which required no advanced
computing or bioinformatics skills. Optimization of our assay, including nucleic acid extraction, depletion
of human DNA, library preparation and target enrichment methods, is ongoing to improve our detection
rate. The detection of additional organisms not evaluated by SOC suggests that the unbiased
metagenomic approach may help identify clinically significant, unsuspected pathogens in oncology
patients.
Session Number: 43
Session Number: 43
Abstract Title:
Case Series of Non-Hacek and HacekGroup Gram Negative Endocarditis At A Large Uk Tertiary
Cardiology and Cardiothoracic Unit
G. Hughes, I. Das; Univ. Hosp. Birmingham, Birmingham, United Kingdom
Abstract Body:
Background: Gram negative organisms particularly non HACEK group organisms are a relatively
uncommon cause of endocarditis comprising <5% of the total cases of Infective endocarditis1. Previous
reviews have identified a number of risk factors including intravenous drug use, prosthetic cardiac valves
and healthcare exposure2. These at risk groups have expanded in recent years to include patients with
central venous lines, haemodialysis patients with tunnelled catheters, the elderly and a growing number
of complex immunocompromised patients3. Given the incidence is low, evidence based guidelines are
limited and often involve combination therapy1,2,3.We present 11 patients managed at a tertiary
referral, regional Cardiology & Cardio-thoracic unit over a 6 year period. Methods: Observational
retrospective case series including patients admitted to our hospital from 2011-2017 using an electronic
database. Results: We found 11 patients in total admitted to our unit over the time period with the
following organisms: S.typhimurium (1), Bartonella sp (2), E.coli (2), Serratia marcescens (1),
Pseudomonas aeruginosa (1), Proteus mirabilis (1), Capnocytophaga canimorsus (1) and Aggregatibacter
sp (2). 7 went on to have their valves extracted and tissue was sent to our lab for culture. Of these 7
patients 2 grew a Gram negative organism on culture (one of which identified a different Gram negative
organism on PCR). 5 of the 7 patients were identified as having a Gram negative organism on molecular
16S rDNA PCR testing. 4 of the 11 who did not have their valve excised were identified as having a Gram
negative endocarditis from blood culture in combination with ECHO findings and the clinical picture. We
identified that 64 valve tissues were sent over the study period for culture and 39 of these for 16S rDNA
PCR. Of the 64 we identified 7 patients (9%) whose valve tissue identified a HACEK or Non-HACEK Gram
negative organism on culture or PCR. This figure is slightly higher than that quoted in the literature. The
reasons for this are currently unclear. 5 of the 11 patients subsequently died though 2 of these were
successfully treated and died of an unrelated cause. Conclusions: Gram negative endocarditis,
particularly involving non HACEK group organisms is rare, carries a high mortality and can be difficult to
manage. Sending valve tissue for culture and broad spectrum molecular 16S rDNA PCR is recommended
as it can often help in the diagnosis.
Session Number: 43
Session Number: 43
Abstract Title:
Causes and Prevention of Needle Stick Injury in Hlth. Care Settings: Cross Sectional Study
B. A. El Awady; Cairo Univ., Cairo, Egypt
Abstract Body:
Background: Needle stick and sharp injuries are one of the major clinical threats experienced by health
care workers. Handling contaminated needles and sharp devices represent high risk of infection by
bloodborne pathogens such as hepatitis C and and HIV. Factors associated with occupational exposure
have not been studied among injured health care workers in Obstetrics and Gynecology hospital in Cairo
University.Objective: Identify needle stick and sharp injuries among healthcare workers and the possible
measures to reduce the risk of re exposure. Methods: cross sectional questionnaire survey of registered
healthcare workers who were exposed to blood and bloody fluids by contaminated needles of other
sharps. Data were collected from 160 health care workers. This study employed analysis of the type of
exposure, the device used, the mechanism of exposure and the department where this injury occurred.
Results: Out of 160 healthcare workers, 62.5 % were nurses, 18% were surgeons, 15% were hospital
housekeepers and 4.5% were lab technicians. Fourty seven percent of exposures were encountered in
operation room and emergency units. Injuries by hollow born needles accounted for 66% of cases.
Manipulating needles in patients, recapping needles after sampling, inappropriate handling of surgical
equipment and improper disposal of sharps were the main circumstances associated with needle stick
injuries. Communication problems when passing sharps, unsafe injection practices and inadequate
policies and procedures were the major contributing factors for sharps injuries. Conclusion: Needle stick
injuries should be recorded in special forms and their causes should be analyzed and checked by
infection control committee. Training and orientation about safe injection practices, appropriate
technique and adequate policies and procedures are the most effective measures to reduce
occupational exposure to needlestick injuries.
Session Number: 43
Session Number: 43
Abstract Title:
Recovery of A Psychrophilic Pseudomonas VeroniI from A Unit of Packed Red Blood Cells Following A
Fatal Transfusion Reaction
J. Farrell; Univ. of Illinois Coll. of Med., Peoria, IL
Abstract Body:
Background: Transfusion reactions related to bacterial contamination of blood products are unusual;
and fatal reactions are exceedingly rare (particularly for red blood cell units). Between 1998 - 2000, CDC
identified rates of 2 deaths/millions units for platelet transfusions, and 0.1 deaths/million units for RBC
units.1 The highest mortality was attributed to Gram negative bacteria.1 Methods: A unit of packed red
blood cells (PRBC) was submitted to our clinical microbiology lab for routine examination following a
suspected transfusion reaction. Blood from the unit of packed red blood cells was collected for Gram
stain, culture in BACTEC blood culture bottles (Becton-Dickinson Microbiology Systems, Sparks, MD,
USA), and aerobic and anaerobic cultures performed on sheep blood, chocolate, and MacConkey agar
plates at 20 oC and 37 oC. PCR molecular identification by Filmarray blood culutre identification (BC ID)
multiplex BioFire PCR panel (Biomerieux, France) was performed on positive BACTEC bottles. Colonies
were identified by Vitek-MS (Biomerieux, France). Results: Heavy Gram negative bacilli (GNB)
contamination of the packed RBC unit was readily apparent on microscopic examination of the Gram
stain. The GNB were identified as Serratia marcescens with BioFire BC ID Filmarray panel, but no viable
organisms were recovered from BACTEC blood culture bottles. There was also no growth in aerobic or
anaerobic cultures of blood, chocolate or MacConkey agars incubated at 37 oC, but a cytochrome
oxidase positive, lactose nonfermenting GNB was recovered on all agars incubated at 20 oC (both
aerobically and anaerobically). The GNB were identified as Pseudomonas veronii by Vitek-MS.
Conclusions: 1. BACTEC culture bottles are NOT reliable for recovery of bacteria from contaminated
PRBC units. 2. Samples from potentially contaminated PRBC units should be cultured at cool ambient
temperatures to maximize recovery of psychrophilic bacteria. 3. The BioFire BC ID Filmarray panel is
NOT recommended for organism identification in cases of suspected PRBC contamination. 4. Whole
genome sequencing may be useful for identification of adaptive mutations in psychrophiles.
Session Number: 43
Session Number: 43
Abstract Title:
Detection of Staphylococcus Species among Postoperative Patients in A Tertiary Care Hospital
O. T. Adekunle1, E. Ojeogwu2, M. Olabode3, H. Y. Akinbusuyi4, G. Iheakor5; 1Obafemi Awolowo Univ.,
Ile Ife, Nigeria, 2Oduduwa Univ., Ile Ife, Nigeria, 3Federal Med. Ctr., Ado Ekiti, Nigeria, 4Olabisi Onabanjo
Univ., Ago Iwoye, Nigeria, 5Imo State Univ.,
Abstract Body:
Background: Surgical site infection (SSI) is defined as an infection that occurs at an incision site within 30
days after surgical operations affecting either the incision or deep tissue at operation site. Postoperative
wound infections have been an important cause of morbidity. The aim of this study is to isolate and
identify the species of Staphylococcus causing postoperative wound infections and also to determine
the antimicrobial sensitivity patterns to conventional antibiotics. Methods: A cross-sectional study was
conducted between October 2016 and February 2017 on 120 surgical site infected patients attending
Obafemi Awolowo University Teaching Hospitals, Ile Ife, Nigeria. The clinical samples were cultured on
media and processed as per standard guidelines. Staphylococcus species were identified with the use of
Microbact™ Staph 12S identification system using a combination of sugar utilization and colorimetric
enzyme detection substrates. Antimicrobial susceptibility testing was performed as per CLSI guidelines.
Results: Out of 120 wound swabs, 74 samples (61.7%) were culture positive, out of which 5 samples
showed more than 1 isolate. 50 samples (38 .3%) yielded no growth after incubation period of 24 hours.
The most common specie of the Staphylococcus isolated were Staphylococcus aureus 29(39.2%), S.
epidermidis 23(31.0%), S. xylosus 7(9.5%) S. hyicus 6(8.1%), S. chromogenes 6(8.1%), S. warneri 2(2.7%)
and S. caprae 1(1.4%). Most of the Staphylococcus aureus exhibited resistance to cefoxitin but all were
sensitive to imipenem. Conclusion: This study showed that Staphylococcus aureus was the most
frequently isolated pathogen from post-operative wound infections. Aseptic surgical practice will help in
preventing post-operative infections.
Session Number: 43
Session Number: 43
Abstract Title:
Identification of Clostridium Botulinum B1 Okra Isolates from A Foodborne Outbreak in China, 2016
Y. Lin1, Y. Jiang1, Z. Gong2, Y. Wang3, Y. Qiu1, M. Jiang1, Q. Chen1, Q. Hu1, X. Shi1; 1Shenzhen Ctr. for
Disease Control and Prevention, Shenzhen, China, 2Southern Med. Univ., Coll. of Publ. Hlth., Guangzhou,
China, 3The Univ. of Shenzhen-Hongkong Hosp.
Abstract Body:
Background: Botulinum neurotoxins (BoNTs) of seven serotypes from A to G is the reason for genetically
diverse of C. Botulinum. Among the species, including A, B, E and F subtypes are prevalently identified
worldwide which cause deadly diseases to the infected. The infection of C. botulinum is commonly
related to foodborne poisoning, with the typical symptoms of dysphagia, dysarthria, ophthalmoplegia
and weakness of limbs. In the study, we review a food poisoning of Clostridium botulinum in Shenzhen,
China. Two poisoning patients involved in the incident had recovered and discharged for the reason of
timely treatment and accurate detection. Methods: In the processing of detection, serum, enema and
suspected food samples were assayed for botulinum neurotoxins by the methods of Detection Kit for
Botulinum A/B/E, detection of morphology, real-time PCR. Enema and suspected food samples were
cultured and isolated for C. botulinum. Mouse bioassay had also been done for detecting enema
samples. Botulinum neurotoxins were identified by specific antitoxins of BoNTs/A, B, E. MALDI-TOF MS
detection was used for confirming, while Gene sequencing technique was used for typing. Results: Cases
of patient’s enema specimens were confirmed for the existence of C. botulinum type B by the methods
of real-time PCR, mouse bioassay and MALDI-TOF MS. The virulence of neurotoxins from the food
poisoning was approximately estimated to 10000 MLD/ml-100000 MLD/ml. Based on phylogenetic
analysis, the isolation of C. botulinum was typed into C. botulinum B1 okra which is far different from C.
botulinum E3 str. Alaska identified in Shenzhen, 2015. Conclusions: It is confirmed that the isolates of C.
botulinum from the food poisoning in Shenzhen, China was typed into C. botulinum B1 okra, with the
virulence of neurotoxins estimated to 10000 MLD/ml-100000 MLD/ml.
Session Number: 43
Session Number: 43
Abstract Title:
Virulence Genotyping and Antibiotic Resistance Profiling of Commensal and Adherent-Invasive
Escherichia coli from the Gut of Inflammatory Bowel Disease Patients and Controls
C. Camprubí Font1, C. Ewers2, M. Lopez-Siles1, M. Martinez-Medina1; 1Univ. of Girona, Girona, Spain,
2Justus-Liebig Univ. Giessen, Giessen, Germany
Abstract Body:
Background To date, no molecular tools are available to identify the adherent-invasive Escherichia coli
(AIEC) pathotype, which is associated to Crohn’s disease. Current techniques, based on phenotypic
screening of isolates, are non-standardisable and time consuming. Several virulence genes (VG), either
its presence or specific variants, have been related with AIEC pathogenicity. Besides, scarce studies have
evaluated the antibiotic (AB) resistance of AIEC. The aim was to study the VG and AB resistance profiles
in a collection of AIEC and non-AIEC strains in order to search for signature traits to assist in AIEC
identification. Methods Fifty-four VG were amplified by PCR, and Sanger sequencing was conducted to
identify fimH, lpfA and chiA point mutations in a collection of 29 AIEC and 37 non-AIEC strains. Strains
were also screened against 30 AB. The χ2 test was used to find differences in the frequency of VG, point
mutations and AB resistance according to AIEC phenotype and phylogroup. Binary regression logistic
test was applied to assess the usefulness of particular genetic traits/AB resistances for AIEC
identification. Results Twenty-five VG reported different prevalence according to the phylogenetic
origin, most of them (24) being mainly present in B2 or D phylogroups. Nevertheless, four VGs were
more frequently found in AIEC strains than in non-AIEC (p<0.05). These were involved in proteolytic
processes, serum resistance and adhesiveness. Moreover, ChiA variable amino acid position A415V was
found specific of AIEC strains, although present only in 20% of AIEC (p=0.049). Interestingly, the gene
sequence of LF82 was shared with 35.5% of AIEC studied and only with the 6.8% of non-AIEC strains
(p=0.027). In addition, AIEC strains were more resistant to ampicillin, piperacillin-tazobactam, ticarcillin-
clavulante, cefazolin and cefuroxime than non-AIEC strains (p<0.042). Binary regression logistic analysis
revealed that typifying E. coli isolates using one VG and an AB resistance was useful to correctly classify
strains according to the phenotype with a 75.5% of accuracy. Conclusions Although there is not a
molecular signature fully specific and sensitive to identify the AIEC pathotype, we propose two features
easy to be tested that could assist in AIEC screening. Moreover, we support that the LF82 ChiA variant
can be of relevance not only for the reference strain but also for a substantial part of AIEC.
Session Number: 43
Session Number: 43
Abstract Title:
Prevalence of Helicobacter Pylori among Patients Attending Private Med. Diagnostic Lab. in Karshi,
Abuja Nigeria
G. Okezie1, P. Onyeka2, G. Mgbowula1, I. Godspower1; 1Decency Amana Med. Diagnosis, Abuja,
Nigeria, 2Argosy Univ., Dallas, TX
Abstract Body:
Introduction: Helicobacter pylori (H. pylori) has been recognized as the primary cause of chronic
gastritis, peptic ulcer disease, gastric carcinoma and gastric mucosal associated lymphoid tissue (MALT).
The prevalence of Helicobacter pylori infection worldwide varies greatly among countries and among
population group in the same country. It is a highly prevalent infection in developing countries with poor
socio-economic status. The discovery of Helicobacter pylori (H. pylori) by warren and marshell in 1983
was a major break-through in the management of dyspepsia. Purpose: The purpose of this study is to
determine the prevalence of H. pylori infection among patient attending private medical laboratory
diagnostic center in karshi Abuja Nigeria. Methodology: Helicobacter pylori antigen rapid diagnostic test
(serological method) was used to determine the prevalence of Helicobacter pylori among three hundred
and fifty (350) patients aged 20-69 years between January 2016 to December 2016 participated in Karshi
FCT Abuja Nigeria. Results: Out of the three hundred and fifty patients, ninety eight 98 (28%) tested
positive for H. pylori while two hundred and fifty two (252) 72% tested negative . A higher H. pylori sero-
prevalence was recorded in the males (20%) than in females (8%).The highest prevalence occurring in
patients 30-39 years of age (15.4%) 11.5% occurrence was found among 20-29 years, while the age
group 60-69years has the least (1.1%). Conclusion: The 28% sero-prevalence of Helicobacter pylori
(H.pylori) in this study is significant. This study has provided additional information on the sero-
prevalence of Helicobacter pylori infection in karshi Abuja Nigeria. Good personal hygiene and good diet
can help reduce or prevent H. pylori. Recommendations are made for the test to be carried out on
patients before emperical treatment with triple regimen and referral to tertiary facilities for diagnostic
endoscopy. This study suggests urgent preventive measures particularly health education of the general
public on Helicobacter pyloric infection.
Session Number: 43
Session Number: 43
Abstract Title:
Monitoring Incidence of V. Cholerae and Related Vibrios in Water Sources and Patients 2016 in Baku and
Baku Districts of Azerbaijan
M. Namiq, S. Sheyda, I. Rita, A. Rakif; Republic Anti-Plague Station, Baku, Azerbaijan
Abstract Body:
Background: Cholera is a devastating diarrheal disease that can result in death within days if left
untreated. The majority of cholera cases are caused by consumption of contaminated water or food.
During 2016, we analyzed samples from patients with intestinal diseases and different water sources in
the Baku region of Azerbaijan, and here we present our results on detection of V.cholerae and other
contaminants. Our aim was to find a release of NAG-vibrion, among others, to the environment that
would indicate the need for continuous monitoring of V. cholerae and related vibrios in water sources.
Methods: Our laboratory carried out bacteriological examination of patients with intestinal diseases,
and a control group -people leading a disorderly lifestyle (alcoholics, drug addicts, persons from
psychiatric hospitals) who are at risk.We collected: 1. fecal and vomit samples from patients diagnosed
with intestinal disease and suspicion of cholera infection as well as control groups; 2. samples of water
from various natural and municipal sources - potable water, sea, lakes, rivers, and others. To test for the
presence of V.cholerae, after sowing the samples on selective and alkaline media, the microorganisms
are also tested for agglutination and specific enzymatic activity. Results: 19,555 samples were analyzed,
among them 19,081 from humans, and 474 from various water sources. Among human samples - 10,620
(55%) from patients with acute intestinal diseases, 546 (3%) from patients with toxic infection, 7,911
(42%) from control groups, and 4 cases of individuals who had a recent contact with sick people or
suspicious cases.V. cholerae has not been found in the human samples, while NAG-vibrio was found in 3
samples and P. aeruginosa - in 26 samples.We detected 92 cases of NAG-vibrio in the water samples,
among them 5 samples from potable water, 65 - from seawater, and 22 - from canals.The 92 NAG-vibrio
positive samples from water sources may be connected to NAG-vibrio positive human cases. In the two
out of three positive cases NAG-vibrio group II was detected, the same as the one isolated from drinking
water. Conclusions: NAG-vibrio is the causative agent of diarrheal disease thus, obviously, there is a
need to expand epidemiological studies of its sources and improve hygienic measures in the areas
where NAG-vibrio have been discovered.
Session Number: 43
Session Number: 43
Abstract Title:
Clinical Impact of A Multiplex Gastrointestinal Pcr Panel in Patients with Acute Gastroenteritis
A. Bateman1, R. Cybulski2, L. Bourassa2, A. Bryan2, B. Beail2, J. Matsumoto3, B. Cookson2, F. Fang2;
1Wisconsin State Lab. of Hygiene, Madison, WI, 2Univ. of Washington, Seattle, WA, 3Harborview Med.
Ctr., Seattle, WA
Abstract Body:
Background: Molecular syndromic panels can increase the detection of infectious etiologies of acute
gastroenteritis. However, the clinical relevance and utility of these panels has not been established.
Methods: We conducted a prospective, multi-center study in Seattle, Washington to investigate the
impact of the BioFire FilmArray Gastrointestinal (GI) panel on clinical diagnosis and decision-making, and
compared the clinical acuity of patients with positive results obtained exclusively with the FilmArray GI
panel versus those detected by conventional stool culture. During a 9-month period in 2017, 1,887
consecutive stool specimens were tested in parallel with FilmArray GI panel and stool culture.
Laboratory data were used to calculate rates of detection and turnaround times, and medical record
review was performed to assess the presence or absence of predetermined clinical features and to
assess the nature and timing of clinical decisions made on the basis of test results. Results: The
FilmArray GI panel detected a pathogen in 35.3% (669/1,887) of specimens, compared to 6.0%
(113/1,887) detected by stool culture. Enhanced yield by FilmArray when stool culture was negative was
most commonly seen with Shiga-like toxin-producing E. coli (STEC; 18 additional cases), Campylobacter
spp. (17 additional cases), Shigella/EIEC (15 additional cases), Yersinia spp. (10 additional cases), and
Plesiomonas shigelloides (6 cases). The median time from collection to result was 18 hrs for the
FilmArray GI panel compared with 47 hrs for stool culture. The median time from collection to initiation
of appropriate antimicrobial therapy was 22 hrs (FilmArray GI panel) compared with 72 hrs (stool
culture). For STEC, the median time to a negative result was 18 hrs (FilmArray GI panel) compared with
75 hrs (stool culture), which allowed for the early discontinuation of empiric antibiotics once the
FilmArray GI panel result was available. Patients with disease diagnosed exclusively by the FilmArray GI
panel had similar clinical features compared to those identified by stool culture and received more
timely and targeted antimicrobial therapy. Conclusions: <u></u> We conclude that the FilmArray GI
panel can substantially increase the speed and sensitivity of laboratory diagnosis and inform clinical
decisions in patients with acute gastroenteritis.
Session Number: 43
Session Number: 43
Abstract Title:
Assessing the Clin. Utility of An Algorithmic Approach to Molecular Testing for C. Difficile At A Tertiary
Care Hospital
A. Balla, A. Theiss, A. Ross, D. Francis, C. Wojewoda; Univ. of Vermont Med. Ctr., Burlington, VT
Abstract Body:
Background: Clostridium difficile is the causative agent for antimicrobial-associated diarrhea and
pseudomembranous colitis and is a major contributor to morbidity and mortality in the United States.
Diagnosis depends on identifying appropriate signs and symptoms which are then supported by
laboratory testing. Methods for identifying organism in stool include molecular platforms, enzyme
immunoassays, and culture. Controversy persists over whether molecular tests are too sensitive at
identifying C. difficile and have raised questions about how additional laboratory information could
inform clinical management and reduce over treatment. The aim of this study was to assess whether
clinical factors were related to toxin status of patients and whether information about toxin status could
potentially inform clinical management of patients. Methods: Stool from patients positive for C. difficile
testing by PCR at the University of Vermont Medical Center between June 2016 and May 2017 were
subjected to additional testing for toxin production by an enzyme immunoassay method. Clinical and
laboratory information including white blood cell count, fever, duration of diarrhea, concurrent laxative
use, prior antibiotic use, history of inflammatory bowel disease, and immune status were collected. The
percentage of PCR+/toxin+ patients and PCR+/toxin- patients was calculated. Data were analyzed for
categorical variables using a chi-square test, a t-test was done to compare means for continuous
variables and a binary logistical regression using the clinical variables was performed. Results: A total of
201 adult patients that were PCR positive for C. difficile were analyzed. Of the 201 samples, 94 (47%)
were toxin positive and 107 (53%) were toxin negative. Although PCR+/toxin+ patients were more likely
to have had a prior C. difficile infection (P=.015), there was no statistical difference between the
additional demographic or laboratory variables that correlated with a positive toxin test result.
Conclusions: We were unable to show that patients with a PCR+/toxin+ result had worse clinical
parameters than those with a PCR+/toxin- results, and concluded that establishing a testing algorithm
that included both PCR and toxin testing would not change the clinical management of patients at our
hospital. One C. difficile infection complication resulting in death occurred in a PCR +/ toxin- patient in
our study further supporting the notion that toxin status does not necessarily equate with clinical
severity of disease.
Session Number: 43
Session Number: 43
Abstract Title:
Pathogen Prevalence Comparison between Cystic Fibrosis Patients with and Without Cystic Fibrosis-
Related Diabetes
D. Chattin, M. Ayshoa Al Gabara; Natl. Jewish Hlth., Denver, CO
Abstract Body:
Background: Chronic respiratory tract infection leading to respiratory failure is the major cause of
morbidity and mortality for patients with cystic fibrosis (CF). Pathogens causing infective exacerbations
must be treated appropriately to minimize lung function attrition. Methods: Two distinct patient
populations were compared to identify trends in recognized pathogens isolated from lung secretions: CF
patients with a diagnosis of CF-related diabetes (CFRD) and CF patients without CFRD. Electronic medical
records from 2008-2017 were scrutinized, and 4,157 bacterial isolates from 5,324 cultures performed on
88 patients with CFRD were compared to 17,766 isolates from 23,831 cultures from 722 patients
without CFRD. Identification of microorganisms was performed using standard clinical microbiology
techniques in accordance with guidelines published by the Cystic Fibrosis Foundation in a medical
laboratory accredited by the College of American Pathologists. Results: Patients with CFRD had a 7%
higher probability of having an organism recognized as a respiratory pathogen isolated than patients
without CFRD, but CFRD patients had nearly twice the chance of being infected with Burkholderia
cepacia, the organism often attributed to end-stage CF disease (growth in 4.3% of cultures from CFRD
patients vs. growth in 2.2% of cultures from non-CFRD patients). Conclusions: The findings from this
study raise the question of whether or not the CFRD disease state impacts the probability of a patient
becoming infected with B. cepacia specifically, and what, if any, are the mechanisms of that process.
One possible explanation for these results is the correlation between increasing age and higher
prevalence of diabetes and the established evidence that age is usually higher when CF patients become
infected with B. cepacia. Due to the impact a diagnosis of B. cepacia infection has on the CF patient, any
factors which impede or promote the growth of that organism will have clinical significance.
Session Number: 43
Session Number: 43
Abstract Title:
Clinical Impact of the Clia-Waived Filmarray Respiratory Panel Ez in An Urgent Care Setting
L. Mortimer, D. Contreras, O. B. Garner; UCLA, Los Angeles, CA
Abstract Body:
Most patients seeking medical care for respiratory illness present at outpatient clinics or the Emergency
Room. Clinical presentations of respiratory infections are usually non-specific and it is impossible to
distinguish viral and certain bacterial respiratory illnesses from signs and symptoms alone. Rapid
multiplex respiratory panel (RP) point of care (POC) testing that identifies the most common causes of
respiratory infections, as well as pathogens that are not easily detected by traditional methods during
the patient visit is expected to improve care on multiple levels. First, implementation of multiplex RPs
should simplify diagnostic ordering and enable physicians to immediately select appropriate therapies in
place of empiric choices. In turn, this should improve antimicrobial stewardship, and help define which
pathogens are circulating in the community. The FilmArray RP EZ is the first CLIA-waived multiplex RP
designed for POC testing that detects 11 viral and 3 bacterial microorganisms commonly associated with
respiratory tract infections from a nasaopharyngeal swab with a 1 hr turn around time (TAT). Our goal
was to evaluate whether a RP in the urgent care setting with a 1 hour TAT would be useful to clinicians
for patient care. The study took place at 3 outpatient sites in the UCLA Health system from Mar. 2017 -
Sept.2017. A total of 103 subjects were enrolled (intervention = RP EZ, n=98; control = standard of care,
n=5). Of the 98 subjects in the intervention arm, 53 tested positive (54%) with 51 testing positive for a
viral pathogen (96%) and 2 tested positive for bacterial pathogen (4%). Of subjects that tested positive
for a virus, 2 had co-infections (4%). One of the patients was a 6 mo old female and was diagnosed with
Rhinovirus/Enterovirus plus Coronavirus. The other patient was a 35 yr female and was diagnosed with
Metapneumovirus plus Parainfluenza virus. The most common infections in the younger population (<5
yr) were Rhinovirus/Enterovirus which accounted for 50% of infections (n=2) followed by Parainfluenza
virus and Coronavirus. Similar trends were seen in patients above 65 yr, the predominant infection was
Rhinovirus/Enterovirus (44.4%) followed by Influenza B (22.2%) and Parainfluenza virus (33.3%). Overall,
a POC test with a 1 hour TAT placed within the clinic dramatically decreases the time to which
actionable results are available. Whether this is fast enough to improve clinical management decisions in
an outpatient setting is still being examined.
Session Number: 43
Session Number: 43
Abstract Title:
Impacts of Housing Style on Influenza-Like Illness Amongst Coll. Students
N. Jandu, C. Annarumo; Gannon Univ., Erie, PA
Abstract Body:
Background: A college campus provides a unique setting for the ease of transmission of various
infectious disease agents, most notably influenza. Transmission can be impacted y housing style,
amongst other factors. Campus housing styles can vary from off-campus to on-campus, with room-
mates or without room-mates, shared bathroom facilities or private facilities. Regardless of housing
style, students will share many other common spaces (i.e. study spaces, dining areas and social rooms).
The aim of this study was to determine if campus housing style impacts the frequency of influenza-like
illness amongst college students. Methods: In this study, an online survey was used to determine the
influenza vaccination status, housing-style and frequency of illness amongst college students. Survey
responses were collected from Oct. 1, 2017 to Jan. 1, 2018. A total of 290-students consented to
completing the online survey. Results: The majority of respondents, 63% (n=183) indicated that they
received the influenza vaccine, while the remaining 37% (n=107) did not receive the influenza vaccine. In
relation to housing-style, the majority of freshmen (56%) live in on-campus suite-style residence with
private bathrooms, the majority of sophomores (67%) live in on-campus apartments with private
bathrooms, some juniors (31%) may still be living in these on-campus apartments, while some juniors
(38%) have transitioned to off-campus housing; and most seniors (61%) are living off-campus.
Regardless of housing, 55% of respondents self-reporting getting sick a few (3-5) times per year. In
comparison, to their previous year on campus, however, 49% of respondents indicated that they were
less sick in subsequent years compared to previous years. Conclusions: College students are susceptible
to influenza-like illness in-part due to close proximity in living arrangements, sharing spaces and other
resources. As students transition through their years of college (from freshmen to senior), changes in
their living arrangement (from on-campus to off-campus) and other lifestyle choices (i.e. improved
hand-hygiene and less eating on-campus) will result in less cases of illness throughout their years on
campus.
Session Number: 43
Session Number: 43
Abstract Title:
Bacterial Detection and Gram Discrimination by A Nested Pcr in Endophtalmitis Patients in Paraguay
M. Samudio, N. Fariña, R. Guillén, F. Rodríguez, S. Abente, C. Duré, J. Barrios; Natl. Univ. of Asuncion,
San Lorenzo, Paraguay
Abstract Body:
Background: Bacterial endophthalmitis is among the most severe complications of cataract surgery. This
surgical procedure is one of the most prevalent in the aged population. Microbiologic diagnosis of
endophthalmitis, based on isolation of the involved microorganisms in culture, is successful in
approximately 22% to 30% of cases in aqueous humor and in 40% to 69% in vitreous. Molecular
techniques have been used to improve the diagnostic rate and reduce the time to diagnosis. The
objective of this study was to evaluate a nested PCR for bacterial detection and Gram discrimination
compared with conventional cultures in vitreous samples from patients with acute endophthalmitis.
Methods: Vitreous samples obtained by puncture from 33 patients with post-surgical or traumatic
endophthalmitis were studied. Samples were divided in two aliquots, one for conventional
microbiological study and the other for PCR. DNA extraction was carried out with the Wizard
commercial kit (Promega). The reaction conditions were as described by Carroll et al. (2000) with some
adjustments. PCR products were visualized by electrophoresis in 2% agarose gel. In the nested PCR,
expected products of 1025bp in gramnegative and 355bp in gramnpositive bacteria were obtained. The
detection sensitivity of the nested PCR was 3.10-5 ng/μl and 6.10-7 ng / μl for gramnegative and
grampositive bacteria, respectively. Results: Out of 33 samples, 8 (24.2%) were positive for bacteria by
conventional microbiological method and 17 (48.6%) by nested PCR, demonstrating the bacterial origin
of the endophthalmitis. The nested PCR showed 100% correlation for bacteria identification and gram
discrimination in the 8 bacteriological positive specimens. Gram-positive bacteria were predominant 6/8
(75%) by culture (see Table 1) and also by the nested PCR 15/17 (88%). Conclusions: This nested PCR
technique shows very good sensitivity, which is useful for a rapid etiological diagnosis in biological
materials with low bacterial load such as intraocular fluids. In addition, it was useful to detect the
bacterial origin of the endophthalmitis and also for Gram discrimination improving sensitivity of the
microbiological conventional method. <table border="1" cellpadding="1" class="DisplayTable"
id="{F5CBC78D-E615-46E4-AA0F-4B0173C58F96}"><caption>Table 1. Bacteria isolated from vitreous
fluid of eight endophthalmitis patients</caption><tr><td rowspan="1" colspan="1">Bacteria</td><td
rowspan="1" colspan="1">n</td><td rowspan="1" colspan="1">Nested PCR</td></tr><tr><td
rowspan="1" colspan="1">Enterococcus casseliflavus</td><td rowspan="1" colspan="1">2</td><td
rowspan="1" colspan="1">G(+), 355bp</td></tr><tr><td rowspan="1" colspan="1">Enterococcus
faecalis</td><td rowspan="1" colspan="1">1</td><td rowspan="1" colspan="1">G(+),
355bp</td></tr><tr><td rowspan="1" colspan="1">Staphylococcus epidermidis</td><td rowspan="1"
colspan="1">1</td><td rowspan="1" colspan="1">G(+), 355bp</td></tr><tr><td rowspan="1"
colspan="1">Pseudomonas stutzeri</td><td rowspan="1" colspan="1">1</td><td rowspan="1"
colspan="1">G(-), 1025bp</td></tr><tr><td rowspan="1" colspan="1">Streptococcus viridans
group</td><td rowspan="1" colspan="1">2</td><td rowspan="1" colspan="1">G(+),
355bp</td></tr><tr><td rowspan="1" colspan="1">Stenotrophomonas maltophilia</td><td
rowspan="1" colspan="1">1</td><td rowspan="1" colspan="1">G(-), 1025bp</td></tr></table>
Session Number: 43
Session Number: 43
Abstract Title:
Comparison of Culture, Sequencing, and Isothermal Rna Amplification Assay for Detection of
Ureaplasma From First Void Urine of Patients with Urogenital Infections
Y. Liu; Peking Union Med. Coll. Hosp., Beijing, China
Abstract Body:
Background: In order to compare different assays of Culture, Sequencing, and Isothermal RNA
Amplification Assay for Detection of Ureaplasma from First Void Urine of Patients with Urogenital
Infections. Methods: Parallel aliquots of each urine specimen were tested for Ureaplasma by culture
only, culture and sequencing, and Isothermal RNA Amplification Assay (IRAA) (targeting 16S rRNA).
Results: From 2015 to 2016, a total of 103 urine specimens were collected from patients with urogenital
infections. Culture only, culture and sequencing, and IRAA, detected Ureaplasma spp in 17.48%
(18/103), 23.3% (24/103), and 25.24% (26/103) of the 103 specimens, respectively. Based on the gold
standard (culture and sequencing), the sensitivity and specificity of IRAA method were 91.67% (22/24)
and 94.94% (75/79), respectively, compared to 75.00% (18/24) and 100% (75/75), respectively, for
culture only method (p>0.05). Of the 24 Ureaplasma positive specimens, 70.83% (17/24) had U. parvum
only, 29.17% (7/24) U. urealyticum only, and none (0/24) contained both. The antibiotic susceptibility
rates of the 18 Ureaplasma spp isolates ranged from 11.1 % (ciprofloxacin) to 100 % (pristinamycin).
Ciprofloxacin and ofloxacin showed the lowest activity, with only 11.11% and 22.22% susceptibility,
respectively. Conclusions: Our findings suggest that IRAA demonstrate high sensitivity and specificity for
the detection of Ureaplasma, time-saving and high throughput while culture showed much higher
specificity, cheaper and also give antimicrobial susceptibility testing results.
Session Number: 43
Session Number: 43
Abstract Title:
Accumulated Experience of the Biofire Filmarray Meningitis/Encephalitis Panel At A Tertiary-Care
Hospital
C. M. Bolster LaSalle, T. E. Grys; Mayo Clinic, Phoenix, AZ
Abstract Body:
Background: The Biofire FilmArray Meningitis/Encephalitis (ME) Panel has been in use in our laboratory
for testing cerebrospinal fluid (CSF) specimens since October 4, 2016. Our setting consists of a 268-bed
tertiary care hospital service, Cancer Center, and clinics that together serve a high percentage of
patients who are immunocompromised due to cancer treatment, stem cell transplant, and solid organ
transplant. Methods: We examined all results for tests performed on the FilmArray ME Panel in our
laboratory from test go-live through December 31, 2017. We collected data including white blood cell
count (WBC) and glucose as well as other testing performed at the same time. Results: A total of 216
tests were performed during the period with 14 specimens (6.5%) testing positive. Overall, 101 samples
(47%) had normal WBC and glucose values, while both values were abnormal in 23 (11%). Culture was
ordered on most samples tested using the ME panel, but was not ordered on 63 samples (29%).
Redundant single-agent PCR testing was ordered for some samples, including 3 (1%) for enterovirus, 39
(18%) for Herpes simplex (HSV), and 6 (3%) for both enterovirus and HSV.Fourteen specimens were
positive, including Streptococcus pneumoniae (3), Enterovirus (3), Varicella Zoster Virus (3), Human
Herpes Virus-6 (3), and Herpes Simplex Virus-2 (2). Five of the positive specimens had correlating
confirmatory results (growth in culture or positive on other PCR testing), six had no correlating results,
and three had negative results on correlating testing (VZV, S. pneumoniae, HHV6). Twelve (86%) of the
positive specimens had glucose and white blood cell profiles consistent for the pathogen type (i.e. low
glucose high WBCs for bacteria, normal glucose, high WBCs for viruses). Overall 13 of 14 (93%) of
positive samples had elevated WBC. The sole sample with normal WBC was positive for HHV-6, and
though the patient had HHV-6 positive in blood sample as well, the physician team felt the HHV-6 was
an incidental finding. Conclusions: The ME Panel has improved the ability of our laboratory to provide
rapid PCR results for 14 infectious agents, improving our turnaround to a few hours instead of 1-3 days
for send out testing. Preliminary data that suggest that the ME panel should not be performed in
samples that have normal WBC count.
Session Number: 43
Session Number: 43
Abstract Title:
Study of Acute Febrile Illness in Adjara Region, the Country of Georgia
M. Chkhaidze, N. Gugushvili, V. Tavadze, N. Kishmaria, N. Chitadze, T. Chikviladze; Natl. Disease Control
Ctr., Batumi, Georgia
Abstract Body:
The Black Sea regions of Georgia, territories bordering Turkey, are high migration and transit areas. The
region provides ideal climate conditional for vectors and the spread of zoonotic diseases. The Port of
Batumi and the port of Poti are reached by vessels coming from Africa, Asia, and Latin America where
some countries have unfavorable epidemic situation; such as India, China, Pakistan and Philippines.
There is unlimited contact between domestic and international carriers there. This leads to a high risk
for spreading of different dangerous infection diseases as well as Especially Dangerous Pathogens
(EDPs). These worldwide emerging and reemerging infectious diseases are not well-studied in the Adjara
and Guria regions. The study proposes to conduct a serosurvey with the population of Adjara and Guria
regions to determine the causative agent of Acute Febrile Illness (AFI). Information on the infectious
causes of undifferentiated acute febrile illness is essential for effective treatment and prevention.
During 2012-2016, Batumi ZDL collected 549 human blood samples from the patients with
undifferentiated acute febrile illness on the Black Sea Coast. Samples were immediately delivered to the
regional laboratory for initial processing. Sera were tested using a commercial enzyme linked
immunosorbent serologic assay (ELISA) kits for the presence of M (IgM) antibodies to Leptospira,
Brucella and Lyme disease. In-kit positive, negative controls and standards, provided by the reagent
manufacturer, were used. 4 fold or greater rise of antibody titers between paired samples were
measured to confirm the recent infection. Out of 549 samples, 15.66% (86/549) were exhibited
presence of Leptospira IgM; 3.46% (19/549) were positive for Brucella IgM and 7.83% (43/549) were
positive for Lyme disease IgM. The study shows the value of the laboratory diagnostics; out of 549 AFI
cases, a causative agent was not identified in 73% of them. For proper treatment and a strong
surveillance system, it is important to enhance the laboratory diagnostic system in the region.
Session Number: 43
Session Number: 43
Abstract Title:
A Retrospective Study of Fifty Cases of Brucellosis in A Tertiary Hosp. from China
L. Zhang, Y. Xu; Peking Union Med. Coll. Hosp., Beijing, China
Abstract Body:
Background: Brucellosis is a zoonosis caused by Gram-negative bacteria, Brucella spp. Brucellosis shows
wide clinical polymorphism, which frequently leads tomisdiagnosis and treatment delays. Especially in
China, most brucellosis cases were misdiagnosed and rarely case reported due to its relatively low
incidence rate and doctors’ lack sense of this disease. The aim of this study was to report brucellosis
cases in our hospital, and to analyze their clinical and laboratory findings. Methods: A total of 50 cases
were diagnosed as brucellosis in Peking Union Medical College Hospital, over a five-year period from
2012 to 2017. A retrospective study was undertaken and patient files were investigated for their history,
clinical and laboratory findings, as well as clinical outcomes. Results: Nearly all the cases were
misdiagnosed at first, while most patients were finally diagnosed for at least 2 months. Of the 50
patients, 39(80%) were male and 10 (20%) were male. The mean age of patients was 52.3 years. Half
cases had a history of raw milk or products consumption and others were found to have no occupational
risk for brucellosis. The most frequently seen symptoms were arthralgia (68%) and fever (80%). The
most frequent laboratory finding was neutropenia (76%), high ESR (71.7), and a high C-reactive protein
level (52%). The STA test was positive in all 20 cases who had been conducted this test. The most
important discovery was that nearly half cases showed coagulation disorders with prolonged APTT time
and higher D-Dimer level. Doxycycline and streptomycin with or without rifampin appeared to be most
common treatment method. Conclusions: Brucellosis may lead to serious morbidity, and it is
increasingly being an important health problem in China. Much morestudies are needed to show their
clinical manifestations and other characteristics, which could help doctor accurately diagnose brucellosis
and give proper treatment.
Session Number: 43
Session Number: 43
Abstract Title:
Outstanding Abstract Award: Genital and Extragenital Chlamydia Trachomatis and Neisseria
Gonorrhoeae Infections in Pediatric Population
P. Uprety1, R. Shivakoti2, A. M. Cardenas1; 1Univ. of Pennsylvania; Children's Hosp. of Philadelphia,
Philadelphia, PA, 2Johns Hopkins Univ., Baltimore, MD
Abstract Body:
Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (GC) are the two most commonly reported
notifiable sexually transmitted disease in the US. Studies in adults have shown that the prevalence of
CT/GC infections is much higher in extragenital sites (e.g. oropharynx or rectum) compared to urogenital
site (e.g. urine). However, similar data on the burden of CT/GC infections by anatomic site is lacking in
pediatric populations. In this study, we evaluated the distribution of CT/GC infections in genital and
extragenital sites in pediatric population. We retrospectively collected data on samples that were
submitted for CT and GC (N= 655) testing to the Infectious Disease Laboratory at Children's Hospital of
Philadelphia from 10/1/2012-10/27/2017. To compare the burden of CT/GC infection by anatomic sites,
we restricted the analysis to include patients between 2 years to 18 years of age, and with all three
samples types (urine, oropharyngeal swab and rectal swab) collected at the same visit for CT (N=148)
and GC (N=154). CT/GC infections were measured using Aptima Combo 2 Assay for CT/GC. Our results
show that the positivity rate for CT was highest in rectum (16.2%) followed by urine (5.4%) and
oropharynx (0.7%), whereas GC was highest in rectum (10.4%) followed by oropharynx (9.7%) and urine
(1.9%) (Figure 1). Most importantly, for CT, a significant percentage of infections were missed if only
urine (68%) or oropharynx (96%) were tested compared to fewer cases missed with rectum samples
(4%). Similarly, for GC, substantial percentage of infections were missed with urine only (88%) compared
to oropharynx (40%) and rectum (36%). Our data shows that the burden of infection for both GC and CT
is highest in the rectum and a significant percentage of infections might be missed if only one anatomic
site is tested, particularly urine. We hope that this data will aid in revising the current testing
recommendation to include extragenital screening for CT/GC infections in pediatric population.<br
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Session Number: 43
Session Number: 43
Abstract Title:
Longitudinal Exploration of Urine Contamination Rates At A Single Med. Center
H. S. Porterfield, B. Doyle, B. Ford; Univ. of Iowa Hlth.Care, Iowa City, IA
Abstract Body:
Multiple variables leading to submission of contaminated urine specimens complicate quality
improvement projects aimed at correcting this costly problem. Evidence-based guidance to reduce urine
culture contamination is scant. We performed an 18-month (January 2016 to June 2017) retrospective
study to establish baseline urine culture contamination rates at an 811-bed Midwestern academic
hospital. A specimen was counted as contaminated if the clinical report was finalized as skin flora,
urogenital flora, mixed flora, or >=3 uropathogens present. Overall and subgroup rates were calculated,
including for midstream urine specimens (MSU) and straight catheter urine specimens (SCU) for each of
the nursing divisions and the ten highest volume units. Divisions and units of interest were then
analyzed using statistical process control methods including p-charts. After the baseline contamination
rate was determined, an on-going prospective study began in July 2017 to assess the successes and
failures of quality improvement interventions. Analysis of urine specimens over 18 months revealed the
contamination rate for all urine specimens to be 40.4% (16188 of 40,047 specimens), for MSU to be
48.0% (14,357 of 29,918), and for SCU to be 17.4% (773 of 4,455). Analysis by month showed that MSU
contamination rates ranged from 42.1% (650 of 1,544) to 52.0% (869 of 1,670). MSU contamination
rates ranged widely across nursing divisions, from 11.8% (4 of 34) for Perioperative Nursing to 55.9%
(3,467 of 6,203) for the Emergency Department. High-volume units had MSU contamination rates
ranging from 63.4% (861 of 1357) for a women’s health clinic to 34.3% (304 of 886) for a urology clinic.
Subgroup analysis showed a noticeable quality gap between MSU specimens from males (19.7%
contaminated) and females (55.1% contaminated), a 35.4% difference. This gap was not demonstrated
in SCU specimens (14.4% for males and 17.6% for females). Statistical process control and p-charts
showed that rates were not within statistical control, with 9 of 18 months showing special cause
variation, suggesting that institutional processes are not well standardized. The prospective phase of the
study has shown no improvement in urine contamination rates related to improvement efforts. The
baseline urine culture contamination rate for all urines is 40.4% and for MSU is 48.0%. Contamination is
35.4% higher in MSU from females compared to males. More focused and directed quality improvement
efforts are needed to improve the quality of urine culture specimens at the institution
Session Number: 43
Session Number: 43
Abstract Title:
Eskape Pathogens are Escaping from Antimicrobials in A Hosp. in Kathmandu
S. K. Mishra1, R. Pandey2, M. Lakhey3; 1Maharajgunj Med. Campus, Kathmandu, Nepal, 2St. Xavier's
Coll., Kathmandu, Nepal, 3Medicare Natl. Hosp., Kathmandu, Nepal
Abstract Body:
ESKAPE pathogens stand for Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae,
Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp. These are commonly
associated with nosocomial infections and have been listed as priority organisms by World Health
Organization (WHO). To our knowledge, researchers have not focused to determine prevalence of
ESKPAE pathogens in Nepalese hospital settings. Therefore, this study was conducted to establish base-
line status of ESKAPE pathogens in a hospital in Kathmandu, Nepal. In this hospital-based prospective
study, a total of 7296 specimens (excluding blood) from both inpatients and outpatients were processed
in the clinical microbiology laboratory for culture and sensitivity following standard methodology as
described by American Society for Microbiology. Out of 7296 clinical specimens, significant growth of
34.5% (n=1602) was seen in urine specimens, 38.0% (n=450) in lower respiratory tract specimens, 60.0%
(n=657) in pus and 2.7% (n=10) in body fluid specimens. Of the growth positive cases in all the
specimens, the prevalence of ESKAPE was found to be 34.7% (944/2721). These ESKAPE isolates
consisted of Enterococcus faecium (4.7%), Staphylococcus aureus (31.0%), K. pneumoniae (36.4%), A.
baumannii (7.7%), P. aeruginosa (19.1%) and Enterobacter spp. (1.1%). Enterococcus faecium and K.
pneumoniae were most commonly isolated from urine specimens; S. aureus and P. aeruginosa from pus
specimens; A. baumannii from sputum and endotracheal aspirate specimens. Nearly 4.5% of the
Enterococcus faecium isolates were vancomycin resistant, 36.9% were methicillin resistant S. aureus
(MRSA). Carbapenem resistance was shown by 12.3% of K. pneumoniae, 39.4% of Acinetobacter
baumannii and 12.9% of P. aeruginosa. Minimum inhibitory concentration of Colistin sulphate was in the
range of 1-2mcg/mL against gram-negative members of multidrug resistant ESKAPE. ESKAPE pathogens
are the leading bugs in our hospital leaving fewer therapeutic options. The resistance shown by the
respective members of the ESKAPE bugs to drugs like methicillin, vancomycin or carbapenems proves
that they are really going to the offense with a strong defense. Inclusion of antimicrobial stewardship
program is today's demand to tackle the future with potentially no effective antimicrobials.
Session Number: 43
Session Number: 43
Abstract Title:
Pseudoxanthomonas Kaohsiungensis Bloodstream Infection
S-F. Kuo; Kaohsiung Chang Gung Mem. Hosp., Kaohsiung, Taiwan
Abstract Body:
Background: Pseudoxanthomonas kaohsiungensis was first reported in 2005, and was recovered from an
oil-polluted site near Kaohsiung city in southern Taiwan, where it seems to an environmental organism.
In June 2017 , a 28-year-old oil refinery worker at Kaohsiung, Taiwan, with 3 months of chest heaviness
presented with one week of fever. A P. kaohsiungensis bacteremia isolate recovered during
hospitalization by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
Methods: P. kaohsiungensis was identified by matrix-assisted laser desorption ionization-time of flight
mass spectrometry (MALDI-TOF MS) (Bruker Daltonik, Bremen,Germany). We also confirmed the
identification by sequence and biochemical analysis. Conventional biochemical characteristics presented
in the API 20NE (bioMe´rieux, Marcy l’Etoile, France) and VITEK 2 GN ID card microtest systems
(bioMe´rieux,Marcy l’Etoile, France). The antimicrobial susceptibility of the organism was assessed using
the Phoenix automated microbiology system (BD Diagnostic Systems, Sparks, MD) Results: The 16S rRNA
gene sequences of this strain shows 99% identity to Pseudoxanthomonas Kaohsiungensis (accession
number NR043070). The results of biochemical analysis are same with Chang JS et al. described. That is
oxidase activity is present. Nitrate is reduced and esculin is hydrolyzed. Glucose, N-acetyl- D -
glucosamine and maltose are utilized. The strain is negative to indole production, glucose fermentation,
arginine dihydrolase,catalase, urease and gelitin hydrolysis. Arabinose, mannose and mannitol are not
utilized. The fever subsided on the third-day of ceftazidime treatment. The symptom of exertional
dyspnea was absent 5 days later. Conclusions: The evidence suggests that this organism is a potential
human pathogen in an immunocompromised host, rather than simply a biosurfactant-producing
bacterium in the environment.
Session Number: 43
Session Number: 43
Abstract Title:
KlebsiellaIsolated from Cases of Nail Plate Discoloration/Pigmentation and their Etiologic Significance
I. Kobayashi1, I. Kanesaka1, Y. Ishimura1, H. Takahashi1, A. Ohno1, A. Katsuse Kanayama1, A. Kaneko2,
K. Ohkusu3; 1Toho Univ., Tokyo, Japan, 2Tokai Univ., Kanagawa, Japan, 3Tokyo Med. Univ., Tokyo,
Japan
Abstract Body:
Background: Bacterial causes of nail infections due to Pseudomonas aeruginosa and enteric bacteria
have been shown to be associated with an underlying disease such as diabetes or in occupations when
there is frequent exposure to water. In a previous report at the ASMmicrobe 2016, we reported another
risk factor associated with nail discoloration/pigmentation. P. aeruginosa and enteric bacteria were
recovered at a high frequency from wearers of acrylic nail gels and other artificial nails over an extended
period. Methods: Using sterile swabs, samples from under fingernails were taken from 24 adult women
users of acrylic gel and other artificial nail with nails showing greenish discoloration. Swabs were
inoculated into BHI semi-solid agar and incubated at 35 C° for 48 hours followed by isolation using
CHROM agar. Microbial identification was performed of Phoenix Automated Microbiology system (BD).
Multiplex- PCR was using for identification of Klebsiella (Garza-Ramos et al. 2015). Results: Gram
negative bacilli were isolated from 16 (66.7%) of the samples taken. Of the 16 isolates, the most
frequently isolated organism was P. aeruginosa at 13 (81.3%) followed by 9 (56.3%) Klebsiella. Of the 9
Klebsiella isolates, BD Phonenix identified 5 as K. pneumoniae and 4 as K. oxytoca. Using a multiplex-PCR
probe assay, 2 of the Klebsiella isolates were identified as K. variicola. Conclusions: Our study shows that
2 of the Klebsiella isolates recovered from discolored/pigmented nail plates were K. variicola which is
naturally associated with plants. As this organism is generally misidentified as K. pneumoniae in clinical
microbiology laboratories, our findings expand the clinical spectrum of infections associated with this
organism.
Session Number: 43
Session Number: 43
Abstract Title:
Escherichia Albertii Masquerading As Typical Enteropathogenic Escherichia coli
E. A. Cebelinski, V. R. Lappi, G. Salo, M. Orth, N. Podemski, S. Jawahir, X. Wang, D. Boxrud; Minnesota
Dept. of Hlth., St. Paul, MN
Abstract Body:
Background: Escherichia albertii is an emerging enteropathogen often misidentified as enteropathogenic
E. coli (EPEC) because both possess intimin (eae) and can be difficult to differentiate by biochemical
reactions. Typical EPEC (tEPEC) possess bfp (bundle forming pilus) whereas atypical EPEC (aEPEC) and E.
albertii do not. BioFire FilmArray Gastrointestinal (GI) panel identifies EPEC by detection of eae but it
cannot differentiate between E. albertii, tEPEC or aEPEC. Here we describe unique isolates of E. albertii
possessing bfp that were identified as EPEC by FilmArray. Methods: Clinical materials positive for EPEC
are required to be submitted to Minnesota Department of Health Public Health Laboratory. Stools
submitted during 2016-2017 were cultured on sorbitol MacConkey (SMAC) and Hektoen (HE) agar.
Mixed colony sweep PCR for eae, bfp, and E. albertii housekeeping genes (mdh, lysP) was performed
from SMAC. Colony PCR from positive culture sweeps was used to identify isolates. Suspect lactose
negative colonies on HE were screened by biochemicals. Isolates identified from HE or colony PCR were
tested by PCR for eae, bfp, mdh, lysP, cytolethal distending toxin (cdtB), and an E. albertii species-
specific PCR (transcriptional activator). Confirmed isolates of E. albertii were characterized by PFGE and
WGS. De novo assembly was performed on sequence reads using CLC Workbench and analyzed using a
pipeline available from Center for Genomic Epidemiology. Results: Six isolates were PCR positive for eae,
bfp, mdh, lysP, cdtB, and transcriptional activator and confirmed as E. albertii by biochemical reactions.
WGS-based serotypes were identified as O131 (n=5) and O182 (n=1). Virulence markers detected in
silico included eae, bfpA, cdtB and perA. One isolate had antimicrobial resistance markers detected
(blaTEM-1B). By PFGE, 5 unique patterns were identified. Two isolates identical by PFGE had co-
detections with STEC O157. The STEC isolates also matched by PFGE though a common exposure was
not identified. Five of 6 specimens had an additional pathogen detected, including Campylobacter,
sapovirus, STEC O157 (n=2) and Salmonella. Conclusions: To our knowledge, this is the first report of E.
albertii possessing bfp, a virulence gene more commonly associated with tEPEC. It is unclear what effect
the presence of bfp has on the virulence of E. albertii. The FilmArray GI panel is an instrumental tool for
identifying EPEC and E. albertii. Going forward, it is important to properly categorize eae+ isolates to
better understand their role in causing illness.
Session Number: 43
Session Number: 43
Abstract Title:
Bacteria of the Candidatus Saccharibacteria Phylum Present in Diverse Clin. Specimens from Humans
D. R. Hoogestraat, N. G. Hoffman, S. J. Salipante, B. T. Cookson; Univ. of Washington, Seattle, WA
Abstract Body:
Background: Among recently revealed bacterial divisions, the Candidatus Saccharibacteria (formerly
TM7) phylum is of interest due to its association with inflammatory disease conditions and the ability to
form epibiotic relationships with known pathogens, which can alter bacterial interactions with immune
cells. While studies of TM7 bacteria to date have focused on cultivation and characterization, the
prevalence of these organisms in diverse clinical samples has not been explored. Methods: V1V2 regions
of the 16S rRNA gene were amplified from DNA extracted from clinical samples submitted to our lab for
characterization of bacterial pathogens. After library preparation, Next generation sequencing (NGS)
was performed on an Illumina MiSeq. Data analysis included (i) quality filtering with exact sequence
variants (SVs) inferred according to the dada2 algorithm, (ii) discarding SVs with poor alignment to a 16S
structural model and (iii) classifying SVs by BLAST identity against NCBI type-strain sequences. SVs with
no matches > or = 90% were placed on a reference tree for likelihood-based phylogenetic assessment
using pplacer. Results: 585 clinical specimens were examined, representing sites within respiratory,
cardiac, lymph, central nervous, and genitourinary systems, among others. 551/585 (94%) samples
contained SVs <90% identical with NCBI type strain 16S sequences by BLAST, revealing 3239 unique SVs
(2.72% of all reads) from aggregate reads across all samples. ~7% of these filtered reads from 85
specimens contained 92 variants with high probability phylogenetic placement within TM7. 83.9% of
reads classified within TM7 were found in respiratory and oral specimens. Of SVs identified, most were
similar to sequences from previously reported human-associated phylotypes. Four SVs did not match
any record in the NCBI nt database >98.5% representing potentially novel species within the phylum.
Conclusions: NGS-based analysis is expanding our understanding of the polymicrobial contributions to
the pathogenesis of infection. This study indicates that majority of samples submitted for clinical testing
contain evidence of species with low 16S sequence similarity to NCBI type strains. We found evidence of
TM7 in a diversity of samples (cerebrospinal fluids, lymph node and bowel biopsies, mandibular tissue,
and respiratory specimens obtained via bronchoscopy, among other types) suggesting a potential role
for TM7 bacteria in the pathogenesis of systemic infections.
Session Number: 43
Session Number: 43
Abstract Title:
Impact of Using A Rapid Diagnostic Test for the Identification and Treatment of Bacteremia and
Fungemia in Four Colombian Hospital´S Intensive Care Unit
S. Reyes1, C. Hernández-Gómez1, C. Pallares1, S. Salcedo2, S. Valderrama3, K. Ordóñez4, M. V. Villegas1;
1CIDEIM, Cali, Colombia, 2Clínica Gen. del Norte, Barranquilla, Colombia, 3Hosp. Univ.rio San Ignacio,
Bogotá, Colombia, 4Hosp. Univ.rio San Jorge, P
Abstract Body:
Background: Blood cultures (BC) are considered the gold standard for diagnosis of sepsis; however, BC
can take up to 72 hours from sampling to the final positive result. In contrast, new molecular
technologies such as peptide nucleic acid fluorescence in situ hybridization (QuickFISH®) identify the
microorganisms directly from positive blood cultures, increasing the accuracy and reducing the time to
final report. Considering the lack of studies evaluating this molecular method in our country, the aim of
this study was to assess the impact associated with the use of QuickFISH in the diagnosis and treatment
of bacteremia and fungemia in Colombia. Methods: We conducted an ambispective cohort study in four
high-complexity intensive care unit hospitals in Colombia between 2016 and 2017. Adult patients with
systemic inflammatory response syndrome and ≥1 positive blood culture were included in the study.
Patients with microorganisms identified by QuickFISH composed the exposed group and those with
pathogens identified by the conventional culture composed the non-exposed group. Variables such as
sociodemographic, clinical, microbiological, duration of treatment and time to final reports were
compared between the groups using descriptive and inferential statistics. Results: A total of 153 patients
were included in the study; 73 patients (48%) in the exposed group and 80 (52%) in the non-exposed
group. The mean age was 60 years old; 83 (54%) men and 70 (46%) women. Bacteremia was the most
common type of infection in 133 patients (87%) and fungemia was found in 20 patients (13%), with no
difference between groups (p=0.09). The mean time required to obtain the Gram stain report was 24
hours in both groups (p=0.257). In contrast, the mean time for the culture report was 55 hours vs 29
hours for the PNA FISH report (p=0.0001). The empiric therapy was changed in 25% of the exposed and
15% of the non-exposed group. The duration of antimicrobial treatment was significantly shorter in the
exposed group compared to the non-exposed group (328 hours vs. 405 hours; p=0.026). The survival
rate was greater in the exposed group (74% vs. 47%; p=0.009). Conclusions: Our findings suggest that
new molecular technologies such as QuickFISH lead to a better clinical outcome compared to the
conventional methods by reducing the time to final reports and duration of therapy in intensive care
unit patients with bloodstream infections. Survival may be associated with a better chance of
appropriate therapy. These results are consistent with the findings in other international settings.
Session Number: 43
Session Number: 43
Abstract Title:
Evaluation of Shotgun Metagenomics for Direct Detection and Identification of Prosthetic Joint
Pathogens in Synovial Fluid
M. Ivy, M. Thoendel, P. Jeraldo, K. Greenwood-Quaintance, A. Hanssen, M. Abdel, N. Chia, J. Yao, A.
Tande, J. Mandrekar, R. Patel; Mayo Clinic, Rochester, MN
Abstract Body:
Background: Diagnosis and treatment of prosthetic joint infection (PJI) through targeted surgical
management and antimicrobial therapy is contingent on pathogen identification. Metagenomic shotgun
sequencing allows unbiased pathogen detection in clinical specimens. In the largest series to date, we
utilized a metagenomics-based approach applied to synovial fluid to define potential microbial etiologies
of failed total knee arthroplasties (TKAs). Methods: Synovial fluid was collected from 168 failed TKAs
[106 PJI and 62 aseptic implant failure (AF)] via preoperative arthrocentesis. Standardized culture and
DNA-based metagenomic shotgun sequencing were performed. A workflow showing sample preparation
and bioinformatic analysis is depicted in Figure 1. Patients were classified as having PJI using IDSA
diagnostic criteria and clinical review. Reads successfully assigned to the genus and species level were
examined independently, and results recorded as percent agreement, with 95% confidence intervals
(CI), of metagenomics to synovial fluid culture. Results: Genus- and species-level analysis of
metagenomic sequencing yielded the known pathogen in 73 (89%) (CI 80.2%- 94.9%) and 68 (83%) (CI
73.0%-90.3%) of the 82 culture-positive PJIs analyzed, respectively, with additional potential pathogens
in two (2%) (CI 0.3%- 8.5%) and four (5%) (CI 0.2%-9.6%) cases. For the 24 culture-negative PJIs tested,
genus- and species-level analysis yielded 19 (79%) (CI 57.9%-92.9%) and 20 (83%) (CI 62.6%-95.3%)
samples with insignificant findings, respectively, and 5 (21%) (CI 4.2%- 37.4%) and 4 (17%) (CI 1.4%-
31.9%) cases identified with potential pathogens. Genus- and species-level analysis of the 62 AF cases
identified potential pathogens in 7 (11%) (CI 4.7%-21.9%) and 4 (6%) (CI 1.3%-12.0%), respectively.
Conclusions: Metagenomic shotgun sequencing can detect pathogens involved in PJI when applied to
synovial fluid and may be useful for culture-negative PJI.<br /><p><a
href="http://files.abstractsonline.com/CTRL/dc/6/869/272/8df/4b2/cab/cea/d3c/c1b/03b/7d/g7158_1.
src="http://files.abstractsonline.com/CTRL/dc/6/869/272/8df/4b2/cab/cea/d3c/c1b/03b/7d/g7158_1.j
Session Number: 43
Session Number: 43
Abstract Title:
Characterization of Clin. Isolates Representing A Novel Genus Within the YersiniaceaeFamily
A. Rossi1, M. Fisher2; 1Utah Publ. Hlth.Lab., Taylorsville, UT, 2Univ. of Utah, Salt Lake City, UT
Abstract Body:
The large volume of isolates submitted to reference laboratories for organism identification offers
unique opportunities for the discovery of novel bacterial species. The formal taxonomic characterization
of a novel species allows subsequent evaluation of its role as a pathogen, harmless colonizer, or lab
contaminant. Here we present the polyphasic characterization of six clinical isolates that could not be
assigned to any known bacterial species. Phenotypic characterization of the isolates included: colony
and Gram stain morphology, biochemical profile and sugar utilization via API 20E and API 50CH panels,
and fatty acid methyl ester profiling by gas chromatography (MIDI Labs). The genomes of the isolates
were sequenced using an Illumina MiSeq platform. Genomic information was used to carry out multi-
locus sequence analysis (MLSA) based on the concatenated atpD, rpoB, gyrB and infB genes. Whole
genome comparisons among the six isolates were also used to determine their average nucleotide
identity (ANI) values in order to assess whether they belonged to multiple novel species. The six isolates,
referred to here as 16-Iso1, 16-Iso2, 16-Iso3T, 16-Iso4T, 13-Iso5, 15-Iso6T, are Gram negative bacilli
sharing a 99.3-99.7% identity to each other and a 97.5-97.7% identity to other members of the
Enterobacteriales by 16S rRNA sequencing. They all displayed several distinctive phenotypic
characteristics such as the presence of multiple colony morphotypes, yellow pigmentation,
temperature-dependent motility and gelatinase activity. Their fatty acid profiles did not yield species-
level identification within the MIDI database (similarity indexes of <0.6 to reference profiles from the
genera Yersinia, Proteus and Alcaligenes), yet confirmed the relatedness of the six isolates (Euclidian
distances <6). A phylogenetic tree based on MLSA indicated that these strains constitute three individual
species within a novel genus of the Yersiniaceae family. Consistent with this observation, the strains 16-
Iso1, 16-Iso2, 16-Iso3T and 13-Iso5 strains displayed ANI values greater than 99% in pairwise
comparisons, indicating they are isolates of the same novel species. The isolates 16-Iso4T and 15-Iso6T
strains represent two additional novel species as indicated by ANI values below 91% when compared to
each other or to any other isolate. This work underscores the diversity of clinically-relevant enteric
bacteria and will allow laboratories to begin to recognize these organisms and ultimately determine
their clinical significance.
Session Number: 43
Session Number: 43
Abstract Title:
Impact of Rapid Influenza Molecular Testing on Diagnosis and Patient Management
N. Mercuro, S. Krupp, R. Tibbetts, E. Hadley, G. Sharma, I. Rubinfeld, J. Bongiorno, K. Callahan, G.
Alangaden, L. Samuel; Henry Ford Hlth.System, Detroit, MI
Abstract Body:
Background: The rapid influenza antigen test is limited by poor sensitivity, which may result in diagnostic
uncertainty and overtreatment. In 2017, the FDA reclassified rapid influenza antigen tests as class II
devices and mandated use of assays with improved performance characteristics by January 2018. The
purpose of this study was to evaluate the impact of on-site CLIA waived rapid influenza PCR
implementation, in conjunction with appropriate training. Methods: This was a multicenter, quasi-
experimental study including patients who were tested for influenza in four Emergency Department (ED)
locations of the Henry Ford Health System between December 2015 and April 2017. A series of
educational interventions and a testing algorithm for influenza was developed and all ED sites were
transitioned from antigen testing to rapid onsite PCR testing in December 2016 Patients who received
influenza antigen testing were compared to those with PCR testing . Microsoft SQL Server 2016® was
used to extract subjects tested for influenza along with baseline demographics, admission information,
diagnoses, and receipt of antiviral. Continuous variables were compared with Mann-Whitney U or t-test,
as appropriate, and Chi-squared was used to assess categorical variables. Logistic regression was used to
determine predictors of hospital admission from the ED. Results: During two consecutive flu seasons,
20157 patients were tested with the rapid antigen (n=12,672) or PCR (n=7,485). Tests were positive in
1,358 (10.7%) rapid antigens vs 1,700 (22.7%) PCRs (p<0.001). Subsequently, 7.3% vs 16.5% (p<0.001)
had primary diagnosis of influenza and there were no differences between sepsis or pneumonia
diagnoses. Patients tested with PCR were more likely to receive oseltamivir (15.8% vs 21.6%) and were
also less likely to receive inappropriate antivirals if tested negative (9.1% vs 6.9%, p <0.001). Admission
rates from the ED for patients tested with rapid antigen and PCR were 34.5% vs 42.3% (OR=1.31 [1.22-
1.48]), respectively. After using logistic regression to control for confounders, PCR use and influenza
positivity were protective against hospital admission, while age, sepsis, and hospital location predicted
hospital admission. Of those admitted, subjects tested with PCR had an overall shorter length of stay
(4.9 vs 4.4 days, p <0.001). Conclusions: The influenza PCR had an improved diagnostic yield and was
associated with greater odds of receiving appropriate antivirals. Results should be validated in a
prospective analysis.
Session Number: 43
Session Number: 43
Abstract Title:
Low Admission Plasma Gelsolin Levels Help Identify Community--Acquired Pneumonia Patients At High
Risk for Severe Outcomes
W. Self1, C. Grijalva1, D. Williams1, T. Stossel2, M. DiNubile3, S. Levinson3, R. Balk4, S. Fakhran5, A.
Bramley6, S. Jain6, K. Edwards1, R. Wunderink7; 1Vanderbilt Univ. Med. Ctr., Nashville, TN, 2Harvard
Med. Sch., Boston, MA, 3BioAegis Therapeutics, N
Abstract Body:
Background: Timely identification of patients with community acquired pneumonia (CAP) at high risk for
adverse outcomes remains challenging. Plasma gelsolin (pGSN) is an abundant circulating protein that
neutralizes actin exposed by damaged cells, limits the spread of injurious inflammation, and enhances
macrophage antimicrobial activity. Decreased pGSN levels at presentation are found in patients with
infectious and non-infectious inflammatory diseases at high risk to subsequently develop complications.
Mouse models of pneumococcal infection have shown improved survival in mice treated with pGSN
without antibiotics. We investigated the association of pGSN depletion with clinical outcomes in a well
characterized cohort of patients hospitalized with CAP. Methods: The CDC funded Etiology of
Pneumonia in the Community (EPIC) Study enrolled adults hospitalized with CAP at 5 centers and
prospectively assessed clinical outcomes. pGSN concentration at hospital admission was measured with
an enzyme-linked immunosorbent assay in a convenience sample of patients enrolled in the EPIC study.
Patients were categorized into 4 hierarchical, mutually exclusive categories based on the maximum
clinical severity observed during their hospitalization: 1) general medical floor admission; 2) intensive
care unit (ICU) admission without invasive respiratory or vasopressor support; 3) invasive respiratory or
vasopressor support for respiratory failure or shock; 4) in hospital death. We compared pGSN
concentration across these categories using the Kruskal-Wallis test. A pGSN threshold of 30 μg/mL was
evaluated as a cut-point to distinguish between survivors and non-survivors. Results: Among 455
patients, the median (mean) [IQR] pGSN concentrations were 38.1 (39.7) [32.1, 45.7] μg/mL. Patients in
higher (more severe) clinical outcomes categories had lower pGSN (p<0.01); median pGSN levels for
each outcome category were: general floor admission (n=242) 40.3 μg/mL; ICU admission (n=144) 36.7
μg/mL; invasive respiratory or vasopressor support (n=56) 35.8 μg/mL; death (n=13) 25.7 μg/mL. Among
88 patients with pGSN <30 μg/mL, 9 (10.2%) died, compared with 4 (1.1%) deaths among 367 patients
with pGSN ≥30 μg/mL (p < 0.01). Conclusions: Lower pGSN levels near the time of admission were
associated with worse outcomes among hospitalized CAP patients, and may help identify candidates for
exploring the potential therapeutic benefits of recombinant human pGSN infusions.
Session Number: 43
Session Number: 43
Abstract Title:
Characterization of Microbial Communities of the Post-Mortem Human Heart and Spleen
L. Harrison1, C. J. Schmidt2, J. Pechal3, E. Benbow3, H. Jordan1; 1Mississippi State Univ., Mississippi
State, MS, 2Univ. of Michigan, Ann Arbor, MI, 3Michigan State Univ., East Lansing, MI
Abstract Body:
An accurate time of death is valuable evidence in criminal investigations, and while traditional methods
can employ a combination of body temperature, state of rigor mortis and presence of invertebrates,
these methods carry a certain degree of uncertainty from several hours to several days. Microbial
forensic evidence is a new concept with applications for establishing prior contact between a suspect
and victim to determining a more accurate time of death. The aim of this study was to establish a time
of migration of certain bacteria into sterile or semi-sterile organs which could provide a consistent,
measurable assay for time of death, and to characterize virulence and antibiotic resistance genes within
the transmigrating bacteria. The eight cadavers used in the study included male and female, white and
black, and spanned ages from 0 to 82 years of age. Punch biopsies from the heart and spleen were
collected from cadavers by the Detroit medical examiner and stored at -80ºC in 5ml tubes containing
RNAlater. DNA and RNA were extracted separately using the TRIzol method and quantified using a Qubit
fluorometer. Whole-genome shotgun DNA and RNA libraries were prepared and sequenced using
Illumina HiSeq technology. Sequences were trimmed, assembled, and OTUs assigned using
Trimmomatic, Mira, and MetaPhlAn programs. Community diversity analysis and PERMANOVA statistical
analyses were completed using Phyloseq and Vegan packages in R. Antibiotic resistance genes were
identified by comparing assembled sequences with the CARD database. The data collected from this
study established the bacterial communities and their function present in the heart and spleen after
different post-mortem intervals and across several demographics. Trends and commonalities between
these subgroups were identified for future experimentation to establish the best bacteria or genes for
predicting post mortem interval. Furthermore, whole genome sequencing allowed virulence gene and
antibiotic resistance gene detection. Results from this study and those from in-progress samples will
provide a clearer profile of bacterial community transmigration into sterile spaces. While this is a very
preliminary study, by measuring diversity and composition of these communities and the
presence/absence of certain microbes it may be possible to find a commonality that could be used in
future time of death estimations. Furthermore, identification of antibiotic resistance genes will
contribute to the compounded knowledge of antibiotic resistant bacteria present within humans.
Session Number: 44
Session Number: 44
Session Title: CPHM04 - Diagnostic Immunology: Using Host Factors to Diagnose Infection
Abstract Title:
Cytokine Profiling in Bcg Vaccinated and Non-Vaccinated Healthy Controls in Tb Endemic Setting
K. Ahmed1, Z. Jamil2, S. Qureshi1, F. Aziz1, R. Hussain3, N. T. Iqbal1; 1The Aga Khan Univ. Hosp.,
Karachi, Pakistan, 2The Aga Khan Univ. Hosp. Karachi, Karachi, Pakistan, 3The Aga Khan Univ. Hosp.
Karachi, Pakistan, Karachi, Pakistan
Abstract Body:
Background: Pakistan has more than 90% of Bacillus Calmette-Guerin (BCG) coverage at birth through
Extended Program on Immunization (EPI). Though, BCG does not provide protection against pulmonary
tuberculosis, it protects children under five from severe form of tuberculosis. BCG is known to have
immuno-modulatory effects, as being live attenuated vaccine, which activates multiple cells of innate
immunity such as Macrophages, Dendritic Cells and Neutrophils. It also trains immune system by
instructing specialized population of T cells. In this study, we compared the cytokine profiles between
vaccinated and non-vaccinated groups to determine if prior vaccination status has any effect on cytokine
secretion, and whether stimulation of BCG using whole blood (WB) and PBMCs has differential response
in cytokine secretion. Methods: Venous blood (16-20ml) was collected from vaccinated and non-
vaccinated adults (n=20), aged between 14 to 40 years in heparinized tube (20IU/ml). 4-5ml of blood
was used for whole blood assay (WBA) and isolation of PBMCs. After counting the cells, whole blood and
PBMCs were stimulated with BCG vaccine (MOI 1.2x106) at 12 hours. Supernatant were tested for
cytokine Th1[IL-2, IFNγ and TNFα], Th2 [IL-4, IL-10], Th17[IL-17A,] using Bio-Plex Pro Human Cytokine
Group I Assay kit (BioRad labs, Hercules CA, USA) on Luminex 200. Results: No difference was observed
in the median level of cytokines in vaccinated and non-vaccinated groups (p>0.05). In terms of
comparison between stimulation of WBA and PBMCs, BCG stimulation in PBMCs showed a higher
median response in IL10 (p =0.013), IL17 (p =0.003) but not in IL2 (p =0.011) and IFNƴ (p =0.004). Of all
the six cytokines, TNFα levels were similar in the two groups (p =0.112), and showed a highest
stimulation index for all six cytokines. Conclusions: A non-significant difference between BCG vaccinated
and non-vaccinated group is probably due to endemicity or continuous exposure to non-tuberculous
mycobacteria. BCG stimulation assay using PBMCs or WB was equally good for the stimulation of most
of the cytokines. A longer stimulation with BCG is required to stimulate Th2 and Th17 cytokines.
Session Number: 44
Session Number: 44
Abstract Title:
Outstanding Abstract Award: Cytokine Profile in Tuberculous Meningitis
J-S. Kwon, J. Park, J. Kim, H. Cha, M-J. Kim, Y. Chong, S-O. Lee, S-H. Choi, Y. Kim, J. Woo, Y-S. Koo, S-A.
Lee, S-H. Kim; Asan Med. Ctr., Seoul, Korea, Republic of
Abstract Body:
Background: Among various types of tuberculosis (TB), the diagnosis of TB meningitis (TBM) is the most
difficult. In this study, we investigated the diagnostic utility of cytokine profile analysis and IFN-gamma
releasing assay (IGRA) in cerebrospinal fluid from patients with suspected TBM. Methods: We
prospectively enrolled adult patients with suspected TBM from March 2015 to August 2017. CSF
specimens were analyzed in terms of 18 cytokines and chemokines. ELISPOT assay for IGRA (i.e., T-
SPOT.TB; Oxford Immunotec) were performed on mononuclear cells from CSF (CSF-MCs). Results: A
total 87 patients suspected TBM were enrolled including 10 TBM patients (2 definite, 8 probable, 32
possible) and 45 non-TBM patients. Excluding 32 patients with possible TBM, 10 patients with TBM and
45 patients with non-TBM were finally analyzed. The levels of adenosine deaminase, IL-12p40, and MIP-
1a from CSF were significantly higher in TBM group than those in non-TBM group (p < 0.05). The receiver
operating characteristics (ROC) curves and their area under ROC curves for the various tests are shown
in Figure. When we determined the optimal cut-off values based on the ROC curve, the sensitivities and
specificities, respectively, of the test methods for diagnosing TBM were as follows; CSF adenosine
deaminase > 6.95 U/L, 70% and 85%, IL-12p40 > 89.8 pg/mL, 70% and 88%, MIP-1a > 8.8 pg/mL, 80%
and 65%, and CSF-MC ELISPOT > 13.5 spots per 250,000 CSF-MC, 30% and 95%, respectively.
Conclusions: IL-12p40 and MIP-1a concentration in CSF are useful adjuncts for the diagnosis of TBM.<br
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Session Number: 44
Session Number: 44
Abstract Title:
Seroprevalence Study of Francisella Tularensis Among Military Recruits in Georgia
N. Chitadze1, R. Chlikadze1, M. Chubinidze1, T. Chikviladze1, K. Dalakishvili2, L. Durglishvili2, R.
Kuchukhidze2, M. Betashvili3, P. Imnadze1, R. Rivard4, M. Nikolich5, T. Akhvlediani6; 1R. G. Lugar Ctr.
for Publ. Hlth.Res. Natl. Ctr. for Disease Contro
Abstract Body:
Background: Francisella spp. cause zoonotic infections and present in glandular, ulceroglandular,
oculoglandular, oropharingeal and pneumonic forms with significant morbidity. F. tularensis is
considered to be a biological threat agent that poses a substantial risk to public health. Mammals
transmit the bacterium to humans directly through bites and scratches as well as through arthropod
vectors and contamination of water and food sources. The disease is considered to be endemic for
Georgia. Small and large outbreaks are periodically reported. Methods: A serosurvey of F. tularensis to
determine risk for exposure and to determine risk factors for infection was conducted among military
personnel from different regions of Georgia as they undergo pre-contract medical and health
assessment at the Military Hospital of the Ministry of Defence of Georgia in Gori. A total of 1000 recruits
were enrolled in this study. Written informed consent was received from each volunteer before
enrollment in the study. Blood samples were obtained from volunteers at the time of their medical
check-up. For each volunteer, previous illness and exposure of risk factors were collected using a
standardized epidemiological questionnaire. Data analysis was done using Epi Info 7 and SPSS 24.
Results: Samples were screened in duplicate for F. tularensis specific antibodies by using a commercial
IgG enzyme linked immunosorbent assay (ELISA). All ELISA positive samples were confirmed by standard
microagglutination test (MAT). Briefly, serial dilutions of serum were incubated overnight with safranin-
stained, formalin-killed live vaccine strain (LVS) cells at room temperature, and a titer was assigned.
Samples with a titer of 1:128 or greater were reported as positive. Testing was conducted at the Lugar’s
Center for Public Health Research, at the laboratory of the National Center for Disease Control and
Public Health of Georgia. Sixty one serum samples (6.1%) were positive for F. tularensis IgG ELISA.
Eighteen (1.8%) from them were confirmed by MAT. None of the study subjects had a history of
tularemia or clinical symptoms characteristic to this disease. Conclusions: This is the first study of
seroprevalence of F. tularensis among Georgian military recruits. It provides an initial assessment of the
potential exposure risk of military recruit population to the tularemia infection. This data will be a
valuable contribution to the scant information currently available in the public health system of Georgia.
Session Number: 44
Session Number: 44
Abstract Title:
Mumps Outbreak in A Military Population and Seroprevalence of Igg-Specific Antibodies for Measles,
Mumps, and Rubella
L. E. Nielsen, D. Kelly, C. Davis, A. E. Marklez, E. P. C. Ager; Brooke Army Med. Ctr., San Antonio, TX
Abstract Body:
Background: In 2016, the CDC reported nearly 6000 US mumps cases despite estimates that 91.9%
received the MMR series. Outbreaks continue, especially amongst those in crowded living conditions,
such as military barracks and university dormitories. The military requires proof of MMR vaccination at
accession, while the Department of Defense (DOD) also requires serum to be archived. Despite 88%
vaccine efficacy for mumps and 97% for mumps and rubella, those completing a childhood series are
considered to have lifetime protection. MMR provides immunity for mumps serotype A, but there is
insufficient information on the cross-reactivity against other serotypes, which includes circulating
serotype G strains. Methods: In May 2017 an outbreak of mumps occurred in a barrack of 252 service
members. Suspected cases were evaluated with a combination of mumps IgG, IgM, viral culture, PCR
and sequencing. This confirmed 7 cases in febrile patients with salivary gland swelling or orchitis. IgG
titers on serum repository samples of all potential exposed service members were completed to
determine the frequency of immunization among those with a complete MMR vaccination record and
frequency of seronegativity. Results: Of the 252 service members tested for mumps IgG, 20.1% of those
were mumps IgG seronegative. Mumps infection was confirmed by convalescent IgM and positive PCR
(n=6) or convalescent IgM alone (n=1) with 4/7 cases having sufficient IgG titers. Sequencing confirmed
mumps serotype G strain in all cases. Rubella and measles seronegativity rates were 24.4% and 27.8%,
respectively. All mumps patients were seropositive for rubella and measles IgG. All combinations of
sereonegativity to measles, mumps, and rubella were demonstrated without a strong predicative
correlation to any one IgG titer. Conclusions: Sequencing confirmed mumps serotype G strain infection.
The mumps IgG assay was not a sufficient predictor of immunity to the serotype G strain. Protective
titers levels from one component of the MMR vaccine cannot be extrapolated to other immunogens
suggesting each must be surveyed independently. Our study suggests seronegativity or waning
immunity to all components of the MMR vaccine is higher than previously reported and the assumption
of lifelong protection by the MMR series is a less than optimal.
Session Number: 44
Session Number: 44
Abstract Title:
Detection of Pertussis Toxin Neutralizing Antibodies from Human Dried Blood Spots Using Real-Time Cell
Analysis
S. Bolcen, L. Cronin, D. Boulay, J. Cloward, M. H. Jenks, G. Rajam, J. Ao, J. Schiffer, H. Li; CDC, Atlanta, GA
Abstract Body:
Bordetella pertussis is the causative agent of the severe respiratory infection commonly known as
whooping cough. Despite global vaccination efforts, epidemic cycles occur every 2 to 5 years with higher
incidence in children <1 year of age. The level of pertussis toxin (PTx) neutralizing antibodies (Abs) is a
critical parameter in patient recovery and for vaccine efficacy evaluation. An in vitro assay to quantify
PTx neutralizing Abs from human serum using real-time cell analyzer (RTCA) technology was developed
to measure the Ab neutralizing capability in a quantitative manner. As serum preparation presents
challenges and higher transportation costs, dried blood spot samples (DBS) obtained during newborn
screening offer an alternative sample matrix for testing with fewer on-site equipment requirements,
easier storage, and lower associated costs. With the pTNA assay developed, DBS elution conditions were
optimized for best recovery of the serum and Ab titers. A matched set of DBS and serum samples were
analyzed for toxin neutralization activity using the RTCA assay. Normalized anti-pertussis toxin Effective
Dilution (NED50) values were compared between paired samples (n=20). Additionally, the DBS eluent
was evaluated for storage stability at 4°C for 7 days and at -20°C for 28 days. To further establish the use
of DBS in the assay, samples from state public health labs (n=130) were paired with a quantitative Ab-
capturing assay to assess correlation between anti- PT binding Ab titers and toxin neutralization
capability (NED50) from DBS samples. Sample matrixes were not found to be significantly different for
use on the RTCA platform (R2 = 0.848, log10 (DBS conc) = 0.823 x log10 (serum conc) + 0.314).
Additionally, the DBS eluent was found to be stable for ≤7 days at 4°C and ≤28 days at -20°C post
elution. When anti-PTx Ab binding activity in DBS eluents was compared to functional antibody titer, a
statistically significance correlation was noted for both study groups (R = 0.576, F(129,129) = 63.60, p <
0.0001). Elution of anti-pertussis Abs from DBS and successful detection of pertussis toxin neutralizing
Abs from DBS eluent using RTCA technology was achieved. Furthermore, while significant variance was
noted in neutralizing power between individuals with comparable antibody titers, the level of Ab
present in DBS eluent was determined to positively correlate with the ability to neutralize toxin present
in the system. DBS provides a viable alternative to serum for use in RTCA systems with no loss of
functionality in the assay.
Session Number: 44
Session Number: 44
Abstract Title:
Kinetics of Serologic Responses to Pertussis Infection
L. Pawloski1, A. Blain1, H. Ju1, C. Miner1, T. Skoff1, M. Tondella1, K. Edge2, A. Brusseau3, S. Peña3, L.
McNamara1; 1CDC, Atlanta, GA, 2Colorado Dept. of Publ. Hlth.and Environment, Denver, CO, 3New
Mexico Dept. of Hlth., Albuquerque, NM
Abstract Body:
Background: Pertactin-deficient Bordetella pertussis strains are the predominant phenotype in the
United States. They have been associated with a longer duration of infection than pertactin-producing
strains in acellular pertussis-vaccinated mice, suggesting that pertactin loss could increase bacterial
persistence and potentially alter immune responses in infected people. While recent development of
calibrated IgG anti-pertussis toxin (PT) tests has improved pertussis detection, test utility is narrowly
defined for 2-12 weeks post-cough onset. We assessed the duration of seropositivity in pertussis cases
and contacts to better understand the immune response of infected individuals in the context of recent
B. pertussis changes. Methods: PCR-positive cases and symptomatic household contacts were identified
through Enhanced Pertussis Surveillance in CO and NM. Blood samples were collected within 7 days of
the initial case report, with additional specimens collected 14-21 days and 2, 4, 6, 8 and 12 months later.
Follow-up was discontinued after two sequential negative serology results. Information on symptoms,
antibiotic use, and other medical conditions was collected at each visit. IgG anti-PT titers were measured
by ELISA with positive, indeterminate, and negative cut-offs of ≥ 94, 49-93, and <49 IU/mL, respectively.
Results: To date, 29 participants (ages 1-77 years) have been enrolled and a mean of 2.7 samples
collected per enrollee (range 1-6). Mean titers (and # samples tested) for visits 1-6 were 315 (26), 388
(23), 378 (9), 86 (9), 52 (6), and 23 (1) IU/mL, respectively. Titers peaked at the second visit (mean: 38
days post-cough onset). Five (17%) enrollees never seroconverted; all five were PCR-positive. By the 4th
visit, 4/9 enrollees remained positive (range 136-156 days post-cough onset), but 3/9 had become
negative (139-169 days post-cough onset). Conclusions: Despite low case numbers, the post-infection
IgG anti-PT profile was similar to previous studies, with titers peaking around 38 days post-cough onset
and decreasing to 86 IU/mL by the 4 month visit. Titers remained positive up to 156 days after cough
onset. Negative titers among PCR-positive cases may be false negatives or could suggest challenges with
PCR specificity or interpretation. These findings suggest that titers can remain elevated beyond 12
weeks of cough. Because titers wane by 6 months, IgG anti-PT serology can be successfully used to
diagnose recent pertussis infection in the US in spite of the prevalence of pertactin-deficient strains.
Session Number: 44
Session Number: 44
Abstract Title:
Evaluation of A Multiplex Fully Automated Treponemal and Non-Treponemal (Rapid Plasma Reagin)
Assay
S. Arbefeville, M. Lynch, P. Ferrieri; Univ. of Minnesota Med. Ctr. and Fairview Hlth.System,
Minneapolis, MN
Abstract Body:
Introduction: In June, 2017 Bio-Rad Laboratories, Inc. received U.S. Food and Drug Administration
clearance for its BioPlex® 2200 Syphilis Total & RPR (rapid plasma reagin) assay. It is the first fully
automated treponemal/non-treponemal multiplex flow immunoassay, simultaneously detecting T.
pallidum and reagin antibodies and an RPR titer. We compared the performance of the BioPlex® Syphilis
Total & RPR assay with the LIAISON® Treponema assay and the manual BD Macro-Vue RPR 18 mm Circle
Test. Methods: 314 serum specimens were tested for treponemal IgG/IgM and RPR with the LIAISON®
Treponema Assay, the BioPlex® 2200 Syphilis Total & RPR assay and the manual BD Macro-Vue RPR card
test. All discordant results were further tested with the Treponema pallidum particle agglutination assay
(TTPA), from Fujirebio Diagnostics, Inc. Results: 112 specimens were reactive, 2 equivocal and 200 non-
reactive for treponemal antibodies with the LIAISON® Treponema Assay. 101 specimens were reactive, 1
equivocal, and 212 non-reactive for treponemal antibodies with the BioPlex® Syphilis assay. 63
specimens were reactive and 251 non-reactive for RPR with the manual BD Macro-Vue RPR card test. 64
specimens were reactive and 250 non-reactive for RPR with the BioPlex® RPR assay. The percent
agreement for the BioPlex® treponemal antibodies assay with the LIAISON® Treponema assay was 95.5%
when equivocal results were included with positive results, and 95.9% when equivocal results were
included with negative results. Sensitivity, specificity, positive predictive value, and negative predictive
value for the BioPlex® RPR assay were 90.5%, 97.2%, 89.1%, and 97.6%, respectively (the manual RPR
assay was considered the gold standard). The 13 specimens that were reactive/equivocal for treponemal
antibodies with the LIAISON® and non-reactive by the BioPlex® were tested by TPPA assay (9 non-
reactive, 3 reactive, and 1 inconclusive for TPPA). Six specimens were reactive by the manual RPR and
non-reactive by the BioPlex® assay, and 7 were non-reactive by the manual RPR and reactive by the
BioPlex® assay. Nine of 13 specimens were TPPA reactive (6 from manual RPR and 3 from the BioPlex®
RPR assay) and had low titers except for one with a titer of 1:8. Conclusion: The BioPlex® 2200 Syphilis
Total & RPR assay performance was comparable to the LIAISON® Treponema assay and the manual RPR
test. Compared to the manual RPR, the automation of RPR testing offered labor savings, objective result
reporting, and improved workflow.
Session Number: 44
Session Number: 44
Abstract Title:
Laboratory Testing of Emerging Lyme Disease in India An Experience of Two-Tiered Serological Testing in
A Tertiary Hlth. Care Centre
E. Vinayaraj, R. Chaudhry, N. Gupta, K. Sreenath, S. Gulati, A. Vaishakh; AIIMS, New Delhi, India
Abstract Body:
Background: Lyme disease is the most common vector borne disease in temperate regions of Northern
hemisphere.A total of 7 Lyme disease cases with typical erythema migrans have been reported and
published in India following tick bite. Ixodes ticks have been identified in North Eastern part of our
country.Presence of Ixodes ricinus species and occurrence of typical erythema migrans following tick
bite support the existence of unexplored Lyme disease in India. In view of the above facts, Lyme disease
becomes an interesting topic among clinicians in India. Here we describe the false positive nature of IgM
assays and importance of two step CDC serological testing for diagnosing emerging Lyme disease in
countries where prevalence is unknown. Methods: 100 Serum samples were collected from clinically
suspected cases of Lyme disease (13 rash with nonspecific flue like illness, 50 mono/oligo arthritis, 24
neuroborreliosis, 8 neuroretinitis, 3 carditis cases) over a period of 2 years and were subjected to ELISA
(recombinant p41, Osp C and VlsE) followed by immunoblot (VlsE, p41, p39 and OspC) based on CDC
two-tier serology criteria for Lyme disease.Positive or indeterminate results were repeated with a
second serum sample 4 - 8 weeks later Results: Out of 100 clinically suspected cases, 21 were positive
and 5 had borderline results for IgM antibodies. 2 samples were positive for IgG antibodies and 2 were
borderline. Among 26 IgM antibody positive cases, 9 turned out to be positive for IgM immunoblot and
had never seroconverted to IgG even after 2 months. IgM immunoblot had clearly excluded the
possibility of Lyme disease in 17 samples. O.tsutsugamushi DNA was detected in one of the samples
which was showing IgM immunoblot bands for Osp C, p41 and p39. Conclusions: Without considering
the duration of clinical illness and history of exposure, interpretation of IgM immunoblot, may lead to
erroneous result despite having more than 2 bands. Specificity of IgM ELISA and IgM Immunoblot is
questionable in patients suffering from Lyme disease having a duration of symptoms > 1 month. Failure
to demonstrate seroconversion and absence of production of IgG antibodies among Lyme suspected
patients without the epidemiological link in non-endemic areas can result in overuse of antibiotics,
thereby contributing to the increased antimicrobial resistance rate and pseudomembranous colitis. The
test result obtained from this study reinforces the two-tiered serological assay and IgM immunoblot
interpretation criteria laid down by CDC in order to reduce the false positive IgM results.
Session Number: 44
Session Number: 44
Abstract Title:
Seroepidemiology of Scrub Typhus among Patients with Acute Febrile Illness Attending Tertiary Care
Hosp. Nepal
P. Hamal; Chitwan Med. Coll., Bharatpur, Nepal
Abstract Body:
Background: Scrub Typhus is an acute febrile illness caused by Orientia tsutsugamushi, transmitted to
humans by the bites of the larva of Trombiculid mites. Scrub Typhus infection is one of the major global
public health problems including in Nepal. The prevalence of which is increase in Nepal after the
earthquake of 2015. Objectives: The study was aimed to determine the seroprevalence of Scrub Typhus
among febrile patients attending Chitwan Medical College Teaching Hospital. Materials and Methods: A
hospital based prospective study was conducted on patients with suspected Scrub Typhus cases in CMC-
TH from 1st July 2016 to 30th June 2017. After taking informed consent, blood samples were collected
from acute febrile illness patients. The detection of Immunoglobulin M (IgM) antibodies to Orientia
tsutsugamushi from the serum sample was done by ELISA. Results: Among 1797 enrolled individuals,
524 (29.2%) were found to be infected by Orientia tsutsugamushi, comprising of 314 (60.0%) females
and 210 (40.0%) males. In relationship to age group, the distribution of Scrub Typhus infected cases
were found to be maximum (37.2%) between 51-60 years of age group, while minimum cases were
found in >70 years of age group. The study showed that, the high prevalence was noted in farmers
(37.8%). Likewise, seasonal variation showed that the majority (57.7%) of the infected individuals were
found in the month of July. Fever was the most common (100%) clinical features seen in the study.
Other clinical presentations were found in a descending order like anorexia (64.7%), headache (54.6%),
abdominal pain (19.8%), vomiting (19.3%), lymphadenopathy (16.6%), jaundice (9.4%), eschar (6.5%)
and seizures (6.1%). Similarly, the laboratory parameters in Scrub Typhus positive cases (46.9%)
presented lower level of haemoglobin, (23.5%) patients showed leucocytosis, (73.7%) patients showed
thrombocytopenia. Likewise, the activity of transaminase enzymes, Alanine Transaminase (ALT) and
Aspartate Transaminase (AST) were also found to be increased in majority (70.0 and 76.1%) of infected
cases respectively. Conclusion: The study showed that, there was high prevalence rate of (29.2%) Scrub
Typhus among patient presented with acute febrile illnesses. Eschar was an important clue to diagnosis
of Scrub Typhus. However, it was less common (6.5%) features in Scrub Typhus infection. ELISA can be
carried out timely and jointly for early diagnosis of Scrub Typhus in patients with acute febrile illness in
developing countries like Nepal.
Session Number: 44
Session Number: 44
Abstract Title:
Host-Derived Markers of Lyme Disease and their Diagnostic Potential
G. P. Joyner1, N. Beeching2, A. Semper3, J. Hiscox1; 1Univ. of Liverpool, Liverpool, United Kingdom,
2Liverpool Sch. of Tropical Med., Liverpool, United Kingdom, 3Publ. Hlth.England, Porton, United
Kingdom
Abstract Body:
Background: Lyme disease (LD) is a multisystem infection caused by tick-borne spirochaetes of the
Borrelia burgdorferii (B.b) sensu lato group. Laboratory diagnosis of LD involves the two-tier serological
approach. The negative predictive value of the test has been challenged, particularly in early LD. Two
recent systematic reviews of serological testing for LD in Europe and N. America concluded that two-tier
testing showed “poor and highly variable sensitivity in the initial stages of disease when an individual is
mounting an immune response to B.b” [1,2]. There is considerable interest, therefore, in the
development of improved diagnostic tests. The main aim of the project is to identify new markers that
could form the basis for improved tests. Methods: A mass spectrometry biomarker discovery study was
undertaken on LD positive and negative residual diagnostic samples from UK LD testing by Public Health
England (UK government body). A control group of healthy subjects serum sample were also included.
To ensure differences were specific to LD, a related-disease control group including serum samples from
syphilis, leptospirosis and chronic fatigue syndrome were included. 50 human samples were compared
by label-free quantitative mass spectrometry. Proteins significantly increased or decreased between
groups were identified, with particular focus on differences between LD positive and negative sera.
ELISAs were used to validate findings in a larger sample set. Results: Several proteins were found at a
significantly higher or lower in abundance in the LD-positive patients compared with those that were LD-
negative. Of particular interest was Lipocalin-2 (LCN2), a protein involved in immunity. LCN2 has
previously been found in increased abundance in mice exposed to B.b. Analysis by ELISA confirmed
elevation of LCN2 in LD-pos samples. Proteins of interest were also identified in other groups.
Leptospirosis sera were found to be highly distinct to other groups with over 100 proteins found to be
significantly increased. Conclusions: Serological testing is the mainstay of diagnostic testing in LD and
many other bacterial infections; however, these tests have been found to be less effective in early
disease. In contrast, host protein markers are not reliant on the mounting of a specific immune response
and are likely to be detectable at earlier stages. Several key differences were also found in the serum
proteomes of other bacterial infections. Further analysis will assess the potential of these host-derived
markers in infection diagnostics.
Session Number: 44
Session Number: 44
Abstract Title:
Evaluation of the Bioplex 2200 Syphilis Total & Rpr Kit
D. Ortiz, M. Loeffelholz; Univ. of Texas Med. Branch, Galveston, TX
Abstract Body:
Treponema pallidum is a spirochete bacterium known to cause syphilis, a sexually transmitted disease.
Infection with T. pallidum is commonly diagnosed using serological assays that detect treponemal
and/or nontreponemal antibodies. In high-volume laboratories, these serological assays have become
automated to produce results faster with lower labor costs. The BioPlex 2200 Syphilis Total & RPR kit is
the first FDA approved, automated assay that simultaneously detects treponemal and nontreponemal
antibodies in serum or plasma. This allows laboratories to produce quicker results and simplify their
workflow by performing a treponemal and nontreponemal assay on a single test system. In this study, a
total of 132 samples were tested by the BioPlex 2200 Syphilis Total & RPR kit and compared to the
predecessor BioPlex 2200 Syphilis IgG and manual RPR assays. Based on laboratory data, samples were
classified as active (44), past (28), unlikely (30), and non-reactive (30) syphilis infections. The positive,
negative, and overall percent agreement between the BioPlex 2200 Syphilis Total and the BioPlex 2200
Syphilis IgG treponemal assays were 76%, 100%, and 77%, respectively. The low positive percent
agreement was primarily due to a non-reactive BioPlex 2200 Syphilis Total for 28 unlikely syphilis
infections that produced BioPlex 2200 Syphilis IgG (+), RPR (-), and TP-PA (-) results. The positive,
negative, and overall percent agreement between the BioPlex 2200 Syphilis RPR and manual RPR
nontreponemal assays were 66%, 99%, and 88%, respectively. Among the discrepant results, the BioPlex
2200 Syphilis RPR failed to identify 15 cases of active syphilis infection in which the manual RPR was
reactive (range 1:1 to 1:4). Compared to the predicate methods, the BioPlex 2200 Syphilis Total & RPR
produced fewer false-reactive treponemal results, and could reduce the amount of additional
confirmatory testing if used as a treponemal screening assay. However, the 15 active syphilis infections
missed by the BioPlex 2200 Syphilis RPR, albeit at low titers, is concerning. Overall, the BioPlex 2200
Syphilis Total & RPR assay is suitable as a treponemal screening assay, but non-reactive nontreponemal
results do not exclude the possibility of a syphilis infection.
Session Number: 44
Session Number: 44
Abstract Title:
Serological Analysis of Immunoglobulin M and G against Cytomegalovirus among End Stage Renal
Disease Patients and their Donors
G. Karki; St.Xavier's Coll., Kathmandu, Nepal
Abstract Body:
Cytomegalovirus (CMV) is one of the major viral pathogen with negative effect in solid organ transplant
recipient. CMV seronegative recipient of kidney from CMV seropositive donor are at higher risk of
invasive disease and organ rejection. Therefore, CMV screening prior to transplantation is needed to
avoid D+/R- setting. In a cross sectional descriptive study, 140 serum samples from Renal failure patients
and their donors were evaluated for CMV specific immunoglobulin M and G using Electro-
chemiluminiscence Immunoassay (ECLIA). Seropositivity to CMV was found to be 96.4% (135/140).
Among them 6.4% (9/140) were positive for CMV IgM. The seropositivity was found to be higher in
donors than ESRD patients (98.6% vs 94.3%).Higher percentage of donors have CMV IgG titer in the
range >500 U/ml whereas higher percentage of ESRD patients have titer in the range 100-250 U/ml.
Statistically there was no association found between CMV serostatus with sex and age groups of both
ESRD patients and donors (p>0.05).This study provide the evidence of high CMV Seropositivity in both
ESRD patients and donors in Nepal. Hence, the study recommended prior screening of blood from
donors before blood transfusion to the high risk patients.
Session Number: 44
Session Number: 44
Abstract Title:
Procalcitonin-Guided Treatment for Inpatients with Pneumonia Reduces Antibiotic Exposure
O. Henig, R. Putler, K. Rao; Univ. of Michigan Med. Sch., Ann Arbor, MI
Abstract Body:
Background: Antibiotic stewardship programs (ASPs) are crucial in reducing antimicrobial resistance.
Randomized controlled trials show procalcitonin (PCT) use reduces antibiotic exposure, but real-world
data are lacking. This observational study evaluated the impact of PCT use on antibiotic duration in
hospitalized patients with pneumonia. Methods: Adults admitted to Michigan Medicine from 2013-2015
with diagnostic codes for pneumonia were included. Data were extracted from the electronic medical
record using structured query. If ≥1 PCT was ordered, treatment was considered PCT-guided. A
PCT<0.25ng/mL was low. Antibiotic exposure was quantified by days of treatment (DOT). Unadjusted
and adjusted models of DOT were built using generalized linear models. A stratified analysis by intensive
care unit (ICU) stay was performed. Results: There were 2213 patients (2574 admissions) included; mean
age was 62.6±17.7 and 47.1% were female. Mean Elixhauser comorbidity score was 4.9±2.5. PCT was
used in 1443 admissions (56%) and was low in 692 of first measurements (48%). ICU stay greatly
increased DOT (+7.35 days) and interacted with several other covariates, prompting us to stratify our
analyses. In the ICU-stay cohort, PCT use associated with fewer DOT (−2.62 days; 95% CI −4.13 to −1.11;
P<.001) after adjustment for covariates (Table). A low first PCT associated fewer DOT (−2.78 days; 95%
CI −4.97 to −0.58, P=.01, unadjusted). Among Non-ICU patients, PCT use did not associate with DOT
(Table). However, a low first PCT associated with fewer DOT (−1.27 days; 95% CI −0.73 to −0.81; P<.001,
unadjusted). PCT-guided treatment was not associated with in-hospital mortality. Conclusions: In nearly
half of admissions, PCT was not used to guide treatment for pneumonia. When treatment was PCT-
guided, this independently associated with fewer DOT in ICU patients. In the entire cohort, a low first
PCT associated with fewer DOT and PCT-guidance did not increase in-hospital mortality.<br /><p><a
href="http://files.abstractsonline.com/CTRL/a8/7/da5/8f9/1b1/47b/586/58c/50d/17b/9c3/0e/g5522_2.
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Session Number: 44
Session Number: 44
Abstract Title:
Development of A Simple Immunoassay to Quantify the Level of Bacterial Alginate in the Sputa of
Patients with Cystic Fibrosis
R. Al Ahmar1, B. Kirby2, H. D. Yu1; 1Marshall Univ., Huntington, WV, 2Progenesis Technologies LLC,
Huntington, WV
Abstract Body:
Background: Chronic lung infections with Pseudomonas aeruginosa is a major cause of morbidity and
mortality in Cystic Fibrosis patients. During early childhood, patients are colonized by multiple bacterial
pathogens including a nonmucoid isolate of P. aeruginosa. Emergence of mucoid isolates is a marker for
the onset to chronic infections. Even though alginate is an important disease marker, it is still not clear
whether there is correlation between the amount of alginate and lung pathology, and whether alginate
can be used as a prognosis marker for treatment. Methods: Using a mouse monoclonal anti-alginate
antibody (QED Biosciences Inc. Catalog: 15725/15726) we directly quantify amounts of alginate in
patient samples through an indirect peroxidase-based ELISA. Microtiter plates were treated with
sample/standard (diluted 1:100 in 0.9% NaCl) and incubated at 37oC for 2 hours. Plates were then
washed and blocked with standard blocking buffer overnight at 4oC. Following that, plates were
washed, primary and secondary (polyclonal HRP-linked anti-mouse IgG) antibody (diluted 1:2000 in 0.9%
NaCl) were added, washed, TMB solution added and the reaction stopped by acid Stop Solution. Results:
A standard curve was first created using pharmacological grade alginate (>98% pure), with a range of
detection was 1024μg/ml. to 8μg/ml. Six complex carbohydrates were tested to determine the
specificity of the antibody; none produced significant reactions. ELISA sensitivity was determined to be
50ng/ml using pure seaweed alginate. We validated this immunoassay by measuring the amount of P.
aeruginosa alginate produced by strains PAO1 (minimal amount of alginate), PAO581(alginate-
overproducing), and PAO581_ΔalgD (no alginate) grown in Pseudomonas Isolation Broth (PIB) and
Synthetic CF Media (SCFM2) and compared the results to the standard Carbazole method. The amounts
of alginate detected by ELISA were comparable to the amounts measured with both methods. Lastly,
three CF patient sputum samples were tested to determine if alginate could be detected directly in the
samples. Alginate was detected in all three samples, indicating that alginate in the sample itself was
quantifiable. Conclusion: This technique utilizes a minimal amount of preparation and manipulation of
the sample, so it could be streamlined and used in hospital laboratories to accurately monitor alginate
levels in the lungs. Further tests will be conducted to verify the results shown including antibody
specificity studies and additional patient samples.
Session Number: 44
Session Number: 44
Abstract Title:
Bacterial and Host Adaptations During Pseudomonas Aeruginosa Interaction with the Ocular Surface In
Vitro and In Vivo
M. M. E. Metruccio1, M. R. Grosser1, V. Nieto1, A. R. Kroken1, D. J. Evans2, S. M. J. Fleiszig1; 1Univ. of
California, Berkeley, Berkeley, CA, 2Touro Univ., Vallejo, CA
Abstract Body:
Background: P. aeruginosa can cause sight-threatening corneal infection in contact lens wearers. To
explore how P. aeruginosa subverts corneal defenses, we employed a global transcriptomic and genomic
approach, combined with high-resolution imaging using a novel murine model of contact lens-induced
corneal infection. Methods: Dual RNA-seq analysis of host and bacteria traversing multilayered human
corneal epithelial cells (hTCEpi) in vitro was performed. In the same conditions, a transposon library was
used to identify genes important for epithelial traversal via Tn-seq. Whole genome sequencing was
applied to P. aeruginosa isolates from keratitis in a murine model of contact lens wear in vivo. Intra-vital
and ex vivo imaging of murine corneas wearing contact lenses was also performed with or without
lenses and/or bacteria. Genomic data were analyzed using the Galaxy platform, Rockhopper, and the
Panther classification system. Confocal image reconstruction and analysis was performed with ImageJ
and Imaris software. Results: Transcript deregulated by ≥ 4-fold in intracellular versus extracellular
bacteria, revealed enrichment of sphingosine catabolism, iron transport and pyochelin biosynthesis
categories (40, 20 and 60-fold respectively, p ≤ 0.05). Type III secretion genes were enriched in bacteria
alone versus extracellular or intracellular (10 and 5-fold respectively). Considering genes deregulated by
≥ 8-fold, bacterial exposure to hTCEpi cells enriched for intrinsic and extrinsic apoptotic signaling, cell
junction assembly, and regulation of leukocyte migration categories (≥ 2.5-fold, p ≤ 0.001). Comparison
of RNA-seq with Tn-seq revealed 40 affected genes in common including iron limitation (pvdL, fptA) and
type III secretion (pscP, pcrV). In vivo, P. aeruginosa inoculated lens wear caused keratitis in 10 of 29
mice. Compared to input, 93 isolates recovered from contact lens related corneal infection in mice (13
days) showed SNPs in genes related to the Pf4 phage with a variant allele frequency from 0.03 to 1.
Confocal imaging revealed biofilm formation on the posterior lens and numerous round host cells
(resembling neutrophils) actively moving behind the lens and within the cornea. Conclusions: Host-
pathogen interactions between P. aeruginosa and corneal epithelium are complex, even more so in the
context of lens wear. The murine contact lens-wearing infection model provides an excellent method to
decipher key bacterial and host elements involved.
Session Number: 45
Session Number: 45
Session Title: CPHM07 - Diagnostic Parasitology: Diverse Approaches
Abstract Title:
Advanced Molecular Detection of Parasitic Pathogens
R. S. Bradbury1, B. Flaherty1, K. Kines1, J. Barratt1, M. Lane2, C. J. Olsen3, M. Sheth1, E. Talundzic1;
1CDC, Atlanta, GA, 2IHRC Atlanta, Atlanta, GA, 3Pacific BioSci.s, Menlo Park, CA
Abstract Body:
Background: Targeted amplicon deep sequencing (TADS) of the 16S rRNA gene is often used to
investigate the microbiome with important epidemiological, diagnostic and pathogen discovery
implications. Attempts to apply TADS to the detection and characterization of parasite communities
have been hampered since targeted amplification of the commonly used 18S rRNA gene at regions that
would allow capture of all parasites using a single primer set results in competitive priming and
preferential amplification of host DNA. This paper describes a novel molecular method to reduce host
18S rRNA template and maximize the amplification of parasite 18S rRNA for detection via next
generation sequencing (NGS). Methods: Restriction enzymes were used to digest selectively a sequence
region of 18S rRNA gene at cut sites present only in the host, but not parasite DNA. Following this, a
single 18s rRNA PCR was performed on the sample and sequenced using the MiSeq. Subsequent reads
were quantified and binned according to their respective parasite and/or host reference sequences.
Results: Enzyme digestion prior to PCR enrichment and Illumina amplicon deep sequencing led to a 50-
60% reduction in human reads and a corresponding 5- to 10-fold increase in parasite reads relative to
undigested samples. This method was validated using 16 species of blood-borne parasites. This single
assay allowed for identification and discrimination of all common parasitic agents found in human
blood, even in cases of multiple infection, and demonstrated a greater than 10-fold lower limit of
detection in digested versus undigested samples. Conclusions: This method provides a novel single test
assay capable of detecting of all known blood borne parasites in a single sample, even in cases with
multiplicity of infection.
Session Number: 45
Session Number: 45
Abstract Title:
Evaluation of Four Typing Strategies for Cyclospora Cayetanensis Using Stool Samples from Past U.S
Outbreaks
F. S. Nascimento, J. Hofstetter, S. Park, E. Van Roey, J. Barratt, E. Talundzic, M. J. Arrowood, Y.
Qvarnstrom; CDC, Atlanta, GA
Abstract Body:
Background: Cyclospora cayetanensis is the causative agent of cyclosporiasis, a foodborne
gastrointestinal illness associated with seasonal outbreaks in the U.S. Past outbreaks have been linked to
fresh produce imported from countries where cyclosporiasis is endemic. However, outbreak
investigations often fail to identify the contaminated food item, which makes it difficult to limit and
prevent outbreaks. A robust molecular genotyping tool to aid in linking cases has yet to be developed.
This study evaluated four typing strategies, based on eleven genetic markers, for their usefulness in
discriminating cyclosporiasis outbreaks. Methods: PCR amplification and Sanger sequencing were used
to characterize the eleven genetic markers from 60 Cyclospora positive stool samples. The samples had
been collected during U.S outbreaks between 1997 and 2017 and included 32 samples epidemiologically
linked to 12 case clusters. The four typing strategies were compared based on amplification rates,
quality of the DNA sequences, and ability to discriminate isolates. Results: A previously described
microsatellite-based MLST method was not effective for typing cyclosporiasis cases due to the common
occurrence of uninterpretable DNA sequences. Conversely, a highly variable region in the mitochondrial
genome was successfully amplified and analyzed in 99% (59/60) of samples and separated the samples
into 13 sequence types. Another mitochondrial marker had a similar analysis success rate but exhibited
less discriminatory power among the samples tested. A typing method based on four nuclear protein-
coding genes divided the samples into 9 groups in agreement with available epidemiologic data.
However, it exhibited evidence of intra-isolate heterozygosity (polymorphic sites) that contributed to a
23% (14/60) failure rate. Conclusions: Two of the evaluated typing strategies presented enough
sequence diversity to distinguish samples from separate epidemiologically implicated outbreaks. While
further evaluation of these five markers is required, they show promise as an efficient typing tool to
improve cyclosporiasis outbreak investigations.
Session Number: 45
Session Number: 45
Abstract Title:
Genetic Population Structure OfSchistosoma MansoniIsolates from Human and Non-Human Primates in
Ethiopia
T. Kebede1, J-F. Allien2, N. Bech3, J. Boissier2, B. Erko1; 1Addis Ababa Univ., Addis Ababa, Ethiopia,
2Univ. of Perpignan Via Domitia, Perpignan, France, 3Univ. of Poitiers, Poitiers, France
Abstract Body:
The epidemiology of Schistosomiasis involves human, non-human primates & rodunts. Significant
numbers of non-human primates, Ch. aethiops (vervet) & Papio anubis (baboon), are infected with
Schistosoma mansoni in Ethiopia but, the role they play in parasite maintenance & transmission is not
known. Genetic diversity & population structure studies have provided insights into the transmission
dynamics of S. mansoni, but there has been no research done in human & non-human primates in
Ethiopia. The objective of this study was to assess the genetic diversity & population structure of S.
mansoni isolates from human & non-human primate hosts living in close proximities in selected endemic
areas of Ethiopia. In a cross-sectional study stool specimens were collected form 911 human &
droppings form 106 non-human primates, from Bochesa, Kimpe & Fincha endemic localities, &
examined for S. mansoni infection using Kato method. In total 2,356 S. mansoni miracidia directly
isolated from 60 humans & 44 non-human primate hosts & DNA extraction, PCR amplification &
genotyping was performed using fourteen microsatellite loci. 201 alleles in total were scored for all of
the 14 loci examined, individual alleles counted for Bochesa human & monkey were 137 & 100, for
Kimpe human & baboon were 103 & 119, & for Fincha human & baboon were152 & 89, respectively. At
a population level, the mean number of alleles per locus, allelic richness, expected heterozygosity in
Hardy-Weinberg equilibrium & pairwise FST values ranged from 6.3-10.9, 4.71-7.38, 0.41-0.55, & 0.19-
26.78%, respectively. The Bayesian STRUCTURE & the PCA had shown three clusters of population.
Genetic diversity of S. mansoni isolates among & within individual subjects from the three endemic
areas at infrapopulation level also showed a value of 2.00-6.93, 1.06-1.93, 34-59% & -0.146-0.236, mean
number of alleles per locus, allelic richness, expected heterozygosity in Hardy-Weinberg equilibrium &
FIS, respectively. Mean number of breeding males & females within an individual host, assuming r&om
mating, was 104 ranging from 10-248. This result showed the occurrence of infection of a single host
with multiple S. mansoni strains & inter- & intra-host genetic variations. The data indicated the
substantial genetic diversity of S. mansoni in the study sites & gen flow across the S. mansoni population
per site. Therefore, non-human primates play a significant role in local transmission & maintenance of S.
mansoni infection even in the absence of human host, important information for schistosomiasis control
programmers.
Session Number: 45
Session Number: 45
Abstract Title:
Assessment of Mfg-E8, Opg & Socs-3 in Human with Cutaneous Leishmaniasis
N. Yentur Doni1, F. Yildiz Zeyrek1, F. Ilhan2, Z. Simsek3, G. Gurses1, S. Topluoglu4, E. Aydin4, A.
Satoskar5; 1Harran Univ., Sanliurfa, Turkey, 2Firat Univ., Elazig, Turkey, 3Bilgi Univ., Istanbul, Turkey,
4Hlth.Inst. of Turkey, Ankara, Turkey, 5The Ohi
Abstract Body:
Background: Milk fat globule epidermal growth factor-factor 8 (MFG-E8) is a peripheral glycoprotein
that acts as a bridging molecule between the macrophage and apoptotic cells, thus executing a pivotal
role in the scavenging of apoptotic cells from affected tissue. Osteoprotegerin (OPG) is a member of the
tumor necrosis factor receptor superfamily of proteins. Numerous recent reports indicate that OPG is
also an important factor in inflammatory pathways and tumor cell survival. Suppressor of cytokine
signaling-3 Receptor (SOCS-3) has a role in preventing the inflammation. However, possible roles of
MFG-E8, OPG and SOCS-3 in cutaneous leishmaniasis (CL) physiology are not known and studied
formerly. The aim of this study to assess the role of MFG-E8, OPG, SOCS-3 in human with cutaneous
leishmaniasis. Methods: Forty six patients with CL and forty four healthy persons without any findings of
CL were included in this study using a case-control design. Written informed consent was obtained from
each participant or his/her parents who attended the Center for Diagnosis and Treatment of Oriental
Sore, Sanliurfa Public Health Directorate. Two slides were prepared from bloodless, serous fluid of the
skin lesions of the patients and stained with Giemsa for the microscopic examination to identify
Leishmania amastigotes. Blood samples were collected from patients and controls in order to assess
MFG-E8, OPG and SOCS-3 levels with The Sandwich Enzyme-Linked ImmunoSorbent Assay method.
Results: All of the slides were determined as positive for Leishmania amastigtes. Statistically there was a
significant difference between MFG-E8, OPG and SOCS-3 serum levels of patients and control groups
(p<0,05). Conclusions: To our knowledge, this study is the first to report the significant association
between case-control groups in terms of MFG-E8, OPG and SOCS-3 serum levels. We consider that
further studies should be carried out to support this idea.
Session Number: 45
Session Number: 45
Abstract Title:
Early Serological Detection of Toxoplasma gondii in Pregnant Females Can Prevent Neonatal Mortality
T. Ijaz1, S. Ijaz2, S. F. H. Gilani1; 1Mayo Hosp., King Edward Med. Univ., Lahore, Pakistan, 2Shalamar
Med. and Dental Coll., Lahore, Pakistan
Abstract Body:
Background: Toxoplasma gondii, a parasite causing toxoplasmosis, a public health problem of zoonotic
potential. Toxoplasma gondii (T. gondii) is transmitted through vertical and horizontal routes causing
trans-placental infection that could leads toward still births, spontaneous abortions and further major
complications in neonates like hydrocephaly, microcephaly, blindness etc. Aims and Objectives: In
Pakistan society human beings have close interaction with the livestock, pets and therefore issue of
toxoplasmosis is considerably important this observational and prospective study was planned with the
objectives, to determine the prevalence and risk factor associated with Toxo gondii infection among the
pregnant women visiting public and private clinics in Lahore Pakistan. Material and Methods: For the
purpose blood samples were collected using SOPs from the women visiting different public and private
clinics from October 2012 to August 2014. A questionnaire was designed to find out the level of health,
hygiene, education and socioeconomic status of pregnant women and their exposure to various risk
factors like eating undercooked meat, salads, drinking water/milk and to assess their knowledge about
toxoplasmosis. A total of 279 blood samples were collected using SOPs, and questionnaires were filled
carefully. Samples were analyzed for presence of IgG and IgM antibodies against Toxo gondii by using
ELISA method. The results and data of risk factors determined by questionnaire was interpreted by using
statistical analysis. Results: From a total of 279 pregnant women 31.5% were found reactive for
antibodies to Toxo gondii IgG while 6.1% were found reactive for antibodies to Toxo gondii IgM,
whereas 2.9% were found reactive to IgG and IgM both. The major associated risk factors identified
were, age of the women, illiteracy, unpasteurized milk, undercooked food, office job, personal hygiene,
trimester of pregnancy, number of pregnancies and hemoglobin level.( p< 0.05) Conclusions: It is
concluded that 31.5 % pregnant women were infected with Toxo gondii. The high prevalence is found
among the women who were, less than 20 years of age, illiterate, drink unpasteurized milk, eat
undercooked food, belong to upper class, do a job, poor personal hygiene, in first trimester of
pregnancy, history of multiple pregnancies and low hemoglobin level. Only 7% were women aware
about the infections while consuming raw/under cooked food, but no one was aware of Toxoplasmosis.
Session Number: 45
Session Number: 45
Abstract Title:
Standardization of An Elona Test for Toxoplasma gondii Rop18 Detection in Human Serum Samples with
Toxoplasmosis
M. Vargas Montes, N. I. Cardona Perez, J. E. Gomez Marin, GEPAMOL; Univ. of Quindio, Armenia,
Colombia
Abstract Body:
Background: ROP18 is a major virulence factor of Toxoplasma gondii, that immunomodulates the host.
Currently, there are reports about the presence of antibodies against ROP18 in serum of humans with
toxoplasmosis; however the direct detection of the protein has not been done yet. Therefore, our aims
were to standardize an ELONA test with AP2039 and AP2056 DNA aptamers to detect the ROP18 protein
in human serum samples with toxoplasmosis and evaluate in silico the affinity of both aptamers to
recognize ROP18. Methods: A direct ELONA was standardized using the AP2039 and AP2056 aptamers,
evaluating incubation conditions, washing buffer, blocking solution, aptamer concentration, streptavidin
and serum dilution. The positive control included total antigen of T. gondii RH strain and the
recombinant ROP18 protein (rROP18). Irrelevant proteins and total lysates from different cell types
were used as negative controls. The direct ELONA was applied in 62 serum samples with different
clinical forms of toxoplasmosis (acute, chronic-asymptomatic, congenital and ocular toxoplasmosis) and
20 T. gondii seronegative samples to stablish the cut off. To perform the in silico analyses, 3-D structures
of aptamers were prepared for docking using AutoDock-Tools 1.5.6. Molecular docking of aptamers with
ROP18 (PDB: 4JRN) was performed using AutoDock-Vina 1.1.2. Results: The optimal ELONA conditions
included overnight incubation of the plate at 4°C, a 1:10 dilution for serum samples, PBS (pH 7.2) with
Tween 20 (0.05%) as washing buffer, blocking solution with PBS and 1% of BSA, 300nM for aptamer
concentration and 1: 10.000 dilution for streptavidin. ELONA test showed that aptamer AP2039 presents
higher affinity to rROP18, as well as the most specific recognition agent (Kd= 62.7 nm). AP2039-ELONA
was positive for 22.6% (14/62) of the samples with toxoplasmosis. A significant association between the
test positivity and congenital toxoplasmosis clinical form (p=0,006) was found, as well as an association
with the severity of the disease in this group (p = 0.03). Docking of AP2039 with ROP18 showed a better
binding energy (-10.2 Kcal/mol) compared with AP2056 (-9.4 Kcal/mol). Conclusions: Conditions of an
ELONA test for ROP18 detection were standardized. ELONA assay on human serum samples showed that
test positivity is correlated with congenital toxoplasmosis. Experimental and in silico analysis showed
that aptamer AP2039 is a promising recognition agent to detect ROP18 in human serum samples with
toxoplasmosis.
Session Number: 45
Session Number: 45
Abstract Title:
A Dual Colour Fluorescence in situ Hybridization (Fish) Assay for Identifying the Zoonotic Malaria
Parasite Plasmodium Knowlesi
J. Shah1, A. Poruri2, F. Mohring3, R. Moon4, R. Ramasamy1; 1ID-FISH Technology Inc., Palo Alto, CA,
2IGENEX Inc., Palo Alto, CA, 3London Sch. of Tropical Med., London, United Kingdom, 4London Sch. of
Hygiene andTropical Med., London, United Kingdom
Abstract Body:
Background: Plasmodium knowlesi is primarily responsible for zoonotic malaria in several Southeast
Asian countries. Precise identification of the parasite in the blood of patients presently relies on an
expensive and elaborate PCR procedure because microscopic examination of blood and other available
field identification techniques lack adequate specificity. Therefore, the use of a simple and inexpensive
dual-colour fluorescence in situ hybridization (FISH) assay, analogous to FISH assays recently described
for Plasmodium falciparum and Plasmodium vivax, was investigated as a potential tool for identifying P.
knowlesi. Method: Methanol fixed thin blood smears were hybridized with two fluorescent labeled
probes Plasmodium genus and P. knowlesi specific probe at 37ºC for 15 minutes. Excess probe was
removed by washing the smear with wash buffer twice for 2 minutes each and a final rinse in rinse
buffer at room temperature. After drying the smear, a drop of mounting medium with DAPI. The smear
was then covered with a glass coverslip and viewed at ×1000 magnification, in an Olympus light
microscope fitted with a light emitting diode light source and appropriate emission and barrier filters
fitted with a light emitting diode light source and appropriate emission and barrier filters Results: The
limit of detection in the P. knowlesi FISH assay was 84 parasites per μl in infected monkey blood and 61
parasites per μl for P. knowlesi cultured in human blood. The P. knowlesi-specific FISH probe detected
only P. knowlesi and not P. falciparum, Plasmodium malariae, Plasmodium ovale, P. vivax or a panel of
other human blood-borne pathogens. A previously described Plasmodium genus-specific probe used
simultaneously in the P. knowlesi FISH assay reacted with all tested Plasmodium species. Conclusion: To
our knowledge, this is the first description of a FISH assay for P. knowlesi that is potentially useful for
diagnosing infections in remote laboratories in endemic countries. Keywords: DNA probe, Fluorescence
in situ hybridization, Malaria diagnosis, Plasmodium knowlesi, Zoonotic malaria.
Session Number: 45
Session Number: 45
Abstract Title:
Malaria Diagnostic Practices in U.S. Laboratories, 2017
C. Prestel1, K. R. Tan2, F. Abanyie2, R. Jerris3, J. Gutman2; 1Emory Univ. Sch. of Med., Atlanta, GA,
2CDC, Atlanta, GA, 3Children's Hlth.care of Atlanta, Atlanta, GA
Abstract Body:
Background: In the United States (US), gold standard for malaria diagnosis is blood smear examination
by light microscopy; rapid diagnostic tests (RDTs) for malaria antigens must be confirmed with
microscopy. Given that malaria is not endemic in the US, the availability and quality of these diagnostic
tests may be limited, possibly leading to delays in diagnosis and treatment, and increased morbidity and
mortality. Methods: A nationwide convenience survey of United States laboratories was conducted from
June 26th to July 24th, 2017 to document challenges with malaria diagnosis. The survey, sent out via the
American Society for Microbiology listservs, explored malaria diagnostic test availability, techniques, and
reporting practices. Results were assessed using the Clinical and Laboratory Standards Institute (CLSI)
guidelines for malaria testing and reporting and compared to results from a similar 2010 survey (1).
Basic statistical analysis was performed using RStudio; Fisher exact test and chi-square test were used to
assess the significance of comparisons.Results: After excluding incomplete and duplicate responses,
results from 175 laboratories were included. Only 1% reported receiving no specimens, 31% reported
receiving 1-10 specimens for malaria per year, while 15% receive 100 or more. The majority (74%)
diagnosed five or fewer cases of malaria per year. Most (91%) had at least one type of malaria diagnostic
test available on-site; 90% of all surveyed laboratories performed blood smears on-site and 40%
performed RDTs on-site. Over half (63%) provided initial blood smear results within 4 hours, and 80%
provided complete results, including speciation, within 24 hours. Of those with RDTs on-site, 94% had
follow up testing with microscopy. Although diagnostic testing for malaria was available 24 hours a day,
seven days a week at 74% of responding laboratories (141), only 12% (17) met CLSI guidelines for
analysis and reporting of malaria testing. This rate of compliance with CLSI guidelines (12%) was higher
than reported in a similar survey in 2010 (3%; p < 0.05). Conclusion: The majority of laboratories
responding to this survey had the capability for timely diagnosis of malaria, however, few were in
compliance with CLSI guidelines. Inexperience might be a factor with this non-compliance; many
laboratories see few to no cases of malaria per year. Although reported adherence to CLSI guidelines
increased since the 2010 survey, updates or a review of standard operating procedures are needed to
improve laboratory compliance to CLSI guidance.
Session Number: 45
Session Number: 45
Abstract Title:
Performance of the Bd Max™ Enteric Parasite Panel in A Large Community Hospital: Evaluation of Labor
Savings and Acceptance As the Primary Parasite Screen
M. Morgan, S. Antonson, F. Lou-Pejero, R. Chan; Cedars-Sinai Med. Ctr., Los Angeles, CA
Abstract Body:
Background:For decades, laboratories have used laborious manual methods to search for enteric
parasites. Depending on the patient population, a great deal of labor has been spent to find a very low
percentage of positives. The BD MAX™ Enteric Parasite Panel (BDEPP) is designed to detect Giardia
lamblia, Entamoeba histolytica and Cryptosporidium species from stool samples. Clinical trials have
proven this panel offers a sensitive, specific, and rapid screen for the most common enteric parasitic
pathogens. We believe it also has the potential to save significant labor in the microbiology laboratory.
Methods: The number of process steps and hands-on-time was averaged for the manual ova and
parasite (OAP) exam and the BDEPP. The acceptance rate of the BDEPP as the sole method for enteric
parasite exam was analyzed for a six-month period in which 1778 stool specimens were submitted for
BDEPP. When a manual OAP examination was requested in addition to the BDEPP, the parasite(s)
detected in the manual OAP exam was noted. Results: Thirty process steps were necessary for the
performance of the OAP examination compared to 10 for the BDEPP. The average hands on time per
manual OAP specimen was 19 min compared to 2.7 min per specimen for the BDEPP (85% decrease in
hands on time). The acceptance rate for the BDEPP as the primary parasite examination was 92.7%. Of
the 1778 stools tested by BDEPP, 130 (7.3%) were also requested for manual OAP and from these 2
Blastocystis hominis and 1 Dientamoeba fragilis were detected. Conclusions: The BDEPP requires
significant less hands on time and process steps compared to the manual OAP examination. The BDEPP
has been accepted by a large percentage of our clinicians as the primary screen for enteric parasites.
Manual OAP performance could not be eliminated but the hours for laboratory operation was greatly
decreased to only 6-8 hrs per week.
Session Number: 45
Session Number: 45
Abstract Title:
Performance of Lab. Professionals Working on Malaria Microscopy in Tigray North Ethiopia
M. Alemu1, D. Tadesse2, T. Hailu2; 1Bahir Dar Univ., Bahir Dar, Ethiopia, 2Mekelle Univ., Mekelle,
Ethiopia
Abstract Body:
Background: Microscopic analysis of stained blood smear is the most suitable method of malaria
diagnosis. However, gaps were observed among clinical laboratory professionals in microscopic
diagnosis of malaria. Objective: To assess the performance of laboratory professionals on malaria
microscopy in Tigray, North Ethiopia. Methods: A cross sectional study was conducted in December
2015 among 46 clinical laboratory professionals. Data was collected via on-site assessment and panel
testing. The slide panel testing was composed of positive and negative slides. The kappa score was used
to estimate the agreement between participants and reference reader. Results: A total of 46 laboratory
professionals have taken part in this study. Most of the study participants were male gender (78%), in
the age of 20-30 years (65.2%) and served for less than five years in the health institutions (52.2%). The
overall sensitivity and specificity of laboratory professionals in detection of malaria parasites was 63%
and 95.7%, respectively. Out of the total 46 laboratory personnel involved in the study, only 1 (2.2%), 2
(4.3%) and 7(15.2%) of the participants correctly reported all the six, five and four slides, respectively.
The overall agreement between the study participants and the reference reader in malaria detection
was 79 % (kappa = 0.62). Participating in refresher training on malaria microscopy (AOR=7, CI= 1.5-36.3)
and malaria epidemic investigation (AOR= 4.1 CI= 1.1-14.5) had statistical significant association with
detection rate of malaria parasites. Conclusion: Clinical laboratory professionals showed low
performance in malaria microscopy. Most of the study participants were graded ‘in-training’ in
laboratory diagnosis of malaria. Table 1. Performance of laboratory personnel in identification of malaria
parasites, North Ethiopia 2016.<table class="AbstractTable" id="{9DA5CD8E-C75C-43A8-A2F6-
EC22A803B674}"><caption class="AbstractTableCaption"></caption><tr><td rowspan="1"
rowspan="1" colspan="2">Participant reader</td><td rowspan="1" colspan="2">Expert reader</td><td
colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="2"></td><td
rowspan="1" colspan="1">+ve</td><td rowspan="1" colspan="1">-ve</td><td rowspan="1"
colspan="1">Sensitivity</td><td rowspan="1" colspan="1">Specificity</td><td rowspan="1"
colspan="1">Agreement</td><td rowspan="1" colspan="1">Kappa</td></tr><tr><td rowspan="1"
colspan="2">Species identification</td><td rowspan="1" colspan="1"></td><td rowspan="1"
colspan="1">P. falciparum (n=92)</td><td rowspan="1" colspan="1">+ve</td><td rowspan="1"
colspan="1">56.5%</td><td rowspan="1" colspan="1">95.7%</td><td rowspan="1"
colspan="1">76%</td><td rowspan="1" colspan="1">0.61</td></tr><tr><td rowspan="1"
colspan="1"></td><td rowspan="1" colspan="1">-ve</td><td rowspan="1" colspan="1">40</td><td
rowspan="1" colspan="1">44</td><td rowspan="1" colspan="1"></td><td rowspan="1"
rowspan="1" colspan="1">P. vivax (n=92)</td><td rowspan="1" colspan="1">+ve</td><td rowspan="1"
rowspan="1" colspan="1">Mixed infection<br />(n=46)</td><td rowspan="1" colspan="1">+ve</td><td
rowspan="1" colspan="1">10</td><td rowspan="1" colspan="1">2</td><td rowspan="1"
colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1"
colspan="1"></td></tr></table>
Session Number: 45
Session Number: 45
Abstract Title:
Prevalence and Determinant Factors of Intestinal Parasites among Yekolo Temari Children Attending
Traditional Ed. in the Ethiopian Orthodox Churches in Northern Ethiopia
G. B. Hailu, D. T. Mengiste, S. B. Dasissa, A. A. Fissiha; Mekelle Univ., Coll. of Hlth.Sci., Mekelle, Ethiopia
Abstract Body:
Background: Yekolo temari are children who are studying traditional education in the Ethiopian
Orthodox Churches. These special groups of children are characterized by migration, begging and
hardship. Objective: To determine the prevalence of intestinal parasites and determinant factors among
Yekolo temari children of the Ethiopian Orthodox Churches in Northern Ethiopia. Method: A cross
sectional study design was employed to assess the prevalence and factors associated with parasitic
infection among Yekolo temari children in 2015.Wet mount and kato-katz techniques were used to
detect S.mansoni and other intestinal parasites. Intensity of infection was estimated from the number of
eggs per gram of faeces. SPSS version 20 was used to analyze data. Result: 361 children participated in
the study with a response rate of 85.6%. Of the study participants, 77.8% were in the age group 16 years
and above. One hundred eighty three (50.7%) children were positive for at least one parasite.
E.histolytica was the predominant parasite followed by S.mansoni which were detected in 108(29.9%)
and 60(16.6%) of study subjects, respectively. Of the study participants, 139(38.5%) and 37(10.2%)
harbored single and dual infections, respectively. The mean intensity of S.mansoni infection was found
to be 118 eggs per gram (epg) of stool and 38(71.7%) of the study participants had light infection (1-99
epg). Majority (82.5%) used to defecate on open fields and 253(70.1%) did not wash their hands after
defecation. Moreover, 308(85.3%) of them reported that they get their food by begging and 58.4%
trimmed their fingers. Significant relationships were observed between parasitic infection and
environmental/behavioral factors. The likely hood of washing hand after defecation was found to be
more protective against parasitic infection by 31.8 % (OR=0.68, 95% CI (1.249, 3.132). Children who used
to wear shoes were less likely to be infected by hookworm by 3.7 times (OR=3.649, 95%CI (0.005,
0.147). The presence of dirty materials on finger nails was also found to be a risk factor for infection by
53% (AOR=0.47, 95%CI (1.043, 2.45). Conclusion: intestinal parasites are very common among this group
of children. Therefore, multiple intervention strategies should be implemented to reduce the burden of
these infections.
Session Number: 45
Session Number: 45
Abstract Title:
Endemic Malaria in West French Guiana: A 7 Years Retrospective Study
M-F. Manca1, A. Jollivet1, C. Kezza1, G. Carles1, C. Misslin-Tritsch1, A. Barrelet1, J. Clouzeau1, J. Miller2,
R. Boukhari1, P. Boex1, L. Musset3, J-F. Carod1; 1West French Guiana Hosp. Ctr., Saint-Laurent-du-
Maroni, French Guiana, 2Microbiol. Technical
Abstract Body:
Background: French Guiana is a French territory located in South America where malaria is endemic. The
laboratory of Western French Guiana Hospital also serves the general practitioners of the area and the
health centers of Western French Guiana. Until 2005, the majority of malaria cases in this area was due
to Plasmodium falciparum. Most cases are now due to P. vivax. The purpose of this study was to
evaluate and analyse the results of malaria diagnosis made by the laboratory of Western French Guiana
hospital between 2011 and 2017. Methods: Malaria was diagnosed using conventional microscopic
analysis by Giemsa staining thin and thick peripheral blood smears plus rapid diagnostic testing
(Immunochromatographic OptiMAL assay) (Biosynex SA, Illkirch,F rance). Since March 2017, LAMP
technology (Illumipro, Meridian Bioscience, Cincinnati, USA) replaced the thick smear. This retrospective
descriptive study was conducted using the laboratory database. The analyses were performed using
Stata®13.1. Results: Between the 1st of January, 2011 and the 18th of December, 2017, 14,476 samples
were examined. Among these, 217 were positive for malaria (1.5%, CI95% 1.3-1.7). The prevalence
remained stable between 2001 and 2016. In 2017, a significant increase was observed: 3% (p<0.05). The
majority of the positive cases were diagnosed in patients consulting at the emergency unit of the
Hospital (80.2%). The median age of malaria patients was 28 years old (IQR: 12) with no difference in
prevalence observed between female and male. No seasonal pattern was found. 47.5% of cases were
due to P. vivax, 41.9% to P. falciparum, and the prevalence of mixed P. falciparum and P. vivax infection
was 9.2%. P. malariae was found in 1.4% of the cases. The place of residence for all cases was along the
Maroni River, in French Guiana (n=54,27%) or Suriname 27% (n=58/217), respectively. of the positives
cases were located in Surinam (border city) and are all (100%) of Brazilian origin. 57% of the positives
cases from Saint-Laurent-du-Maroni were also Brazilian nationals. Conclusion: The analysis of the
laboratory database showed a stable occurence of infections mostly related to P. vivax but also
associated with P. falciparum. Most of the cases were related to Brazilian nationals. This may be
correlated to illegal gold mining activity which is mainly handled by Brazilians “garimpeiros”. The
implementation of Molecular biology, enhancing the sensitivity of the diagnosis, may play a role in
increasing the number of diagnosed cases.
Session Number: 45
Session Number: 45
Abstract Title:
Cysticercosis in Madagascar: Unprecedented and Unreported Very High Taenia Solium Antigen
Prevalence Rates Amongst Schoolchildren
J-F. Carod1, M. Frédéric2, A-L. Parmentier2, M. Desmaret2, M. Rakotondrazaka3, R. Ramahefarisoa3, D.
Ménard3, P. Dorny4; 1West French Guiana Hosp. Ctr., Saint-Laurent-du-Maroni, French Guiana,
2Besançon Univ. Hosp. Ctr., Besançon, France, 3Pasteur Inst.
Abstract Body:
Introduction: Taenia solium (Ts) cysticercosis is a neglected zoonotic disease particularly prevalent in
Madagascar. Few data are available for children, current data mainly rely on antibody prevalence. We
sought to determine the Ts-antigen seroprevalence amongst school children from various cities in
Madagascar (excluding the capital), and evaluated associated risk factors. Methods: In seven cities in
Madagascar, the presence of cysticercosis in school children was investigated using the
B158/B60antigen (Ag)-ELISA. The analysis were conducted in Stata 10. To take into account the
hierarchical structure of the data, the relationship between the individual serological status and the
individual and collective factors was analyzed using a multilevel logistic regression model. Each factor
was first analyzed in a univariate model. When significant at p<0.20, factors were introduced in
multivariate analysis using a backward stepwise procedure. A random parameter was finally introduced
at level 1 in order to control for heteroscedasticity of the HRV parameters. Results: A total of 1751
schoolchildren were included in this study. The age ranged from 3 to16 years, the M/F sex ratio was
1.02, 53%(n=924 children) were classified as belonging to an ethnic group of Asian origin (Merina and
Betsileo ethnic groups), whereas the 47% others (n=827 children) were of African origin. The average Ts
Antigen prevalence was 27.7%, and ranged from 10 to 37%. The equatorial sites had the lowest
prevalence ranges (10-21%) while rates above 30% were found in the southern desert as well as in the
highlands. The overall prevalence based on Ag detection was 27.7% [95%CI: 10-37%]. Risk factors
associated with Ag positivity were age, biotope, altitude and annual average rainfall. Conclusion: These
results highlight the high prevalence of active cysticercosis in Madagascar among school children in the
urban setting. This high prevalence as well as the risk factors unraveled point to the emergency to
implement appropriate Public Health measures on a national scale.
Session Number: 45
Session Number: 45
Abstract Title:
Haemoparasites in Pregnant Women Attending Antenatal in Parts of Jos Plateau State Nigeria
J. A. Yohanna1, M. E. Ike1, S. T. Joseph1, S. O. Okodhi2, F. I. Omini3; 1Univ. of Jos, Jos, Nigeria, 2DEE
Med. Ctr., Jos, Nigeria, 3Hasiya Bayero Paediatrics Hosp., Kano, Kano, Nigeria
Abstract Body:
Haemoparasites in the tropics are endemic in Nigeria. Some transmission through the vertical route has
been maintained through those with asymptomatic infections. Five Hundred and ten (510) blood
samples were taken from volunteer pregnant women attending some hospitals in Jos were obtained and
serological analysis with test kits for antibodies and antigens for the various hemoparasites were carried
out. Thick and thin film (Staining methods) were used for each blood sample on greese-free slides using
the Giemsa technique. Results indicated that with serology, infection with Plasmodium falciparum was
298(58.4%) while with the thick and thin method it was 166(32.5%). Infection with Plasmodium malariae
56 (11%), Microfilariae 6(12%), HIV 116(22.8%), HBsAg 178(34.9%) while HCV 76(14.1%). All pregnant
women of all categories were susceptible to haemoparasite infection. All age groups, trimesters and
occupations had significant infection ranging from 0.087-1.000>0.02. The highest infection was with
Plasmodium infection and the least was with microfilariae. Infection with HBsAg was significant while
with HCV wasn't. More literate women were less infected than the less literate. The results indicate the
need for sensitization campaigns on predisposing factors, and basic transmission method should be
carried out-before pregnancies and screening of women during pregnancy so they can be treated.
Session Number: 45
Session Number: 45
Abstract Title:
Study of Opportunistic and Other Intestinal Parasitic Infections in Relation to Cd4 Count among Human
Immuno Deficiency Virus (Hiv) Positive Patients
S. Humagain; St. Xavier's Coll., Kathmandu, Nepal
Abstract Body:
Background: Human Immunodeficiency Virus (HIV) infection is a significant health problem facing in Asia
and Africa. Gastrointestinal infections are very common in patients with HIV infection or AIDS where
diarrhea is a common clinical presentation caused by intestinal parasites. The main objective of this
study was to establish the prevalence of intestinal parasitic infections among HIV positive people in
Nepal visiting National public health laboratory (NPHL), Kathmandu. Methods: A total of 202 stool
samples from the HIV positive patients were examined by Formalin- ether sedimentation and Sheather-
sucrose floatation followed by modified acid fast (Ziehl-Neelsen) staining techniques during the period
of June to November 2015. Results: Among 40 total infected patients, 33 (82.5%) were found to be
infected with single parasite. The prevalence of parasites showed that Giardia lamblia was the most
predominant one i.e. 9 (27.27%), followed by Entamoeba histolytica 6 (18.18%), Trichuris trichiura 5
(15.15%), hookworm 4 (12.12%), Ascaris lumbricoides 4 (12.12%), Cryptosporidium 3 (9.09%) and
Cyclospora 2 (6.06%) respectively. Co-infection of hookworm and E. histolytica 2 (28.57%) and T.
trichiura and A. lumbricoides 2 (28.57%) were the most predominant followed by G. lamblia and
Cryptosporidium 1 (14.29%) and T. tichiura and E. histolytica 1 (14.29%). Co-infection with
Cryptosporidium, hookworm and G. lamblia 1 (14.29%) was also observed. The prevalence of diarrhoea
in infected cases was significantly higher (p<0.05) than the non-infected cases. Significant difference
existed in HIV patients with or without the use of Anti-Retroviral Therapy (ART) and in cases with or
without the use of anti-parasitic drugs with p <0.05 in both the cases. There was no significant
difference between the occupation and the parasitic infection (p>0.05) and between the CD4 count and
the parasitic infection (p>0.05). Opportunistic intestinal infection was observed only in AIDS patients
(CD4 count <200 cells/µl). Conclusions: The result indicates that parasitic infection in HIV patients are
mainly due to illiteracy, poor hygiene and lack of access to the portable drinking water. Government and
related health sector should take the preventive action for the control of HIV/AIDS and proper screening
of intestinal parasitosis in HIV patients.
Session Number: 45
Session Number: 45
Abstract Title:
Role of Stray Dogs of Slum Area in Visceral Leishmaniasis in Eastern Nepal
K. Bantawa, D. Subba Limbu, P. Subba; Central Campus of Technology, Tribhuvan Univ., Nepal, Dharan,
Nepal
Abstract Body:
Background: Visceral leishmaniasis (VL) is a fatal parasitic vector-borne disease, also known as kala-azar.
Worldwide incidence of VL is 500,000 cases per year among them about 90% of cases occur in India,
Nepal, Bangladesh, Sudan and Brazil. During the past 5-6 years, the number of reported cases has
increased on the Indian subcontinent. With the goal to reduce annual incidence of VL in endemic areas
to <1 case per 10,000 people by 2015, Kala-azar elimination program was launched by governments of
Bangladesh, India and Nepal supported by WHO. Leishmania donovani, the etiologic agent of VL, is still
considered as anthroponotic but no clear conclusions have been made regarding animals as risk factors
or reservoir hosts. Identification of reservoir host is essential for controlling the kala-azar. In 2008, L.
donvani was found in person (6.1%), cows (5%), buffaloes (4%) and goats (16%) in endemic areas of
Dharan. To determine the possible role of homeless stray dogs of slum area, this study as performed in
active VL transmission area of Dharan city of eastern Nepal. Methods: All dog surveys were done by
veterinarians in March 2017. Blood samples were collected from 64 (42 female and 22 male) stray dogs
found in slum areas of Dharan-11, 15, 16 and 17. The samples (3ml) were collected by venipuncture into
tubes containing Na2EDTA. All tubes were immediately stored in ice box and transferred to the
laboratory of Central Campus of Technology. DNA extraction was done by phenol- chloroform method.
All DNA samples were allowed to come at ambient temperature and stored in TS1 buffer. All DNA were
analyzed by PCR (Sigma Aldrich PCR kit catalog number P0476) specific for small ribosomal (115 bp)
genes of Leishmania spp. using sense primer 18s-L-F 5'CGTAGTTGAACTGTGGGCTGTGC-3' and antisense
primer 18s-L-R 5' ACTCCCGTGTTTCTTGTTTCTTTGAA-3'. To confirm that amplified DNA corresponded to
Leishmania spp., set of positive samples from BPKIHS were sequenced. Result: The rate of Leishmania
positivity in homeless dogs was 7.82% (5/64), female dogs accounted for 9.53% (4/42) and male dogs for
4.54% (1/22). Statistical analysis showed that there is no significant relationship between gender of dogs
and L. donovani. Conclusion: The findings indicate that both domestic and stray dogs may act as
reservoir host for L. donovani and can play role in the transmission cycle of VL. Regular screening of dogs
and other domestic animals for VL should be implemented in control programs of VL.
Session Number: 45
Session Number: 45
Abstract Title:
Detection of Anthroponotic Cryptosporidium Parvum Subtype in Rabbits (Oryctolagus Cuniculus) in
Nigeria
A. B. Ayinmode, V. I. Agbajelola; Univ. of Ibadan, Ibadan, Nigeria
Abstract Body:
Background: Rabbits are commonly reared by household farmers in Ibadan, Nigeria as a source of meat
and also for experimental purposes, but there is no information available on Cryptosporidium genotypes
occurring in rabbits in Nigeria. Methods: Faecal samples were collected from 107 rabbits and examined
by acid-fast staining technique for the presence of Cryptosporidium oocysts. The microscopic-positive
samples were genotyped to determine the Cryptosporidium species using the sequence analysis of the
18S rRNA gene and subtyped by sequence analysis of the 60-kDa glycoprotein (gp-60) gene.
Phylogenetic analysis were also conducted. Results: The results showed an infection rate of 3.7% (4/107)
by acid-fast technique and all the positive samples were confirmed as C. parvum by 18S rRNA gene.
While the gp60 gene and phylogenetic analysis revealed the presence of C. parvum subtype IIcA5G3
subtype commonly found in humans and are known to be anthroponotic. Conclusions: The findings of
this study suggest that natural infection of C. parvum is possible in rabbits and underscores the need to
investigate the probable role of animal host in the epidemiology of anthroponotic Cryptosporidium
infection. This is the first report on genetic characterisation of Cryptosporidium in rabbit in Nigeria.
Session Number: 45
Session Number: 45
Abstract Title:
Status of Soil Transmitted Helminthiasis and Water, Sanitation and Hygiene Resource in Ogun State,
Nigeria
H. O. Mogaji1, G. A. Dedeke2, B. S. Bada2, S. O. Bankole2, U. F. Ekpo2, Spatial Parasitology and Health
GIS group, Abeokuta, Nigeria; 1Federal Univ. Oye-Ekiti, Ekiti, Nigeria, 2Federal Univ. of Agriculture
Abeokuta, Ogun State, Nigeria
Abstract Body:
Background: Soil Transmitted Helminthiasis (STH) is one of the major public health problem in Nigeria.
Its transmission is governed by access to water, sanitation and hygiene (WASH) resources. Efforts
towards complimenting deworming programme with provision of WASH resources have been hampered
by lack of evidence on the precise relationship between improved WASH and STH's reduction. There is
thus a need for evidence on the status of WASH resources and its interplay on STH transmission and
control in Ogun State, Nigeria. Methods: A community based cross-sectional study was conducted in
Ogun State, between July 2016 and November, 2017. A total of 33 communities were selected across
the 20 local government areas in the State using a systematic grid sampling method. Field visitations
were made to 1,499 consenting households for WASH assessment. The status and condition of WASH
resources were carefully assessed and scored using the WHO/UNICEF set standards, and an overall
cumulative score was recorded. Of the 1,499 households, only 1027(68.5%) provided fresh faecal
samples for laboratory diagnosis of helminths ova using ether concentration technique. The WASH and
STH data were compiled into a georeferenced database and subjected to descriptive statistic,
correlation and chi-square statistic; confidence limit was set at 95%. Results: Findings revealed an
overall prevalence of 17.2% (177/1027) for STH infection. By Species, Ascaris lumbricoides infection was
the most occurring 140(13.6%), followed by Hookworm 47(4.6%) and Trichuris trichiura 17(1.7%). An
overall cumulative score of 53.0% was also recorded for WASH resource condition. By categories,
cumulative scores of 64.1%, 32.8%, 69.0% and 49.3% was recorded for water, sanitation, household
hygiene and individual hygiene respectively. Pearson correlation coefficient (r = -0.006) was recorded,
indicating a negative relationship between WASH score and STH status, however there is no significant
relationship (p>0.05).Conclusion: Findings showed that sanitary condition across the state was very
poor. The overall prevalence for STH infection was also below mandatory treatment levels. However, a
negative relationship, between WASH resource condition and STH prevalence reflects the potentials
improved WASH resource condition may have on STH reduction. This finding calls for a need to
compliment deworming programmes with provision of improved WASH resources.
Session Number: 45
Session Number: 45
Abstract Title:
Trematode Infections in Freshwater Snails and Rainfall Patterns in Ibadan, Nigeria
O. Olayinka; Univ. of Ibadan, Ibadan, Nigeria
Abstract Body:
Background: The extent and intensity of parasitism is known to be affected by climatic conditions.
Climate changes, among other factors, have been confirmed to exert more influence on helminths and
thus, on the diseases caused by helminths. Freshwater snails serve as specific intermediate hosts for
trematodes of medical and veterinary importance and snail ecology is important in the transmission of
the parasites. Snail borne trematodiases are heavily dependent on the environment for dissemination
and are greatly affected by climate. Correlating rainfall patterns, snail abundance and trematode
infections have been known to aid in the design of control models for snail borne diseases. Method:
Fresh water snail samples were collected from August to December in years 2012 and 2014 to
determine abundance and prevalence of trematode infections. Rainfall data for the study period was
obtained from the Department of Geography, University of Ibadan. A total of 550 fresh water snail
samples were collected in 2012 and 480 in 2014. The snails identified were Physa, Lymnaea, Bulinus and
Melanoides species. Examination for trematodes’ cercariae was carried out using the crushing method.
Results: In 2012, 18 snails out of 550 (3.3%) were infected with trematode species with evidence of
cercariae release while 0% was recorded in 2014. The total recorded rainfall for the study period in 2012
and 2014 was 35.2 mm and 303.4 mm respectively. Conclusion: This result showed that rainfall has an
effect on the prevalence of snail-borne infections by cercariae of trematode parasites.
Session Number: 45
Session Number: 45
Abstract Title:
Detection of the Rat Lungworm, Angiostrongylus Cantonensis, in Giant African Snails in Trinidad
K. Kines1, Y. Qvarnstrom1, J. Seetahal2, L. Gyan2, V. Lashley2, C. Wharwood2, A. Balfour2, S. Rahaman3;
1CDC, Atlanta, GA, 2Ministry of Agriculture, Land and Fisheries, Trinidad, Trinidad and Tobago, 3Ministry
of Hlth., Trinidad, Trinidad and Tobago
Abstract Body:
Background: Angiostrongylus cantonensis, the rat lungworm, is the most common infectious cause of
eosinophilic meningitis in humans. A. cantonensis is endemic in Southeast Asia and the Pacific Basin, as
well as in areas of Africa, South America and the Caribbean. The known geographic range of A.
cantonensis has expanded in recent decades, partially due to habitat expansion of the rat and snail
hosts. Lissachatina fulica, the giant African snail, is an invasive and highly destructive agricultural pest
species recently introduced in Trinidad. Giant African snails are known intermediate hosts for A.
cantonensis and there is growing concern about public health implications of this introduction. Although
human angiostrongyliasis cases have been reported from several other Caribbean islands, no human
cases have been detected yet in Trinidad. An environmental investigation was initiated to determine the
prevalence of A. cantonensis infection in giant African snails in Trinidad. Methods: Giant African snails
were collected from multiple locations on the island of Trinidad in 2013, 2014 and 2017. DNA was
extracted using the QIAGEN PowerSoil kit and real-time PCR was used to detect the presence of A.
cantonensis larvae in the mantle tissue of the snails. Results: A total of 211 giant African snails were
tested for A. cantonensis. Infected snails were detected for all three sampling periods: 12% (7/58) in
2013, 2% (2/85) in 2014 and 16% (11/68) in 2017. Three of the 9 locations sampled in 2017 had A.
cantonensis-positive snails. Conclusions: These results revealed that A. cantonensis is present in giant
African snails in Trinidad. Giant African snails are classified as a notifiable pest in Trinidad and
eradication efforts are ongoing, with a special focus on locations where A. cantonensis was detected.
Session Number: 45
Session Number: 45
Abstract Title:
Phytochemical Screening and Antimicrobial Activity OfJatropha TanjorensisLeaf Extract
I. Omorogiuwa; Sch. of Hlth.Technology, Benin City, Nigeria
Abstract Body:
This study was designed to phytochemically screen Jatropha tanjorensis leaf and investigate the
antimicrobial activities of its extracts. Phytochemical screening and antimicrobial testing (minimum
inhibitory concentration and minimum bactericidal concentration) were performed on six groups of
microorganisms namely Escherichia coli (20), Pseudomonas aeroginosa (20), Staphylococcus aureus (20),
Klebsiella species (20), Proteus mirabilis (20) and Candida albican (20) using standard laboratory
methods. Results were presented in figures and tables and statistical analysis was done using the
student’s t test and P value <0.05 was considered statistically significant. The phytochemical screening
revealed the presence of bioactive principles such as alkaloids, flavonoids, tannins, cardiac glycosides,
saponins, glycosides and steroids. Escherichia coli, Pseudomonas aeroginosa, Staphylococcus aureus,
Klebsiella species and Proteus mirabilis were sensitive (P<0.01) to glacial acetic acid leaf extract. The
minimum inhibitory concentration (MIC) for staphylococcus aureus using 12.08µg glacial acetic acid
extract of Jatropha tanjorensis was 23.75±1.37 and this was significantly higher (P<0.001) than 10µg of
ceftazidime, cephalexin, cefuroxime with MIC of 19.20±1.06, 21.31±1.44, 19.31±1.11 respectively. The
same pattern was observed for E. coli and Proteus mirabilis and Klebsiella species. However, the MIC on
Pseudomonas aeroginosa using glacial acetic acid extract was significantly lower (P<0.001) than the
commercially prepared antibiotics. Overall, the medium used for the extraction plays a role in its
sensitivity because glacial acetic acid has a higher extractability index and sensitivity against the
microorganism studied.
Session Number: 45
Session Number: 45
Abstract Title:
Assessment of Antimalarial Drug Resistance-Associated Mutations in Plasmodium Vivax In Northern
India
U. Ghoshal, P. Ranjan; Sanjay Gandhi Postgraduate Inst. of Med. Sci., Lucknow, India
Abstract Body:
Introduction: The growing trend of complicated malaria with the global expansion of antimalarial
resistance is one of the biggest challenges today.The wide distribution of complicated vivax malaria
across several endemic regions suggests the introduction of drug resistance associated mutations within
their genome. Regular screening of markers is extremely important.There are scanty data on drug
resistance vivax malaria from the northern India. Therefore, weassessed the drug resistant mutations in
vivax which might contribute to map the proper treatment guidelines. Method: Of 2186 febrile patients
with clinical suspicion of malaria screened between Jul-2013 to Feb-2015, 561 patients fulfilled inclusion
criteria. Plasmodium species were identified by microscopy and genus and species-specific nested PCR.
PCR was performed for single nucleotide polymorphisms (SNPs) followed by sequencing to identify drug
resistance markers in P. vivax. Result: Plasmodium vivax was detected in 31/561 (5.52%) cases using
PCR. Pvmdr-1 sequence analysis showed that T→M mutations at 958 position andY→F mutations at 976
position were present in 26/31 (83.87%) and 6/31 (19.35%) samples respectively. Double (1076L with
958M) and triple mutants (958M, 976F and 1076L) were also present in 19/31 (61.29%) and 6/31
(19.35%)samples respectively. Further, for the first time in India we observed that 8/31 (25.8%) P. vivax
samples had K10 insertion (AAG codon for lysine) at 10th position of first exon. However, we did not
found any other allelic variation among 14 exons of the Pvcrt(o) gene in any sample. Conclusion: This
study provides new data concerning drug resistant mutations of P. vivax from northern India. Though we
did not assess drug resistance in-vitro, 10/26 (38.5%) individuals harboring mutated vivax strain showed
clinical resistance. Keywords: Single nucleotide polymorphisms, drug resistance
Session Number: 45
Session Number: 45
Abstract Title:
Invitro Anti Parasitic and Anti Bacterial Effects of Anti Malarial and Anti Typhoid Drugs among Patients
Attending Univ. Clin. and Lab. Animals in Nigeria
A. O. Nwosu; Univ. of Jos Hlth.Services, Jos, Nigeria
Abstract Body:
Background: Malaria and Typhoid are the most important parasitic and bacterial infectious disease in
humans. Plasmodium species are the causative agent of Malaria, while that of Typhoid is Salmonella
typhi and Paratyphoid by Salmonella Paratyphi A, B, or C. Enteric fever is a severe human bacterial
disease, Like Malaria, there is a popular believes that Typhoid fever is also endemic and quite prevalent
in Nigeria. While both diseases and most especially Typhoid are going extinct in the wilder world, their
case in Africa remains an alarming one as they have been recorded to constitute a major case of hospital
admissions in Africa,also as an indication of neglect, control of the environment. This study investigated
and established the activities and effects of some commonly used anti-malarial and anti-typhoid drugs,
Via; Artemether/Lumefantrine, Artesunate Amodiaquine and ciprofloxacin, Cefuroxime and
Azithromycine on Plasmodium and Salmonella species. This study aiming at assessing and evaluating the
dual curative effect of these drugs, describe and documents the findings as evidence base data for their
eradication in Nigeria and Africa at large. Methodology: Testing the effect of 5µg/disc incorporated anti-
malarial drugs on Salmonella species, using In-vitro culture sensitivity disc method and In-vivo culture on
Albino Mice. Likewise, that of 10, 30 and 15µg/disc, of the anti-typhoid drugs on Plasmodium species
using modified In-vitro assays method and In-vivo culture methods. Results: In-vitro result obtained
showed that, out of 250 studied cases, 24(9.6%) of them had a co-infection of Malaria and Typhoid,
while only Malaria infection was 133(53.2%) and Typhoid only was 47(18.8%). The sensitivity test
showed that Artemether/ Lumefantrin was the most effective anti-malarial 14/24(58.3%) of the co-
infection cases while the most effective antityphoid was Ciprofloxacin 17/24(70.8%) followed by
Azithromycin 15/24(62.5%). In-vivo effect of these drugs and their synergistic activities were also
demonstrated. Conclusion: This research revealed the curative activities and dual synergistic effects of
these drugs. Evidence that Artemether/ Lumefantrine and ciprofloxacin were discovered to be a single
drug with dual curative effects, proving that, a single drug can alleviate the economic hardship caused
by these diseases. The statistical analysis of variance reveals that there was no statistical significant
difference as the P > 0.05.
Session Number: 45
Session Number: 45
Abstract Title:
Efficacy of Cotrimoxazole in Controlling Malaria Parasitaemia and Bacterial Infections in Hiv-Infected
Pregnant Women in Jos, Nigeria
M. A. Ali1, E. B. Agbo2, M. M. Suleiman2, S. Oguche1, J. Musa1, O. Joseph-Anejo1, P. M. Lar1; 1Univ. of
Jos, Jos, Nigeria, 2Abubakar Tafawa Balewa Univ., Bauchi, Nigeria
Abstract Body:
Although Cotrimoxazole is being used for antimalarial prophylaxis among HIV-infected individuals, there
is insufficient data on its dual effectiveness and safety in HIV-infected pregnant women in Nigeria. The
efficacy of Cotrimoxazole (CTX) on malaria parasitaemia and bacterial infections in 235 HIV-infected
pregnant women in Jos was studied. Study population was divided into 2 groups; Group A took CTX
while group B took SP (standard for antimalarial prophylaxis in pregnancy). Peripheral blood samples
were examined for malaria parasitaemia using the thick and thin blood smears. CD4+ count and PCV
were determined using flow cytometry and capillary tube methods respectively. Bacteria were isolated
using cultural methods and biochemical tests. Cotrimoxazole reduced malaria parasitaemia by 89.4%,
while SP gave 79.01% reduction. Mean CD4+ count (cells/µl) was significantly (P<0.05) increased from
223.55 to 300.54, and decreased from 570 to 534.4 with CTX and SP respectively. Mean PCV was
significantly (P=0.015) increased from 33.09% to 33.20% after prophylaxis with CTX, but insignificantly
(P=0.154) reduced from 33.11% to 32.90% with SP. No malaria parasites were detected in the cord
blood of group A infants while 3.2% of group B infants were infected. Placental malaria was 5.9% among
group A and 7.5% among group B. Enteric bacteria isolated include Salmonella sp, Shigella sp, Proteus
sp, Enterobacter sp, Klebsiella sp, Citrobacter sp and E. coli while respiratory tract bacteria include
Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pyogenes, Haemophilus influenzae,
Streptococcus pneumoniae and Proteus species. All the Shigella, and Salmonella sp isolated were
resistant to Cotrimoxazole, while Enterobacter (77.78%) and Staphyllococcus aureus (42.86%) were the
most sensitive to the drug among the experimental (A) group. Mild anaemia was recorded in the
placenta of both groups. Prophylaxis with Cotrimoxazole is associated with more reduction in malaria
parasitaemia with reduced effect on associated respiratory tract and enteric bacteria compared to
standard SP.
Session Number: 46
Session Number: 46
Session Title: CPHM08 - Diagnostic Public Health Microbiology: Surveillance and Bioterrorism
Abstract Title:
Virminer: A Web Tool for Mining Viral Signals in Metagenomic Data
T. Zheng1, J. Li1, M. Sommer2, G. Panagiotou3; 1the Univ. of Hong Kong, Hong Kong, Hong Kong,
2Technical Univ. of Denmark, Copenhagen, Denmark, 3Leibniz Inst. for Natural Product Res. and
Infection Biology, Hans Knöll Inst. (HKI), Jena, Germany
Abstract Body:
It is well known that as key components of microbiome, phages have a major impact on the function
and structure of bacterial communities through horizontal gene transfer, impacting community
composition and altering phenotypes such as virulence or biofilm formation. Traditionally, phages were
considered as a genetic reservoir for bacterial adaptations during stress such as antibiotic treatment.
However in a recent study, researchers proposed the different view that antibiotic resistance genes
(ARG) frequencies are vastly overestimated in phage genomes. The inconsistent findings indicate that
the role of phage in microbiome remains poorly understood. To gain a better understanding, here we
developed VirMiner, a web tool that provided comprehensive phage analysis from metagenomic raw
reads processing to phage contig identification and relevant downstream analysis in metagenomic data.
Notably, VirMiner built the random forest classifier model that characterized phage contigs using
different metrics including functional information, relative abundance, and contig length for
identification of metagenomic phage contigs, which improved the predictive performance compared to
other two tools, VirSorter and VirFinder. VirMiner has higher sensitivity and F1 score than VirSorter and
VirFinder Futhermore, VirMiner provides the function of phage host relationship prediction using CRT
tools. Additionally, if users provide metadata file specifying conditions (“Control” or “Treatment”),
differential abundance analysis for inter-group comparison would be performed. Application of VirMiner
to a set of human gut metagenomes treated with antibiotics before, during and after treatment
revealed that phage capsid, virulence protein families and protein transportation-related proteins have
significantly differential abundance in the patients during treatment.
Session Number: 46
Session Number: 46
Abstract Title:
Tb Portals Genomic Analysis Portal (G-AP): A Genomic Data Exploration Platform for Translational
Research
M. Harris, J. Yu, K. Wollenberg, A. Gabrielian, E. Engle, J. Taaffe, D. Hurt, A. Rosenthal, M. Tartakovsky;
Natl. Inst. of Allergy and Infectious Diseases, Rockville, MD
Abstract Body:
Background: In silico drug resistance prediction using tuberculosis (TB) whole genome sequencing (WGS)
data is becoming increasingly important as diagnosis of TB moves towards direct sequencing of patient
samples. As such, open-access tools that enable the exploration of Mycobacterium tuberculosis genomic
data are crucial for both research and clinical practice. The TB Genomic Analysis Portal (G-AP) is
designed to support analysis and integration of genomic data collected by the NIAID international TB
Portals Program (TBPP) and other projects. The TBPP is focused on collecting data from multi-drug
resistant (MDR) and extensively drug resistant (XDR) TB patients1. To date, the TBPP has sequenced
over 600 TB genomes and to our knowledge has the largest fully characterized MDR/XDR repository of
patient cases2. Methods: G-AP provides functionality for real-time TB drug resistance prediction, the
identification of novel variants, and TB genome exploration. G-AP GWAS functionality is implemented
using the open-source PLINK 1.9 software. All TB Portals genomic data is publically available in the
National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) repository. Using G-
AP, we conducted a GWAS analysis using 60 drug sensitive patients as controls and 84 XDR patients as
cases. Results were visualized using the dynamic Manhattan plot of significant variants. A detailed in
silico drug resistance analysis using a sample from an XDR patient was conducted using the integrated
tools TB Profiler3, Mykrobe Predictor4, and KvarQ5. Reports from the resistance prediction algorithms
are presented in an integrated report for ease of comparison. Results: The GWAS analysis identified two
highly significant missense variants, katG S315T (p-value 1.1e-34) and rpoB S450W (p-value 4.1e-23).
Investigation of an individual sample from a patient who died from XDR TB using the three available in
silico drug resistance prediction algorithms consistently predicted resistance to isoniazid, rifampicin,
streptomycin, ethambutol and floroquinolones. Resistance predictions for pyrazinimide were not
consistent with both TB Profiler and KvarQ predicting resistance and Mykrobe Predictor predicting
sensitivity. Conclusions: G-AP is an effective tool for the analysis of TB WGS data. We have
demonstrated the use of the system to identify significant variants associated with drug resistance and
to perform in silico drug resistance prediction for a patient with XDR TB. This tool is an important
resource for both TB research and clinical practice.
Session Number: 46
Session Number: 46
Abstract Title:
Use of Mock Microbial Communities As A Means to Optimize A High-Throughput Microbiome
Sequencing Lab. Pipeline
M. S. Clancey, C. P. Cartwright; Lab. Corp. of America, Burlington, NC
Abstract Body:
Variability in the efficiency of nucleic acid extraction is commonly identified as one of the largest
contributors to bias in microbiome studies. The development of standardized means for assessing the
performance of extraction methods is, therefore, necessary to optimize pre-analytical sample
processing; ultimately improving the reproducibility of future studies exploring the role of microbiome
dysbiosis in human disease. We describe in this study an approach using mock microbiome communities
to objectively assess the performance of 3 nucleic acid extraction methods widely utilized in microbiome
analysis. Each method was initially used to extract DNA from a mock microbial community of known
composition, comprised of both gram positive and gram negative organisms, in quadruplicate. After
extraction, DNA was subjected to previously optimized laboratory and bioinformatics workflows
analyzing 3 separate hypervariable regions of the 16S gene: V1-V3, V3-V4, and V4. A modified
GreenGenes database that included accessions for each of 8 organisms included in the mock community
was used. Success was defined by observed/expected ratio obtained from extraction methodology and
hypervariable region amplicon approaches and the theoretical 16S relative abundance. Significant
variation in relative diversity of the bacterial population was seen irrespective of the region used for
organism identification, with method 1 being clearly superior to other approaches. The use of artificial
mock communities and objective performance-assessment algorithms could be valuable tools in
developing and standardizing methods for recovering nucleic acid from clinical samples prior to
microbiome analysis.<br /><p><a
href="http://files.abstractsonline.com/CTRL/a6/8/8e5/5d7/c05/488/8a0/495/488/586/7ed/f8/g4751_1.
src="http://files.abstractsonline.com/CTRL/a6/8/8e5/5d7/c05/488/8a0/495/488/586/7ed/f8/g4751_1.j
Session Number: 46
Session Number: 46
Abstract Title:
Changing Landscape of Diagnostic Testing for Bacterial Enteric Diseases among Clin. Lab. — Foodborne
Diseases Active Surveillance Network 2015-2017
L. Ray1, D. Eikmeier2, N. Spina3, E. Wilson4, K. Wymore5, J. Huang1, A. Geissler1; 1CDC, Atlanta, GA,
2Minnesota Dept. of Hlth., St. Paul, MN, 3CDC, Atlanta, NY, 4Colorado Dept. of Publ. Hlth.and
Environment, Denver, CO, 5California Emerging Infections P
Abstract Body:
Background: Increased development of culture independent diagnostic tests (CIDTs) and uptake by
clinical laboratories are changing the landscape of enteric pathogen detection and public health
surveillance. In 2012, the Foodborne Diseases Active Surveillance Network (FoodNet) began collecting
data on cases of bacterial enteric pathogens diagnosed by CIDT and conducting bi-annual clinical
laboratory surveys. Surveys assess enteric pathogen detection methods, diagnostic testing practices,
and in 2015, use of reflex culture (Cx). Methods: We compared data collected from the FoodNet
laboratory surveys and Campylobacter, Listeria, Salmonella, Shiga toxin-producing Escherichia coli
(STEC), Shigella, Vibrio, and Yersinia cases reported to FoodNet during 2015 and 2017. Results: During
2015 and 2017, 687 and 676 laboratories were surveyed, respectively. The proportion of laboratories
using CIDT alone in 2017 compared with 2015 increased >5-fold for Salmonella (3% vs 18%), >4-fold for
Vibrio (5% vs 22%), Yersinia (4% vs 19%), and Shigella (4% vs 17%), and almost 2-fold for Campylobacter
(15% vs 29%). The proportion of laboratories performing reflex Cx decreased for Campylobacter (22% vs
18%), Salmonella (62% vs 59%), Shigella (75% vs 50%), and Yersinia (33% vs 25%), increased for STEC
(18% vs. 28%), and didn’t change for Vibrio (31%). The first reported use of CIDT for Listeria detection in
FoodNet occurred in 42 laboratories in 2017. Among these, 63% reported using CIDT concurrently with
Cx, 22% performed reflex Cx, and 15% used CIDT alone for Listeria detection. Compared with 2015, the
proportion of laboratories using antigen-based tests decreased slightly (37% vs 34%) whereas use of
DNA-based syndrome panels increased (3% vs 13%). Among infections reported during 2015 (n=21,837)
compared with 2017 (n=19,505), the proportion diagnosed by Cx and CIDT (10% vs 22%) and by CIDT
alone (16% vs 28%) increased. The proportion of infections diagnosed by CIDT in 2017 compared with
2015 increased for Yersinia by 61%, Vibrio 34%, Shigella 30%, Campylobacter 24%, and Salmonella 19%.
Conclusions: The percentage of bacterial enteric infections diagnosed by CIDTs is increasing and use of
Cx is decreasing. As a result, data on species, subtype, and antimicrobial resistance of these pathogens
are decreasing. The declining availability of patient specimens for reflex Cx for pathogen isolation limits
the ability of public health officials to monitor trends, detect outbreaks, monitor antimicrobial
resistance, and attribute illnesses to specific sources.
Session Number: 46
Session Number: 46
Abstract Title:
A Framework for Bacterial Whole Genome Sequencing Accreditation and Quality Assurance
S. A. Ballard, A. Gonçalves da Silva, T. Tomita, M. L. Sait, S. Heaton, V. De Petra, T. Seemann, D. A.
Williamson, B. P. Howden; The Univ. of Melbourne at the Peter Doherty Inst. for Infection and
Immunity, Melbourne, Australia
Abstract Body:
Background: The Next-Generation Sequencing: Standardization of Clinical Testing workgroup (CDC) has
developed guidelines for validation of human genomic testing1. Recently these guidelines were used to
design a template for bacterial whole genome sequencing (WGS) validation2. The Microbiological
Diagnostic Unit (MDU PHL) is a public health reference laboratory in Victoria, Australia and provides
WGS-based analysis of bacterial isolates for gene detection and typing under ISO 15189 accreditation via
the National Association of Testing Authorities, Australia. To date, there are no national standards for
bacterial WGS in public health microbiology in Australia. Accordingly, the aim of this study was to design
a modular validation that explores the impact of variations in DNA extraction, sequencing modes and
sequencers in order to set standards for quality control and enable accreditation of bacterial WGS to ISO
15189 in Australia. Methods: A panel of 15 different organisms was chosen to validate the entire
workflow. Genomic DNA was prepared using the QIAsymphony (Qiagen) and Janus Chemagic
Workstation (PerkinElmer) and a column kit (Qiagen). Dual indexed libraries were prepared using
Nextera XT and sequenced on the Illumina NextSeq and MiSeq as described by the manufacturer’s
protocols. Data was analysed with a custom pipeline (github.com/MDU-PHL/mdu-tools). Outputs
included species identification, assembly metrics, yield, Q-score, and MLST. Sequence reactions (897)
were performed to determine precision, accuracy, sensitivity, specificity and limit of detection. Results:
Accuracy of sequencing relative to the reference genome was >99.99%, sensitivity and specificity was
99.5% and 100%, respectively, with a limit of detection for MLST calculated at 50x coverage of the
genome. Precision ranged from 95% to 100% depending on the extraction method, age of sample,
sequencing mode and sequencer. Conclusion: For next generation sequencing the reportable range has
been previously defined as the portion of the genome for which sequence information can be reliably
derived for a defined test system1. This platform validation provided evidence of the ability to deliver
quality sequence data, meeting ISO 15189, with the reportable ranges specifically addressing MLST,
species determination and genome-wide high quality SNPs. Our protocols form a blueprint for bacterial
WGS accreditation in Australia, and internationally.
Session Number: 46
Session Number: 46
Abstract Title:
Nctc3000 the World’S Largest Collection of Bacterial Reference Genomes
S. Alexander1, M. Fazal1, N. Grayson2, K. Oliver2, A. Deheer-Graham1, J. Parkhill2, J. Russell1; 1Publ.
Hlth.England, London, United Kingdom, 2Wellcome Trust Sanger Inst., Cambridge, United Kingdom
Abstract Body:
Background: NCTC3000 is a collaborative Whole Genome Sequencing (WGS) project between Public
Health England, the Wellcome Trust Sanger Institute and Pacific Biosciences. The project aims to
sequence 3000 Type and Reference strains from the National Collection of Type Cultures (NCTC). NCTC is
the world’s oldest bacterial strain collection (established for providing strains to third parties) and
contains over 5600 strains of medical and veterinary importance. The NCTC3000 project is an open
source community resource initiative, established to ensure that high quality reference genomes are
publically available to scientists globally via a website which will combine both strain metadata and WGS
data. Methods: High Molecular weight (HMW) DNA was isolated from 3250 strains using either the
Qiagen midi or Epicentre Masterpure extraction kits. DNA quality (>60kb) and quantity (>3 µg) was
assessed using the Agilent 2200 TapeStation and Qubit® dsDNA BR Assay Kit respectively. Whole
Genome Sequencing (WGS) was performed using the PacBio Single Molecule, Real-Time (SMRT®) DNA
Sequencing technology followed by genome assembly and automated annotation with Prokka. Results:
HMW DNA was extracted from over 3250 NCTC strains, representing 1074 different species, from 187
different bacterial genera. To majority of the available type strains (96%) were included within the
sequencing project. Genomes from 1255 strains have been assembled, annotated and are now
publically available (<u>http://www.sanger.ac.uk/resources/downloads/bacteria/nctc/</u><u></u>). Of
the strains with assembled genomes 92% have been assembled into <5 contigs and 23.1% have evidence
of one or more plasmids in the WGS dataset. A comparative analysis of the Agilent TapeStation
electropherogram peaks with the WGS data however, reveals that small plasmids (<10kb) maybe
missing from the final WGS data in approximately 10.9% (86/783) of strains analysed. Conclusions: The
WGS data generated by the NCTC3000 project represents a significant public health resource which can
be used to advance the scientific knowledge of these important pathogens. This work was funded under
the Wellcome Trust Grant no: 101503/Z/13/Z
Session Number: 46
Session Number: 46
Abstract Title:
Whole Genome Sequencing Analysis of A Stec Serotype O145:H25 Isolated from A Child with Hus in
Davidson County, Tennessee, Usa
J. Guerra1, C. Zhang1, J. Bard1, D. Yergeau1, N. Halasa2, D. Payne3, O. Gomez-Duarte1; 1Univ. at
Buffalo, The State Univ. of New York, Buffalo, NY, 2Vanderbilt Univ., Nashville, TN, 3CDC, Atlanta, GA
Abstract Body:
Shiga toxin-producing Escherichia coli (STEC) are a leading cause of foodborne illness in the Unites States
and severe complications, including hemolytic uremic syndrome (HUS). Although, O157 is the
predominant STEC serogroup in the US, non-O157 serogroups are estimated to cause more than 50% of
all STEC infections. STEC O145 serogroups are among the top non-O157 serogroups associated with
severe human disease worldwide. The objective of this study was to report a case of HUS due to
O145:H25 STEC isolated in Tennessee and to analyze the strain’s whole genome sequencing (WGS).
Strain EN1I-0044-2 was a clinical isolate from a case of bloody diarrhea and HUS in 2012. This subject
was enrolled in the New Vaccine Surveillance Network (NVSN) case and control study at the Davison
County, Tennessee. Stool from this patient was processed for E. coli isolation and detection of
diarrheagenic E. coli. Isolates were subjected to molecular based testing for virulence genes, as well as
phenotypic testing (serotyping and antibiotic susceptibility). Sequencing was carried out on the strain of
interest by using Illumina platform. A 2 years old female from Davidson county with acute
gastroenteritis, bloody diarrhea and dehydration was enrolled in the study as a case. Her clinical course
was complicated with: HUS, encephalopathy, pericardial effusion with tamponade, and respiratory
failure. The STEC EN1I-0044-2 strain isolated from this patient’s stool, was positive for eae and stx2 and
classified as O145:25 serotype. STEC O145:H25 strain EN1I-0044-2 had a genome size of 5,276,096 bp
containing 5,320 genes. The backbone chromosome was interrupted by several mobile genetic elements
(MGEs), including prophages-like elements (16), integrated elements (11) and insertion sequences (50).
The stx2A gene encoding Shiga toxin was carried in a 79.7 Kbp prophage. Phylogenetic analysis based on
concatenated nucleotide sequences of 341 orthologous genes showed that strain EN1I-0044-2 had
identical evolutionary lineage with STEC O145:H25 strains, isolated in 2003 and 2004 in the US from HUS
patients. Interestingly, this evolutionary lineage is not shared by STEC O145:H28 serotype strains. STEC
O145:H25 strain EN1I-0044-2 is a highly virulent strain isolated from a severe infant HUS case in
Tennessee. WGS analysis shows that the STEC O145:H25 strain EN1I-0044-2 belongs to the same STEC
O145:H25 serotype lineage originally detected in 2003 and 2004 among US isolates from HUS patients,
indicating that this serotype is still circulating and capable of leading to new outbreaks
Session Number: 46
Session Number: 46
Abstract Title:
Evaluation of A Gene-By-Gene Subtyping Approach for Legionella pneumophila
S. Park1, J. Caravas2, B. H. Raphael2, J. Winchell2; 1Association of Publ. Hlth.Lab., Silver Spring, MD,
2CDC, Atlanta, GA
Abstract Body:
Background: Comparison of L. pneumophila isolates recovered from environmental samples and those
recovered from clinical specimens from patients with Legionnaires’ disease can help confirm sources of
exposure. Traditionally, CDC has used a Sequence Based Typing (SBT) method to assess the genetic
relatedness of isolates recovered during outbreak investigations. We evaluated a recently-described
gene-by-gene 50 loci subtyping approach that uses genomic sequence data to understand its potential
usefulness for outbreak investigations. Methods: A total of 544 publicly-accessible genomes and
assemblies were screened for the presence of the 50 core loci using BLAST. A minimal coverage of 75%
and 85% sequence identity were required to call a locus present. Genome alignments and pairwise
distance calculation for all 50 loci were conducted with Muscle (ver.3.8.425) and Raxml (ver.8.2.9),
respectively. We detected unique alleles for each loci and generated a neighbor joining tree. Results:
Nearly all of the genome sequence assemblies contained all 50 loci. Among the 544 genomes assemblies
analyzed, 200 unique allele profiles were observed. The most frequent subtype contained 85 genomes
composed entirely of ST47 isolates. More than half of the genomes analyzed had a unique subtype,
suggesting these 50 loci are useful for strain typing. Moreover, the 50 gene subtyping method detected
known outbreak-related clusters among a group of 30 previously reported L. pneumophila serogroup 1
strains. Conclusions: A recently described gene-by-gene subtyping approach for L. pneumophila
performed well for characterization of publicly accessible genomes and preliminary data suggests that
the method is useful for source attribution studies. The findings and conclusions in this presentation are
those of the authors and do not represent the official position of the Centers for Disease Control and
Prevention.
Session Number: 46
Session Number: 46
Abstract Title:
Ictyping: Rapid Universal Bacterial Strain Typing for Real-Time Infection Control
A. E. Budding1, M. van der Bijl1, M. Boon2, D. Heemskerk1, T. Binsl3, L. Poort2, M. Degen2, P. H. M.
Savelkoul1; 1VU Univ. Med. center, Amsterdam, Netherlands, 2IS-Diagnostics, Amsterdam,
Netherlands, 3Clinicageno Ltd, Amsterdam, United Kingdom
Abstract Body:
Background: Bacterial strain typing is essential for the process of tracking and tracing the spread of
bacterial strains. To contain outbreaks as they occur, a rapid typing technique is essential. To be
effective on a larger scale, such a method should be cheap and applicable locally in any hospital. Data
should be interchangeable between hospitals and should ultimately be comparable to other typing
methods. Here we demonstrate a universal bacterial strain typing approach and coined it infection
control Typing, or icTyping in short. Methods: The technology underlying icTyping is an amplified
fragment length polymorphism (AFLP) approach. We have redesigned all steps of the process and
combined it with an automated software pipeline for fully standardized data-analysis with minimal
hands-on time. First, we created in-vitro reference databases for the species Clostridium difficile (CD),
Staphylococcus aureus (SA) and Pseudomonas aeruginosa (PA), with strains collected in the VU medical
center from 2011-2017. Secondly, we created an in-silico database of all available whole-genome
sequences (WGS) of these species in public repositories and performed a virtual AFLP on all sequences.
Next, all common fragments were defined per species in vitro and in-silico and fragments were
matched. Here we demonstrate the setup and efficacy of this novel approach for the three bacterial
species. Results: In total 552 strains of CD, 1360 strains of SA and 534 strains of PA were used to build
the in-vitro database. Whole genome sequences of 954 strains of CD, 7968 strains of SA and 1675 strains
of PA were retrieved from public repositories. icTyping showed highly comparable results between the
two laboratories. Detected variation was almost entirely attributable to low-level background noise that
could easily be adjusted for by the software. By linking in-vitro fragments to in-silico fragments, icTyping
results can be directly compared to WGS results and via this route to all other typing methods.
Conclusions: icTyping is a rapid and reproducible typing technique that is applicable to any bacterial
species. Data is comparable between laboratories and can be matched to WGS data. In conclusion,
icTpying holds great promise as a cheap universal typing method that can be used locally in an acute
setting and may be a valuable addition to WGS approaches.
Session Number: 46
Session Number: 46
Abstract Title:
Evaluation of Fecal Nucleic Acid Preservatives for Culture-Independent Pathogen Subtyping Using
Metagenomics
Y. Gao1, K. C. Dillon2, J. R. Hensley2, B. A. Aspinwall2, E. Trees3, J. Besser3, H. Carleton3, A. J. Williams-
Newkirk1, A. D. Huang3; 1IHRC, Inc., Atlanta, GA, 2Oak Ridge Inst. for Sci. and Ed., Oak Ridge, TN, 3CDC,
Atlanta, GA
Abstract Body:
Background: Culture-independent diagnostic testing (CIDT) provides fast, cost-effective diagnostics for
clinical laboratories and is replacing culture-based methods to identify foodborne pathogens. However,
CIDT methods do not yield the pathogen isolates critical for outbreak surveillance and investigation.
New direct-from-specimen surveillance techniques will rely on high-quality samples with well-preserved
fecal nucleic acids. Currently, stools are typically collected and stored at room temperature in selective
growth media such as Cary-Blair. This can alter the fecal community composition, making downstream
analyses unreliable. This study evaluated available preservation methods for fecal nucleic acids for
metagenomic and subtyping studies. Methods: Two preservation methods, the OMNIgene®•GUT kit
(DNA Genotek) and Stool Nucleic Acid Collection and Preservation Tubes (Norgen Biotek), were tested
using samples created with 5 clinically healthy donor stools that were homogenized, pooled, and spiked
with 10^6 CFU/mL of Shiga toxin-producing Escherichia coli (STEC) and Salmonella enterica strains.
Samples were incubated at room temperature either for the entire testing duration, or were shifted to
40oC for 72 hours during the first week. Samples were tested on days 0, 7, 14, 21, and 28 for fecal DNA
yield and quality, pathogen recovery using qPCR, and bacterial community composition by 16S rRNA V4
sequencing. Unpreserved stools frozen at -20oC were used as positive controls, and stools in Cary-Blair
were used as negative controls. All samples were set up as triplicates. Results: By all measures, samples
preserved with either kit were most similar to frozen, unpreserved positive control samples. While
spiked-in STEC and S. enterica pathogen levels increased by 3 logs in room temperature unpreserved
and Cary-Blair stools as detected by qPCR, pathogen levels in OMNIgene- and Norgen-preserved
samples remained unchanged at all temperatures and time points. Nonmetric multidimensional scaling
of the Jaccard dissimilarity matrix of the operational taxonomic units identified from the 16S rRNA
sequencing showed that samples preserved in OMNIgene or Norgen kits in all conditions tested have
similar bacterial community composition and membership to the Day 0 and frozen unpreserved
controls. Conclusions: Both the OMNIgene and Norgen kits are promising methods for fecal nucleic acid
preservation. Future studies will test additional variables such as longer storage time, low temperatures,
and complex disease state stool types.
Session Number: 46
Session Number: 46
Abstract Title:
Comparison of Subspeciation of Salmonella by Phenotypic Methods versus Genetic Methods
C. G. Lane, B. A. Dinsmore, A. C. Lauer, M. Van Duyne, E. A. Arnold, IV, P. I. Fields; CDC, Atlanta, GA
Abstract Body:
Historically, the genus Salmonella has been characterized by a set of distinguishing phenotypic traits
that allow delineation between the 2 species, Salmonella enterica and Salmonella bongori, as well as the
6 subspecies described for Salmonella enterica: enterica (I), salamae (II), arizonae (IIIa), diarizonae (IIIb),
houtenae (IV), and indica (VI). As tools for identification and characterization of bacterial pathogens
transition from traditional microbiological methods to whole genome sequencing methods, such as
Average Nucleotide Identity (ANI), the discriminatory power available for subspecies delineation is
expected to increase. Over 600 well-characterized strains from the Centers for Disease Control and
Prevention’s National Salmonella Reference Laboratory were chosen, covering the known phylogenetic
and antigenic diversity within the genus. The sequence data for these strains, along with corresponding
conventional identification data, was used to determine the precision of subspeciation by phenotypic
methods compared to genetic methods. Harvest was used to build a multiple core-genome alignment
from the generated WGS data, which was then used to produce a core genome phylogeny. Based on this
analysis, the majority of strains clustered according to their phenotypic subspecies identification, and
less than 5% of genomes showed discordant subspecies identification results compared to their
phenotypic profile. Over 70 genomes from strains that had one or more atypical phenotypic traits
compared to their designated subspecies profile clustered as expected in the genetic analysis despite
phenotypic variation. Nearly 2% of genomes were determined to be atypical variants belonging to a
different subspecies than phenotypically designated. For example, four of the biochemical tests that
define subspecies VI result in different reactions by different serovars. Four of the eighteen sequenced
genomes (22%) designated as subspecies VI based on phenotypic profile showed a higher genetic
similarity to subspecies I, II, or IIIb. This is likely due to the large margin of variability within the
phenotypic definition of subspecies VI. Conversely, three genomes belonging to subspecies IIIb that did
not have any atypical phenotypic traits for their designated subspecies had higher genetic similarity to
subspecies II. While there is high concordance between conventional and genetic identification methods
down to the subspecies level in Samonella, whole genome sequencing promises greater resolution.
Session Number: 46
Session Number: 46
Abstract Title:
Characterisation of Candida Auris on Chromid® Candida Agar Medium
L. Dévigne1, A. Foray1, J. Roche2, S. Orenga1, S. Ghirardi1; 1bioMérieux, La Balme Les Grottes, France,
2bioMérieux, Marcy l'Etoile, France
Abstract Body:
Background: Candida auris is an emerging multidrug-resistant fungus that causes a wide range of
infections sometimes associated with high mortality rates. Its early detection allows to take appropriate
infection control measures and to adapt antimicrobial therapy. The aim of this study is to identify the
biochemical profile of C. auris thanks to VITEK®2 YST cards and to characterize C. auris on chromogenic
agar culture media. A second step allows to screen enzymatic substrates which could discriminate
specifically C. auris. Methods: Twenty (20) strains of C. auris and 60 strains form other species of
Candida coming from bioMérieux (bMx) and ATCC collections were characterized by VITEK®2 YST cards
and color of colonies on chromID®Candida (CIDCan) (bMx), CHROMagarTMCandida (CMACan) (Becton
Dickinson) and BrillianceTMCandida (BrilCan) (Oxoid) media. Subsequently, synthetic enzymatic
substrates targeting the most relevant activities were incorporated in variations of the CIDCan medium.
The screening of these substrates was performed with 17 strains of Candida, including 4 C. auris using
calibrated suspensions. Reading was performed after 24 and 48 h incubation. Results: On CIDCan, the
isolated colonies of C. auris became pink after 48 h incubation. Conversely, this species was colorless on
CMACan even at 48 h. On BrilCan, the coloration of colonies was brown since 24 h of incubation. Based
on VITEK®2 biochemical profiles and literature data analysis it appears that pink and brown color of C.
auris colonies for CIDCan and BrilCan, were respectively due to β-glucosidase and phosphatase activities.
Data found in literature and VITEK®2 YST results showed that C. auris is able to hydrolyse D-Raffinose, as
well as few other Candida species. The screening of enzymatic substrates has shown that some
phosphatase enzymatic substrates combined with the β‑glucosidase and hexosaminidase activities
could allow the characterization and pre-identification of C. auris by a specific coloration of colonies.
Conclusions: This study highlights metabolic activities of interest to differentiate C. auris. These are β-
glucosidase, phosphatase activities and D-raffinose hydrolysis. This biochemical profile allowed to
screen some enzymatic substrates and to define interesting combinations of substrates which could
allow the pre-identification of C. auris.
Session Number: 46
Session Number: 46
Abstract Title:
Lead Acetate Test to Differentiate Tartrate Fermenting SalmonellA
N. A. Momin, S. Van Duyne, B. A. Dinsmore, A. C. Lauer, C. Lane, P. I. Fields; CDC, Atlanta, GA
Abstract Body:
The ability to ferment tartrate is a useful phenotypic marker to differentiate Salmonella. In particular,
this phenotype is critical in identifying some clinically relevant pathotypes within serotypes such as
Paratyphi B. Several methods have been used to measure tartrate fermentation; the Lead Acetate Test
produces a small, but highly notable precipitate when tartrate is fermented, making it easy to interpret.
To determine the method that unambiguously distinguishes between tartrate fermenting and non-
fermenting strains, we tested four parameters of the standard Lead Acetate Test, previously described
by Alfredsson et al.1972. This assay was modified to include the following four inoculation methods: one
µl size inoculating loop full of bacteria from Blood Agar Base, one µl loop full of bacteria from Luria Agar,
31 µl of inoculated saline solution at a three McFarland Standard, and three drops of inoculated nutrient
broth at a two McFarland Standard. At 37°C, the incubation period ranged from two to six days. The
volume of lead acetate added ranged from 300µl to 500µl. In all, 36 different assay conditions were
tested per isolate. Seventeen Salmonella enterica strains covering two serotypes, Paratyphi B and
Typhimurium, were tested, representing negative, positive, and delayed positive phenotypes. We found
that with 500µl of lead acetate, 33% of strains inoculated from Blood Agar Base produced a true positive
result at an incubation period of less than 3 days, while 25% of strains inoculated from Luria Agar
produced a true positive result at an incubation period of less than 3 days. Following analysis, the results
indicated that an incubation period of six days at 37°C with 500µl of lead acetate was 100% concordant
with the expected results, and definitely distinguished between tartrate-fermenting strains from non-
fermenting strains. For the Lead Acetate Test to effectively identify tartrate-fermenting strains,
reference laboratories that utilize this method should consider up to a six day incubation period
inoculated from Blood Agar Base with 500µl of lead acetate for a true negative result.
Session Number: 46
Session Number: 46
Abstract Title:
Moistened Gauzes Followed by A Broth Enrichment Step is A Very Sensitive Methodology for
Environmental Mrsa Screening
M. Aires-de-Sousa1, S. Rodrigues2, T. Conceição2, H. de Lencastre2; 1Escola Superior de Saúde da Cruz
Vermelha Portuguesa, Lisboa, Portugal, 2Inst. de Tecnologia Química e Biológica da Univ.e Nova de
Lisboa, Oeiras, Portugal
Abstract Body:
Background: Methicillin-resistant Staphylococcus aureus (MRSA) can survive for long periods on
inanimate objects, and therefore environmental surfaces constitute an important reservoir for
dissemination. However, there is no standardized method for the detection of MRSA from
environmental surfaces. The aim of the present study was to evaluate different screening methods to
detect environmental MRSA contamination. Methods: A total of 294 samples were obtained from
inanimate surfaces at a hospital in Luanda, Angola and a hospital in São Tomé and Príncipe, by three
different methodologies: (i) sterile swabs moistened in saline solution; (ii) sterile cotton gauzes
moistened in Tryptic Soy Broth; (iii) commercial premoistened sterile sponges (polywipes). After a broth
enrichment step, all samples were plated onto Tryptic Soy Agar and chromogenic selective media for S.
aureus and for MRSA. The S. aureus isolates were characterized by PFGE, spa typing, MLST, and SCCmec
typing. Results: Comparing the three screening methods, gauzes were the most effective (16 S. aureus
out of 98 samples; 16.3%), followed by polywipes (4/98; 4.1%) and swabs (3/98; 3.1%). Moistened
gauzes were the most sensitive method (p<0.00001) while screening with swabs was the least efficient
(p=0.00002). The majority of the MRSA isolates (75%) belonged to the main clonal types previously
found among patients and healthcare workers in the same hospitals: ST5-IVa (n=7; 44%) and ST88-IVa
(n=5; 31%). Conclusions: The finding of MRSA on environmental surfaces is dependent on the screening
methodology. Moistened gauzes followed by a broth enrichment step proved to be a very sensitive
methodology.
Session Number: 46
Session Number: 46
Abstract Title:
Evaluation of Biol. and Immunological Stability of Clostridium Difficile Glutamate Dehydrogenase and
Toxin At Different Storage Conditions in Stool Specimens
L. Irby, J. L. Sarver, J. H. Boone; TECHLAB, INC., Blacksburg, VA
Abstract Body:
Background: The stability of Clostridium difficile glutamate dehydrogenase (GDH) and toxin in stool is
important for accurate diagnosis. Package Inserts for enzyme immunoassays (EIAs) detecting GDH and
toxin recommend 4⁰C storage for 72 hours then frozen until tested. Previous studies have reported a
decrease in toxin-positive results by biological and immunological activity after collection and
speculated that C. difficile ribotype 027-positive samples may have more reproducible toxin-positive
results due to higher amounts of free toxin. The aim of this study was to evaluate the stability of GDH
and toxin in stool over 2 weeks at three storage conditions using various diagnostic methods and
determine the impact of ribotype on positive test results. Methods: Unlinked stool specimens collected
from patients suspected of C. difficile infection were included. GDH and toxin-positive samples by EIA
including the C. DIFF QUIK CHEK COMPLETE®, C. DIFF CHEK™ -60, and C. DIFFICILE TOX A/B II™
(TECHLAB, Inc.) tests were confirmed by cytotoxic neutralization assay (CNA) for biological activity of
toxin B (T=0). Samples were then tested by EIA and CNA at 4 hours, 2, 5, 7, and 14 days. The storage
conditions analyzed included -20⁰C, 2-8⁰C, and room temperature (RT). Samples were assessed
following a single freeze-thaw after storage at -20⁰C for one to six months. Toxigenic isolates of C.
difficile were identified using PCR ribotyping. Results: A total of 71 CNA-positive fecal specimens were
included with a median Bristol Stool score of 5. 97% (69) of samples remained positive for both GDH and
toxin by EIA after a single freeze-thaw cycle. Seven samples were tested over 14 days (2-8⁰C and RT) and
all samples remained positive for GDH through the testing period. Six samples remained biologically
active by CNA and toxin detectable by EIA when stored at both 2-8⁰C and RT over 14 days. One
specimen remained toxin-positive by EIA and CNA through day 14 at 2-8⁰C and was negative for a single
EIA test by day 2 at RT while remaining positive by CNA until day 5 at RT. PCR ribotyping (N=64) showed
80% of isolates were non-027 ribotype and these samples showed consistent toxin-positive results by
EIA following freeze-thaw. Conclusions: C. difficile GDH and toxin were consistently detected by EIA and
CNA when stored at -20⁰C and 2-8⁰C for 14 days, with toxin detectable by EIA a minimum of 2 days at
RT. The finding that toxin was positive in most specimens when stored at RT, demonstrated the stability
of toxin in stool specimens. Ribotype did not impact the detection of toxin after freezing.
Session Number: 46
Session Number: 46
Abstract Title:
Reproducibility Analysis of Quantitative Culture Method for Bacterial Pathogens in Bronchoalveolar
Lavage and Sputum Specimens
L. Rowland1, B. Jones1, S. Varnum1, M. Dickens1, C. Zapata-Allegro2, D. Roberts1, L. Haller1, C.
Zimmerman1; 1MRIGlobal, Palm Bay, FL, 2Biofire Diagnostics, Salt Lake City, UT
Abstract Body:
Background: Lower respiratory tract infections (LRTI) occur when pathogenic microorganisms enter the
respiratory system. An estimated 4 million LRTI occur in the United States each year 1. Bronchoalveolar
lavage (BAL) and sputum specimens are routinely collected from patients with suspected LRTI and
screened for the presence or absence of bacterial pathogens. During screening, specimens are plated on
one or more agar plates to determine pathogen loads either quantitatively or semi-quantitatively. This
plating method is often used as a reference to measure the performance of new diagnostic methods.
While it is a robust method for detecting the presence or absence of a pathogen, the quantitative
precision is unknown. Therefore, a reproducibility study was conducted to determine the quantitative
variability associated with plating these specimen types. Methods: A total of 32 specimens (21 BAL, 11
sputum) resulted in 288 randomized, distinct plating events. Each specimen was separated into nine
aliquots; three technicians (selected from a pool of six) were randomly assigned three aliquots. Each
aliquot was plated across four media types. Each technician identified the pathogen/s present using
unique colony morphologies followed by analysis on the VITEK® 2 XL (BioMerieux). CFU/mL variation
was analyzed on a logarithmic scale. Missed detections were considered to be one log10 below the level
of detection. The variation of CFU/mL between replicates in each specimen was defined as the
difference between each isolate and the mean isolate log10 CFU/mL. Results: Significant differences
were observed in CFU/mL between technical replicates (9 aliquots) of the same specimen. The
distribution of the variation is negatively skewed with a standard deviation of 0.88 log10 CFU/mL.
Within the distribution, 41% of replicates had greater than 0.5 log10 CFU/mL difference from the mean
and 21% of replicates had greater than 1.0 log10 CFU/mL difference from the mean. Conclusions: This
study demonstrates that there is considerable variability between replicate plating events of lower
respiratory specimens. The range in colony counts among technical replicates is attributed to the
heterogeneous nature of the specimens, sensitivity of the method, and viability of the organism. This
variation should be taken into consideration when using culture plating as a reference method to
measure quantitative or semi-quantitative performance of a diagnostic method. This study was designed
and funded by BioFire Diagnostics.
Session Number: 46
Session Number: 46
Abstract Title:
The Recovery of Nontyphoidal SalmonellaFrom Cidt-Positive Stool Specimens
K. C. Dillon1, J. R. Hensley1, A. J. Blackstock2, M. Patel1, E. Trees2, J. Besser2, H. A. Carleton2, A. D.
Huang2, A. J. Williams-Newkirk3; 1Oak Ridge Inst. for Sci. and Ed., Oak Ridge, TN, 2CDC, Atlanta, GA,
3IHRC, Atlanta, GA
Abstract Body:
Background: Nontyphoidal Salmonella is estimated to cause 1,000,000 infections each year in the US
with more than 19,000 hospitalizations and 380 deaths. US clinical laboratories are adopting culture-
independent diagnostic tests (CIDTs) to detect Salmonella and other pathogens in human stool because
they can provide clinical information faster than traditional microbiological techniques. CIDTs can
jeopardize public health surveillance networks like PulseNet because they do not yield the isolates
required for subtyping, virulence determination, and antimicrobial resistance testing. An optimized
standard protocol for isolate recovery from CIDT-positive stool specimens would reduce barriers to
isolate availability for public health surveillance. This study examined the impact of transport
temperature, plating media, transport media, and transport time on the recovery of two Salmonella
serovars from human stool. Methods: 5 stool samples from clinically healthy, anonymous donors were
homogenized and pooled to make 1 standard stool. The standard stool was divided and seeded with
either Salmonella enterica ser. Newport or Oranienburg at 104 or 102 CFU/mL. Each seeded stool was
aliquoted and stored at -80oC until use. For both experimental replicates, 10 aliquots of stool (2 with
each serovar at each seed level and 2 unseeded negative controls) were used to inoculate vials of each
transport media (Cary-Blair Transport Medium or Gram-Negative broth). Half were held at each
transport temperature (4°C or 22°C) and sampled at different transport times (4, 8, and 14 days) and
inoculated onto two plating medias (Hektoen Enteric Agar or Xylose Lysine Deoxycholate Agar). Plates
were incubated at 37°C for 24 hours. Up to 3 colonies morphologically consistent with Salmonella and a
sweep were collected from each plate. Colony and sweep identity was confirmed by qPCR. Results: At
22oC Salmonella was recovered from 100% of the seeded stool samples. At 4°C Salmonella was
recovered from 90% of 104 CFU/mL samples, but only 2% of samples at 102 CFU/mL (46% overall).
Recovery rates did not differ by serotype, transport time, plating media, or transport media.
Conclusions: Results from this study strongly support the transport of Salmonella CIDT-positive stool
specimens at 22oC to optimize isolate recovery even at low pathogen loads. Future work will examine
screening approaches and enrichment media used in conjunction with the optimal conditions identified
here to allow recovery from samples with lower Salmonella levels and/or after longer transport times.
Session Number: 46
Session Number: 46
Abstract Title:
Assessment of Analytical Reagent Methanol As An Alternative Fixative for Preparing Smears for Direct
Sputum Smear Microscopy
A. Flores, T. Bella, J. Chua, J. Dupalco, J. Ogsimer, L. Pableo, L. Ramos, F. Riparip, A. Sy; Univ. of Santo
Tomas, Manila, Philippines
Abstract Body:
Background: Heat fixation is considered as the standard method for the preparation of sputum smears
for direct sputum smear microscopy. Although there are already advances in sputum smear microscopy
it remains a common method because of its simplicity, inexpensiveness, and accessibility especially
among resource limited areas such as the Philippines. However, inexperienced laboratory personnel are
prone to overfix the smear through prolonged application of heat which can denature the protein
components of acid fast bacilli thus leading to distortion and poor quality of staining. Objectives and
Methods: This study seeks to assess the effectiveness of analytical reagent methanol (ARM) as a fixative
for direct sputum smear microscopy in terms of size, evenness, thickness, cleanness, staining, and AFB
grading with adherence to the criteria set forth by the Philippine National Tuberculosis Program (NTP).
Ten known positive sputum samples were obtained from a tertiary medical center in the Philippines.
The volume, color and consistency of the sputum samples were noted and recorded. Two smears
following the Philippine NTP guidelines were prepared from each specimen. One set of smears were
fixed using heat while the other set using ARM. The staining procedure performed was in accordance to
the Philippine NTP guidelines. The stained smears were coded and randomized and were evaluated
macroscopically and microscopically by a DSSM proficient medical technologist. The results were then
statistically analyzed using Goodman and Kruskall’s Gamma Test and Wilcoxon Signed Ranked Test.
Results: Results show that there is no significant difference between the heat fixed and ARM fixed
smears in terms of size (p=0.00), evenness (p=1.000), thickness (p=0.171), cleanness (p=0.000), and
staining (p=0.000). Thus, analytical reagent methanol showed comparable macroscopic assessment with
the standard heat fixation. Likewise, there is also no significant difference between the heat fixed and
ARM fixed smears in terms of microscopic reading (p=0.000). Conclusion: Analytical reagent methanol
can effectively fix sputum smear thus it can be used as an alternative fixative for Direct Sputum Smear
Microscopy
Session Number: 46
Session Number: 46
Abstract Title:
Bacteria in Med. Professionals' White Coats: are They Little Moppet in the Pocket Or are They Offensive
Bugs Exhausting Drugs?
S. K. Mishra, S. Maharjan, K. Parajuli, J. Sherchand; Maharajgunj Med. Campus, Kathmandu, Nepal
Abstract Body:
Microbial transmission from patient to patient has been linked to transient colonization of medical
professionals' attires. Contamination of these professionals' clothing including white coats might play a
big role in transmission of microbes. To our knowledge, there are no reports on nursing uniform
contamination in health care facilities of our country. This study was carried out to determine the
degree of contamination by bacterial agents on the white coats in a tertiary care hospital in Nepal. To
achieve this, sterilized uniforms, with polyester cotton patches of 10 cm X 15 cm size attached to right
and left pockets, were distributed to 12 nurses of six different wards of the hospital at the beginning of
their shift. Worn uniforms were collected at the end of the shifts and brought to laboratory for analysis.
Then the fabric patches were subjected for total colony count of microorganisms, identification of
selected bacterial pathogens, as prioritized by World Health Organization (WHO), following standard
methodology as described by American Society for Microbiology (ASM). A total of 12 swatches were
sampled and 50% were found to be contaminated by pathogenic bacteria. The average colony growth
per square inch of the patch was 524 and 857 during first and second workdays, respectively. After the
second shift, bacterial colonies on the fabrics increased by 63.6%. The isolates detected on patches after
first shift were Staphylococcus aureus (16.6%), Escherichia coli (8.6%), Pseudomonas aeruginosa (8.6%),
and Acinetobacter spp. (16.6%). Additional bacteria identified included Bacillus spp., Micrococcus spp.
and Coagulase-negative staphylococci (CONS). White coats of nurses from the Department of Pediatric,
Medicine, Maternity, Intensive care unit (ICU), Post operative ward were found to have been
contaminated by pathogenic bacteria while no pathogenic bacteria were isolated from surgical
department. One S. aureus isolate from Maternity ward was found to be methicillin resistant (MRSA).
This study showed that pathogens belonging to WHO list of critical priority and high priority have been
isolated from white coats of nurses, thus posing risk of transmission to patients. Proper maintenance
and handling practices of the white coats should be undertaken to minimize the degree of bacterial
contamination and to prevent cross contamination of healthcare associated pathogens in hospital
setting. Effort should be made to discourage usage of white coats outside clinical areas.
Session Number: 46
Session Number: 46
Abstract Title:
New Sampling Approach to Detect Environmental Microbial Contamination in Italian Hosp. Wards
M. Ferrari, A. Anesi; ASST of Lodi, Lodi, Italy
Abstract Body:
Background: Hospital-acquired infections are often connected to contamination of inanimate surfaces
near the patients. Up to day, there is not standardize and efficient methods to evaluate the microbial
contamination and consequently assess the efficacy of the cleaning procedures. The sampling of the
surfaces can be performed using contact plates or swabs. Contact plates are used for sampling of flat
surfaces. Swabs are used for sampling of articulated surface. The Aim of the present study was to
investigate a new sample device for surface monitoring, the FLOQSwabTM in combination to SRK
solution (Hygiene Monitoring System 958C, Copan Italia) to evaluate the efficacy of the sanitization
method used to clean surfaces in Hospital wards. Nylon flocked swab (FLOQSwabTM) improve the
collection and the release of the sample from the surfaces thanks to its innovative technology. Materials
& Methods: The following Hospital wards were considered for the monitoring: Dialysis Center (n=5
sampling points); Gynecology Surgery Room (n=14 sampling points) and Orthopedic Radiology (n=5
sampling points). Cleaning procedure: identified sampling points were cleaned using a disinfection
system (HyperDRYMist® technology). Sampling was performed in parallel before and after the cleaning
procedure with a new device and the traditional swab. To standardize the area to be sampled, a square
cardboard frame 10 x 10 cm (COPAN Italia) was used to define the area for testing. The flocked swab
was transferred in its transport medium tube (1mL of SRK solution) and the traditional swab in 1ml of
saline solution. The whole 1 ml was used to inoculate Tryptic Soy Agar plate at 35°C up to 3 days. The
bacteria identification was performed by mass-spectrometry. Results: The efficacy of the sanitization
procedure was evaluated on the difference in colonies count detected on the surface before and after
sanitization. In all wards considered, the use of HMD has allowed to identify more bacteria species then
the traditional swab. In all the sampling points, HMD was able to detect on the different surfaces the
“thrue microbial load”, the rayon swab reported an underestimation of the microbial load in all analyzed
sampling sites. Conclusions: Use of Nylon Flocked swabs has improved swab sampling device and SRK
solution as preservation medium allowed to adequately assess the microbial contamination on the
surfaces sampled and thus properly evaluate the effectiveness of disinfection system used.
Session Number: 46
Session Number: 46
Abstract Title:
Antibiotic Susceptibility of L.PneumophilaIsolates from Water Sys. of A Tertiary Hlth. Care Center, India
K. Sreenath, A. Valavane, R. Chaudhry, P. Samanta; All India Inst. of Med. Sci., New Delhi, India
Abstract Body:
Background: Legionella pneumophila, the causative agent of Legionnaires’ disease (LD) is ubiquitously
distributed in aquatic environments.Studies were reported from India regarding the environmental
reservoirs of L.pneumophila, but their antimicrobial susceptibility has received no attention. We
conducted a study to detect L.pneumophila and to analyze the antimicrobial susceptibility of strains
isolated from water systems of a tertiary health care center. Methods: A total of 104 water samples (58
potable and 46 non potable) were collected from various locations of a tertiary care hospital in India
during a period of 3 years. Standardized culture procedure was followed for isolation of Legionella spp.
Identity of L.pneumophila isolates were confirmed by mip gene PCR, similarly L.pneumophila serogroup
1 (Lp1) was identified by real-time PCR targeting wzm gene. Susceptibility testing of isolates against
seven antimicrobials including azithromycin, erythromycin, rifampicin, levofloxacin, ciprofloxacin,
doxycycline and tetracycline were performed by E-strip methodology on buffered charcoal yeast extract
agar(BCYE-α). Results: We detected L.pneumophila in 22/104 (10 potable and 12 non potable) water
samples and identified Lp1 in 13 (5 potable and 8 non potable) out of 104 (12.5%) samples. Among the
isolates obtained, 27 (17- Lp1 and 10- L.pneumophila serogroup 2-14) were tested for antibiotic
susceptibility. Of the antimicrobials tested, rifampicin and levofloxacin exhibited highest in vitro potency
against all tested strains. MIC ranges of antimicrobials azithromycin, erythromycin, rifampicin,
levofloxacin, ciprofloxacin, doxycycline and tetracycline were 0.032-1, 0.023-0.75, 0.016-0.032, 0.016-
0.094, 0.064-1.5, 0.125-1.5, 0.064-8 μg/ml respectively. The MIC50 for azithromycin, erythromycin,
rifampicin, levofloxacin, ciprofloxacin, doxycycline and tetracycline were 0.094, 0.125, 0.032, 0.047,
0.094, 0.5, 1.5 μg/ml and MIC90 were 0.38, 0.25, 0.032, 0.064, 0.25, 1, 2 μg/ml respectively.
Conclusions: We report the presence of L.pneumophila in 21.1% of water systems of a tertiary health
care center and formulated legionella risk management in this setting. Macrolides and fluoroquinolones
are the drug of choice for legionellosis and few studies have reported therapeutic failures in patients
with LD. It is demonstrated that antibiotics used for legionellosis continue to exhibit good activity
against L.pneumophila isolates from environmental sources in our region.
Session Number: 46
Session Number: 46
Abstract Title:
Detection and Quantification of Aflatoxin B1 in Cultured Fish and Feed in Lagos State
U. C. Uba, O. I. Olatoye; Univ. of Ibadan, Ibadan, Nigeria
Abstract Body:
Background: Aquaculture production in Nigeria has experienced an explosive growth as demand for fish
as a cheap source of protein has exceeded supply. This growth, no doubt, has led to the dependence
and use of cheaper plant based ingredients. The danger posed by aflatoxin B1, a toxic metabolite of
Aspergillus sp in Fish and fish feed to man as a potential carcinogen, teratogen and mutagen cannot be
over emphasized thereby leading to a high risk of transfer of feed contaminants via contaminated
aquaculture produce, to consumers as residues. Hence this study is aimed at detecting the presence and
quantifying the levels of Aflatoxin B1 in fish and fish feed in Lagos State. Methods: A total of 270 Catfish
samples were collected from 27 randomly selected high capacity commercial farms and 27 feed samples
from Lagos State were collected for analysis using High Performance Liquid Chromatography (HPLC).
Results: The result showed that the prevalence of Aflatoxin B1 in fish and feed samples were 11.1% and
29.6% with mean concentrations of 0.07 ± 0.03µg/kg, (Range 0.05 - 0.10µg/kg), 0.34 ± 0.25µg/kg, (Range
0.07 - 0.69µg/kg) respectively. The result showed a higher concentration of aflatoxin in feed than in fish
although this was not statistically significant (t = -1.87, p = 0.10). Conclusions: The study revealed the
presence of Aflatoxin B1 residue in fish and feed sample in Lagos although this was below the
recommended EU mrl of 5 µg/kg and 20 µg/kg respectively. This study revealed that most of the catfish
and feed produced in Lagos State, Nigeria were unsafe as they contained aflatoxin B1 residues that are
highly hazardous to consumers’ health. Although AFBI was within the maximum residues limit constant
consumption could predispose the comsumers to sub-chronicty toxicity. Hence, there is need for
regulatory protection of consumers by food safety education of producers and regular surveillance of
fish feed to ensure that manufacturers adhere to strict standards so that aflatoxin in fish feed are within
safety limits.
Session Number: 46
Session Number: 46
Abstract Title:
Genome Typing of Bordetella pertussis from Clin. Specimens Via Metagenomics Approach
Y. Peng, M. R. Weigand, A. Simon, D. Kania, M. Williams, L. Pawloski, M. L. Tondella; CDC, Atlanta, GA
Abstract Body:
Background: Whole genome sequencing (WGS) of Bordetella pertussis, the causative agent of pertussis,
is crucial for a better understanding of the disease epidemiology and clinical relevance of currently
circulating strains. However, with PCR being the most frequently used test for pertussis diagnostics,
fewer B. pertussis isolates are available for WGS. Therefore, a culture independent WGS pipeline is
needed for genome-based diagnostics and comprehensive molecular characterization of B. pertussis
directly from clinical specimens. Methods: Total DNA was extracted from 16 B. pertussis positive clinical
specimens using MagNA Pure and quantified via Qubit. Multiple displacement amplification (MDA) was
performed on low input samples. For metagenomics sequencing, Illumina libraries were prepared by
NEB Ultra II kit. Further target enrichment was performed using custom RNA baits following a modified
in solution hybridization protocol. Libraries were sequenced on a MiSeq. Sequencing reads belonging to
B. pertussis were subjected to genome typing via wgSNP and/or wgMLST analysis. Results: The MDA
procedure using specific and random hexameric primers was able to produce microgram (ug) level DNA
from as little as 0.1 ng input. The additional RNA baits enrichment recovered as high as 30% of the reads
belonging to B. pertussis, providing adequate sequencing data (as high as >100 x coverage) to perform
the downstream bioinformatics analysis: taxonomic analysis, genome subtyping, molecular typing of
genes encoding vaccine immunogens, macrolide resistance surveillance and co-infection studies. Whole
genome phylogenetic reconstruction based on 4,927 SNPs and /or 3,681 coding genes clearly showed
sufficient subtyping information to compare B. pertussis genomes recovered from clinical specimens
with reference isolate genomes. Conclusions: A culture independent WGS pipeline has been developed
for genome-based diagnostics and comprehensive molecular typing of clinical specimens. The WGS data
generated by this pipeline will be applied to the novel genome level typing, and may lead to new
insights into the causes of pertussis resurgence and how to develop more efficacious vaccines. The
findings and conclusions in this report are those of the authors and do not necessarily represent the
official position of the Centers for Disease Control and Prevention.
Session Number: 46
Session Number: 46
Abstract Title:
Outbreak of Listeria Monocytogenes in South Africa: Whole-Genome Sequencing Analysis of Isolates
A. M. Smith, K. H. Keddy, N. P. Tau, S. L. Smouse, S. T. Duze, N. Ramalwa, A. Ismail, M. Allam, N.
Govender, K. M. McCarthy, J. Thomas; Natl. Inst. for Communicable Diseases, Sandringham, South
Africa
Abstract Body:
Background: Globally, there is growing concern about the increasing prevalence of Listeria
monocytogenes associated with foodborne outbreaks. Data concerning the prevalence and
epidemiology of L. monocytogenes in South Africa are lacking. A marked increase in listeriosis cases in
South Africa was noted in mid-2017. As of 05 January 2018, a total of 727 laboratory-confirmed clinical
listeriosis cases was documented for 2017. We describe whole-genome sequencing (WGS) data for L.
monocytogenes isolates associated with this outbreak. Methods: Standard microbiological techniques
were used to confirm identification of L. monocytogenes. For WGS analysis, raw sequencing data
generated on Illumina MiSeq equipment were analyzed using tools available in the CLC Genomics
Workbench Software; trimmed reads were assembled using the ‘De novo Assembly Tool’. Assembled
WGS data were analyzed using bioinformatics tools and on-line analysis pipelines available at the Center
for Genomic Epidemiology (CGE), Technical University of Denmark (http://cge.cbs.dtu.dk/services/).
Results: For the 727 laboratory-confirmed clinical listeriosis cases documented throughout the country
in 2017, most were from the Gauteng (61%, 442/727), Western Cape (13%, 92/727) and KwaZulu-Natal
(7%, 51/727) provinces. Isolates were mostly recovered from blood culture (70%, 507/727) and
cerebrospinal fluid (24%, 176/727). WGS data were available for 296 of these clinical (human) isolates;
data were also available for an additional 107 non-human (food/environmental) isolates. Among human
isolates, 15 multi-locus sequence typing (MLST) subtypes (STs) were identified; 89% (264/296) of isolates
belonged to a single ST (ST6). Isolates in this ST6 cluster were highly related, showing <20 single
nucleotide polymorphism differences following phylogenetic analysis. Among non-human isolates, 20
STs were identified; no non-human ST6 isolate has yet been confirmed. Conclusions: The majority (89%)
of the outbreak isolates are highly related and associated with a single MLST subtype ST6, a subtype
associated with unfavourable outcomes in patients with meningitis. This suggests that most cases in this
outbreak were exposed to a single contaminated food or multiple foods contaminated at a single
source. Presently, the source of the outbreak is unknown, so it is uncertain which food/s may be
implicated. Investigation into listeriosis cases will continue with trace back and further investigation of
any Listeria-positive food/environmental samples.
Session Number: 46
Session Number: 46
Abstract Title:
Superior E. coli Shiga Toxin Gene Detection for Foodborne Publ. Hlth. Surveillance with Sting Algorithm
S. B. Im1, L. Rishishwar2, A. T. Chande1, H. F. Espitia1, H. A. Carleton3, I. K. Jordan1; 1Georgia Inst. of
Technology, Atlanta, GA, 2Applied Bioinformatics Lab., Atlanta, GA, 3Ctr. for Disease Control and
Prevention, Atlanta, GA
Abstract Body:
Background: PulseNet, the national foodborne pathogen surveillance network, utilizes PCR to determine
gene presence during routine characterization of foodborne illness causing pathogens. PulseNet’s recent
adoption of whole genome sequence (WGS) technologies creates a need for analogous in-silico methods
but with 90,000 foodborne illness cases per year, computational approaches must be fast, accurate and
comprehensible. Escherichia coli (E. coli) is commonly found in the gut of mammals as a commensal
bacterium but can become pathogenic to humans if the necessary virulence factors are acquired. The
Shiga toxin gene is a distinct virulence determinant of Shiga toxin-producing E. coli (STEC). Molecular
detection of Shiga toxin gene(s) (stx1 & stx2), intimin (eae) and hemolysin A (ehxA) are imperative to
foodborne outbreak investigations. This study seeks to demonstrate the capabilities a novel algorithm,
STing for gene detection. Methods: 5000 Enterobacteriaceae strains, sequenced and made publically
available by the Enteric Diseases Laboratory Branch and PulseNet affiliated laboratories, were selected
for this study. The sample set includes 4989 E. coli, 5 E. albertii, and 6 Shigella spp. Each strain has been
tested, by PCR, for stx1 and stx2 presence; and 2831 have been tested for eae, and ehxA. Gene
detection was performed using the software package, STing, a k-mer based allele detection tool that
works on minimally processed, raw sequence data. Strains were analyzed using a reference database of
stx1, stx2, eae, and ehxA gene sequence variants developed at the Center for Genomic Epidemiology.
Gene presence was determined by STing based on allelic coverage, that is, the percentage of allele
length covered by the k-merized sequence reads. The allele coverage threshold was determined for each
gene, by selecting the lowest allelic coverage percent value that returned the greatest Matthew’s
Correlation Coefficient (MCC). The classifying power of STing was evaluated using both MCC and
accuracy (ACC). Results: STing predicts gene presence with 99% accuracy for stx1, stx2, eae, and ehxA;
while PCR was able to detect gene presence with 98% accuracy for stx1, stx2, and eae, and 97% accuracy
for ehxA. Conclusions: Our initial results look promising as a rapid, accurate, and easy-to-use alternative
to traditional PCR detection methodologies. Using STing’s assembly-free, sequence-based approach we
see significant reductions in false positive and false negative predictions which can better inform
foodborne outbreak response.
Session Number: 46
Session Number: 46
Abstract Title:
Development and Evaluation of Ngs Standards for Virome Research
B. Benton, J. Lopera, S. King, D. Mittar, R. Shabman; ATCC, Manassas, VA
Abstract Body:
Compared to the abundance of studies, applications, and publications on the human bacterial
microbiome, there are limited reagents and publications focused on the “virome.” Next-generation
sequencing (NGS) has enabled virus genome sequencing on a large scale at an affordable cost. However,
the complexities involved in the NGS methodology, and the diversity of viral genomes pose a significant
challenge to NGS assay standardization. Therefore, there is a critical need for standardized reference
materials across the research and diagnostics communities to serve as controls in assay development.
To support this need, we have developed a viral panel comprising quantified genomic nucleic acids
prepared from diverse viral RNA and DNA. We included members of the Orthomyxoviridae, Flaviviridae,
Picornaviridae, Reoviridae, and Herpesviridae families, since these families differ in their genomic
organization, envelope properties, and genome size. We normalized input viral nucleic acid copy
numbers by droplet digital PCR to a concentration of 2 x 104 genome copies per virus, and subjected the
panel to library construction and NGS on the MiSeq® (Illumina) sequencing platform. The data from both
the RNA and DNA virus library construction generated approximately 24 million paired-end reads with
only 7-8% virus-specific reads. DNA sequencing demonstrated an abundance of 94.84% of herpesvirus
and RNA sequencing efficiently recovered Flavivirus (80% abundance), Herpesvirus (16% abundance),
and Orthomyxovirus (3.8% abundance). While we normalized input genomic copies by ddPCR,
abundance by NGS varied significantly and Reovirus and Enterovirus were not detected. This finding may
be attributed to the difference in their genome organization, thereby warranting library preparation and
sequencing assay optimization. This proof-of-concept study highlights the need for, and utility of, such
virome standards to allow researchers to optimize their sequencing methods to recover sequences from
diverse viral families.
Session Number: 46
Session Number: 46
Abstract Title:
High Sensitivity Pathogen Detection in 16s Ngs Data Using Dual-Stage Mirrored Call Design for
Background Subtraction
P. P. Khil, J-H. Youn, J. Ho, C. D. Spalding, J. P. Dekker, K. M. Frank; NIH Clinical Ctr., Bethesda, MD
Abstract Body:
Broad-range bacterial detection directly from primary specimens remains challenging, despite continued
clinical demand for such testing. PCR amplification of 16S rDNA followed by sequencing can provide
sensitive pathogen detection, but Sanger sequencing is not well suited for polymicrobial specimens, and
16S NGS approaches frequently lack sensitivity to detect pathogens at low abundances. Ubiquitous
bacterial DNA contamination from reagents and environmental sources presents a further problem and
complicates interpretation. We developed a high-sensitivity clinical 16S NGS assay that utilizes a novel,
dual-stage strategy for efficient background contaminant subtraction. The assay employs nested PCR
and a touchdown amplification achieving per-reaction sensitivity down to a single rDNA genomic copy in
the presence of up to 100 ng of human DNA. Because we found that DNA contamination is unavoidable,
we added a two-stage strategy for background DNA subtraction. We first defined OTUs detected in
>25% of 66 independent culture-negative samples to remove these contaminating species from the
report. The 11 species included environmental Pseudomonas sp., Stenotrophomonas sp.,
Achromobacter sp. and Acidovorax sp. To remove sporadic contaminants present at even lower
frequency or introduced during PCR setup, we added a mirrored-call strategy: each sample was split and
identification of pathogen in both halves of the sample was required. In 46 culture-negative samples,
this strategy reduced the low-level contamination rate and resulted in zero false-positive calls. For assay
development and validation stages, 920 libraries were prepared and 984 samples were sequenced on an
Illumina MiSeq, including primary and spiked samples with mono- and polymicrobial mixtures in a
variety of sterile fluid and respiratory matrices. In total, we used 167 primary samples from 8 sources for
assay development and 101 sample for pre-validation and validation. Overall, we find that the 16S NGS
assay allows sensitive detection of a broad variety of bacterial species at concentrations <100 CFU/ml
and as low as 5 CFU/ml.
Session Number: 46
Session Number: 46
Abstract Title:
Fully Integrated Automation of Ngs Library Preparation for Foodborne Pathogens
H. Zhu1, B. Kim1, R. Yasmin1, G. Kastanis2, M. Allard2, R. A. Montagna1; 1Rheonix, Inc., Ithaca, NY, 2Ctr.
for Food Safety & Nutrition/FDA, College Park, MD
Abstract Body:
Background: The identification of foodborne pathogens is important to prevent and track major
outbreaks and to identify sources of contamination in food processing plants. Next Generation
Sequencing (NGS) is a powerful tool to identify such organisms, but considerable effort is required to
isolate DNA and prepare the DNA libraries. To simplify the process, DNA isolation and library prep were
integrated on a single instrument with sequencing performed on MiSeq instruments. Methods: FDA’s
Center for Food Science & Nutrition (CFSAN) provided 12 strains of Salmonella as well as its proficiency
panel (PP) consisting of 4 strains of Salmonella and 2 strains of E. coli O157:H7 used to evaluate the
ability of various labs to correctly identify these pathogens using NGS. Nextera DNA Preparation
reagents were used on the Rheonix Encompass workstation to prepare the NGS libraries. In addition
sequencing results from six strains of Salmonella and six strains of E. coli were evaluated after combining
DNA isolation and library preparation on the same instrument. Results: Purified, well-characterized DNA
was initially used to optimize the library preparation portion of the fully automated process. Sequence-
ready DNA libraries were prepared from these DNA samples on the Encompass workstation within
approximately 3 hours, which included about 30 minutes of hands on time. Sequencing of these libraries
by CFSAN on MiSeq instruments yielded excellent quality metrics. In the case of the 12 strains of
Salmonella received from CFSAN, quality scores exceeded 30 with depth of coverage exceeding 112X.
For the PP samples, the quality scores for both forward and reverse reads exceeded Q30 with the depth
of reads in the range of 30X - 40X. In addition, 95-98% of the reads could be mapped to the reference
genomes. Then, additional studies were performed starting with cultured Salmonella and E. coli
microbes to optimize the combined DNA isolation and library preparation on a single instrument.
Sequencing of these libraries on MiSeq instruments yielded similar results and quality scores.
Conclusions: The ability to reduce the hands-on time and improve quality metrics using automated DNA
isolation and subsequent NGS library preparation will help to reduce costs, improve turn-around time,
and provide consistently reliable data. Automation of the entire process on a single instrument will also
significantly reduce the amount of training required as well as the capital equipment costs and required
laboratory space as compared to manual processes using multiple pieces of equipment.
Session Number: 46
Session Number: 46
Abstract Title:
Genome Sequence of the Threonine-Production Escherichia coli Strain Atcc98082
J. Yang, S. Yang; Inst. of Plant Physiology and Ecology, Shanghai Inst.s for Biological Sci., Chinese
Academy of Sci., Shanghai, China
Abstract Body:
Background: Escherichia coli ATCC98082 is a threonine-hyperproduction strain. Here we report the
complete genome sequence of E. coli ATCC 98082. Methods: To obtain the genome sequence, the
sequencing was performed on Pacbio RSII with Single Molecule Real-Time (SMRT) technology. In total,
323,963 reads ( 1,500,462,470 bases) were obtained The data was assembled with Canu program and
the average coverage was about 300 folds. Annotation of the genome was finished with Prokka
program. Results: The sequencing result finally determined a 4,613,541 bp chromosome and a 454,520
bp plasmid, comprising 4,289 and 497 coding CDS respectively. Mutations are detected by comparing
the genome sequence of ATCC 98082 with E.coli MG1655. Several mutations, which are likely related
tothreonine production, were manually identified, including ilvA, tdcE, aspA, tdcE and dapB . An
interesting finding is that ATCC 98082 contains a 454 kb plasmid. To our knowledge, it is one of the
largest plasmid reported in E.coli. In sum, in this study, the complete genome sequence for ATCC 98082
is sequenced, which paves ways for further comparative analysis of this strain and its derivative strains.
Session Number: 46
Session Number: 46
Abstract Title:
Wgmlst Scheme Dev. and Validation for Improved Molecular Typing of Bordetella pertussis
D. Kania1, K. E. Bowden1, Y. Peng1, H. Pouseele2, C. DeBolt3, M. Williams1, M. Tondella1, M. Weigand1;
1CDC, Atlanta, GA, 2Applied Maths, Sint-Martens-Latem, Belgium, 3Washington State Dept. of Hlth.,
Shoreline, WA
Abstract Body:
Background Multi-Locus Sequence Typing (MLST) provides allele-based characterization of circulating
bacterial pathogens, such as Bordetella pertussis the causative agent of whooping cough. However,
current MLST schemes for B. pertussis seldom reveal diversity among the small number of gene targets
and thereby fail to capture the true molecular epidemiology of the population. The resulting
homogeneity depicted by MLST conflicts with greater genome-wide diversity indicated by pulsed-field
gel electrophoresis (PFGE) and whole-genome single nucleotide polymorphisms (SNPs). Methods To
improve discriminatory power of allele-based molecular typing of B. pertussis, we have developed a
whole-genome MLST (wgMLST) scheme from 214 reference-quality genome assemblies sequenced at
CDC using the BioNumerics platform. An initial scheme comprising 3,681 coding sequences and covering
95% of the genome was evaluated with 1,200 isolates sequenced on the Illumina Hiseq and Miseq
platforms. Isolates for scheme validation included known outbreaks, replicates collected from individual
patients, and the 2012 Washington state epidemic that was previously characterized by current
molecular tools. Results The wgMLST scheme demonstrated both concordance with whole-genome SNP
analyses and improved resolution within predominant strain types defined by existing molecular
methods. The scheme accurately differentiated outbreak and sporadic cases collected from the same
geographic region and time period in a retrospective comparison and clustered replicate isolates
collected from individual patients at a single time point. Additionally, a re-analysis of 285 isolates from
the 2012 Washington state epidemic with wgMLST reconstructed the population structure of circulating
disease with increased resolution, revealing new clusters of related case isolates. Conclusion These
results illustrate the utility of wgMLST for improving B. pertussis characterization to aid molecular
epidemiology of pertussis disease resurgence. Scheme development on the BioNumerics platform will
facilitate distribution, enabling much needed standardization in the international pertussis research
community.
Session Number: 46
Session Number: 46
Abstract Title:
Revising Lab. Diagnosis of Neisseria Gonorrhoeae
A. Roditscheff, B. Sakem, L. Risch, T. Bodmer; labormedizinisches zentrum Dr Risch, Liebefeld,
Switzerland
Abstract Body:
Background: Neisseria gonorrhoeae (NG) is a very successful human pathogen, accounting for over 100
million new sexually transmitted infections each year, according to WHO. Transmission is sustained by
the acquisition of antibiotic resistance traits, rendering standard treatment regimens ineffective, and by
the existence of a pool of asymptomatic carriers - most often women. Thus, in many clinical
microbiology laboratories, urogenital specimens are often screened for NG by adding selective culture
media, even in the absence of clinical information. Here, we address the question what effect the
systematic implementation of liquid-based microbiology (LBM), i.e. liquid transport media in
combination with flocked swabs, in optimizing our workflow for detecting NG in urogenital specimens
might have. Methods: In May 2013, liquid based microbiology (LBM) was introduced as the standard
transport medium for bacteriology. This implementation allowed for simultaneous culturing and NAA
testing. Between June 2013 and November 2017, 9'575 liquid-based urogenital swabs were collected
from women, aged 18 to 87 years, in absence of clinical information. The specimens were cultured on
selective chocolate agar PolyViteX VCAT3 plates. NAAT was performed, using Abott RealTime CT/NG
assay. Results: Overall, NG was found in 0.40 % (38/9'575) of all specimens. Among the positively tested
specimens, 100 % were detected by NAAT (38/38) and 57.90 % (22/38) were detected by culture. Most
discordant results between negative culture and positive NAAT were found in vaginal swabs (10/15) and
cervical swabs (3/9). Conclusions: Our findings indicate that an alternative workflow, i.e. liquid-based
microbiology in combination with NAA testing, improves the detection rate of NG in our setting. Reflex-
testing of NAAT positive specimens by culture remains important for antimicrobial susceptibility testing
and strain collection (strain typing).
Session Number: 46
Session Number: 46
Abstract Title:
Outbreak Analysis by Fourier Transform-Ir Spectroscopy
I. Burckhardt1, K. Sebastian1, F. Guenther2, A. H. Dalpke1, S. Zimmermann1; 1Univ. Hosp. Heidelberg,
Heidelberg, Germany, 2Philipps Univ. Marburg, Marburg, Germany
Abstract Body:
Background: Fourier transform infrared (FT-IR) spectroscopy has been successfully used for
differentiation and classification of microorganisms at the species and subspecies levels in veterinary
and food microbiology already for some years. Different biomolecules (e.g. proteins,
lipopolysaccharides) can contribute to specific spectrometry patterns, which opens the possibility for
fast and reliable strain typing in infection control. Pulse-field gel electrophoresis (PFGE) or whole
genome sequencing (WGS) are standard methods, but they are time consuming and expensive.
Methods: We used a bench-top FT-IR spectrometer (IR Biotyper, Bruker) to investigate multi-resistant
Gram-negative strains from smaller hospital outbreaks from recent years. The clonal similarity of these
strains has been proven by pulse-field gel electrophoresis (PFGE), random amplified polymorphic DNA
PCR (RAPD-PCR) and characterization of their carbapenemase enzyme by PCR. Isolates were grown over
night on blood agar plates, homogenized by vortexing in Eppendorf cups containing inert metal cylinders
and spotted on a silicon target plate. A wavelength spectrum from 4000 to 600 [cm-1] was measured.
Wavenumbers from 1300-800 [cm-1] were used for strain typing analysis. Results: The results obtained
by FT-IR spectroscopy were concordant to conventional methods for an MDR-Acinetobacter baumannii
(OXA-23), a Pseudomonas aeruginosa (VIM+) and a Klebsiella pneumoniae (KPC) outbreak. Hierarchical
cluster analysis (HCA) was used for clonal similarity calculation showing a high discriminatory power.
Results can be easily visualized as dendrogram or distance matrix plots. Including an overnight
incubation of the strains the workflow could be finished within 24 hours with very good results.
Conclusions: FT-IR spectroscopy seems to be a very promising tool for a quick outbreak analysis, at least
for Gram-negative rods. The technology can be used as a first step tool in a suspected outbreak, to
select between a random accumulation of isolates on a ward and nosocomial transmission. A result
proofing the latter would qualify for additional analysis, e.g. by PFGE or WGS. Further studies have to
proof the accuracy of the method for strain typing from different hospitals or different countries. We
have not yet performed experiments for Gram-positive multi-resistant bugs like vancomycin-resistant
enterococci or methicillin-resistant Staphylococcus aureus.
Session Number: 46
Session Number: 46
Abstract Title:
Microbiological Survey of Commercial Tattoo and Permanent Makeup Inks Available in the United States
S. Kim, S. Nho, O. Kweon, P. C. Howard, C. E. Cerniglia; Natl. Ctr. for Toxicological Res./US FDA,
Jefferson, AR
Abstract Body:
Background: Tattooing and use of permanent makeup (PMU) have dramatically increased over the last
decade, with a concomitant increase in ink-related infections. The aim of this study is to determine
whether microorganisms are present, and if so, the number and their identification, in the tattoo and
PMU inks commercially available in the United States. Methods: We surveyed 85 unopened tattoo and
PMU inks, purchased from 13 companies. Companies were located across in the United States (US),
which included East (36 inks from 6 companies), South (8 inks from 2 companies), East (19 inks from 2
companies), and Southeast (13 inks from 3 companies). We incubated 100 µL of ink samples on
trypticase soy agar plates for bacterial growth, 7H10 Middlebrook medium for mycobacterial growth,
and Sabouraud dextrose medium for fungal growth. We identified genus and species of the bacteria
isolated from tattoo and PMU inks based on 16S rRNA gene sequence. Results: Out of 85 ink samples, 42
inks were contaminated with microorganisms (49%). There was a range of difference in the percentage
of contaminated inks, according to companies, from no contamination to 78% of contamination (7 out
of 9 surveyed inks from Company 6). However, contamination of inks was geographically even across
the country. Thirty-three inks were contaminated with bacteria, 2 inks with fungi, and 7 inks had both
bacterial and fungal growth. In 26 inks, microbial concentrations ranged between 101-103 CFU/ml, but
higher counts (>103 CFU/ml) were found in 16 inks. We identified 83 bacteria by their 16S rDNA
sequences, including 20 genera and 49 species. Strains of Bacillus spp. (53%) were dominant, followed
by Lysinibacillus fusiformis (7%) and Pseudomonas aeruginosa (5%). Thirty-four (41%) possibly clinically
relevant strains were identified, including P. aeruginosa, Dermacoccus barathri and Roseomonas
mucosa, some of which have been previously reported to be associated with human skin infections.
Conclusion: The results indicate that commercial tattoo and PMU inks on the US market surveyed in this
study contain a wide range of microorganisms, including pathogenic bacteria. Microbial contaminants in
tattoo and PMU inks are an emerging safety concern for public health. This study provides evidence that
microbial contamination of tattoo and PMU inks available in the US is highly prevalent and highlights the
importance of monitoring these products for potentially pathogenic microorganisms. 1
Session Number: 46
Session Number: 46
Abstract Title:
Small Molecule Inhibitors of Zika Virus Replication Identified from A Drug Repurposing Screen
M. Xu, Jennifer Kouznetsova, Ruili Huang, Wenwei Huang, Misha Itkin, Paul Shinn, Samuel G. Michael,
Anton S; Natl. Ctr. for Advancing Translational Sci., NIH, Rockville, MD
Abstract Body:
Background: Zika virus infects human neural progenitor cells and astrocytes resulting in increased
caspase-3 activity and cell death. Methods: We have developed a high throughput caspase-3 activity
assay and a cell viability assay in 1536-well plates using Zika virus infected human cells. Results: A
screening of approved drugs bioactive compound identified a neuroprotective compound that protects
neuronal cell death caused by ZIKV infection. We also found two groups of compounds that inhibited
ZIKV replication in human cells. Conclusions: These lead compounds can be further studied and
developed for treatment of Zika virus infections.
Session Number: 46
Session Number: 46
Abstract Title:
Core-Genome Multilocus Sequence Typing (Cgmlst) of Acinetobacter Baumannii Using Ridom
Seqsphere+
S. A. Cunningham1, M. P. Murphy1, R. A. Bonomo2, K. M. Hujer3, A. M. Hujer4, B. Kreiswirth5, N. M.
Zinsmaster1, R. Patel1; 1Mayo Clinic, Rochester, MN, 2Louis Stokes Cleveland Dept. of Veteran Affairs
Med. Ctr., Cleveland, OH, 3Case Western Reserve Unive
Abstract Body:
Background: Whole genome sequencing is rapidly replacing traditional typing methods for investigation
of infectious diseases outbreaks. A. baumannii (AB) is a significant culprit in outbreaks of multidrug
resistant bacterial healthcare associated infections. We studied a well characterized collection of AB
isolates, associated with a single medical facility, using cgMLST. Methods: 72 isolates, previously typed
by PCR electrospray ionization mass spectrometry (PCR ESI-MS), were provided by the Antimicrobial
Resistance Leadership Group (ARLG) were studied. DNA was extracted using the Quick-DNA™
Fungal/Bacterial Miniprep Kit and normalized to 0.2 ng/µL with a Quantus fluorometer. Next generation
sequencing libraries were prepared with Nextera XT and dual-indexed. Sequencing was performed on
the MiSeq platform utilizing the 2 X 250 bp paired-end chemistry with a maximum pooling capacity of 9
libraries per flow cell (target coverage of 200X). Reads were processed for adapter and index cleaning
utilizing the MiSeq reporter software in real-time. Adapter-clipped read files were imported into
SeqSphere+ software. Velvet de novo assembly and cgMLST were exercised within the software suite
following default settings. Minimum spanning trees were generated from the typing data table. Results
of the two typing methods were compared. Results: There were 14 PCR ESI-MS types, of which 6 were
represented by single isolates and the remainder by 2, 4, 4, 6, 9, 11, 15 and 15 isolates (Figure). All
cgMLST results clustered with those of PCR ESI-MS, with the exception of a single isolate (ARLG-1299).
Within the PCR ESI-MS clusters, most cgMLST isolates differed by ≤9 allelic differences, but there were
sub-clusters with 10-245 allelic differences from one another. All but one of the 14 PCR ESI-MS types
differed from one another by ≥368 allelic differences. Conclusions: Results of cgMLST typing of A.
baumannii matched those of PCR ESI-MS, with cgMLST being more discriminatory.<p><a
href="http://files.abstractsonline.com/CTRL/86/7/1ba/693/6c7/48b/cb6/c6f/733/658/ba9/11/g6624_1.
src="http://files.abstractsonline.com/CTRL/86/7/1ba/693/6c7/48b/cb6/c6f/733/658/ba9/11/g6624_1.j
Session Number: 46
Session Number: 46
Abstract Title:
Use of Whole Genome Sequencing and Ft-Ir Analysis for Detection of Virulence Factors in the Zoonotic
Pathogen Arcobacter Butzleri
Y. Yamauchi1, S. Boutin2, K. Sebastian2, Y. Uehara1, S. Zimmermann2; 1Juntendo Univ. Hosp., Tokyo,
Japan, 2Univ. Hosp. Heidelberg, Heidelberg, Germany
Abstract Body:
Background: The genus Arcobacter was previously known as aero-tolerant Campylobacter. Today more
than 20 species are known, but only a few cause infections in humans. A. butzleri is described as the
most pathogenic one, but even within this species not all isolates seems to be pathogenic. We
performed a study looking accurately for Arcobacter spp. in 1650 fecal samples using genus specific PCR
and culture. The detection rate was about 1%, comparable to the incidence of Campylobacter in the
cohort. Methods: To investigate the correlation between putative virulence genes and clinical symptoms
whole genome sequencing of 50 Arcobacter strains was performed using HISeq Illumina sequencing.
Briefly, after sequencing reads were trimmed for good quality and assemble with SPAdes. The
assembled contigs were annotated using RAST (Rapid Annotation using Subsystem technology). Rapid
large scale pan genome analysis was performed using ROARY pipeline. The presence of antibiotic
resistance genes was verified by phenotypic susceptibility testing. In addition we used metabolic
fingerprinting by Fourier transform infrared (FTIR) spectroscopy to phenotypically analyze changes in
metabolic profiles and surface patterns of the bacteria. Results: Whole genome sequencing analysis
revealed various patterns of different metabolic genes (amino acids, respiration). Environmental genes
(regulating LPS patterns, flagellum and chemotaxis) also showed some variability. We focused on
virulence factors, e.g. cadF, ciaB or hec to correlate these to the clinical findings of the patients. Analysis
of resistance genes resulted in a more frequent detection of fluoroquinolones resistance while
macrolides resemble are higher susceptibility profile. The FT-IR analysis grouped the Arcobacter isolates
in several distinct subgroups. Yet, an unweighted analysis of the complete FT-IR spectrum did not
parallel the clinical patterns of the hosts. Conclusions: Arcobacter butzleri isolates depict not a
homogenous genetic profile. Significant differences were found in metabolic gene clusters as well as in
antibiotic resistance markers. In an ongoing research approach we did not yet found a single marker
defining pathogenicity within the Arcobacter group. FT-IR is a promising tool for the clustering of
Arcobacter species and analysis of metabolic profiles, but further studies are necessary to develop more
standardized protocols.
Session Number: 46
Session Number: 46
Abstract Title:
Pocket-Sized Electrocoagulator Compatible with Nanogene Assay for Cyanobacterial Detection
E-H. Lee1, B. Chua2, A. Son1; 1Ewha Womans Univ., Seoul, Korea, Republic of, 2Korea Univ., Seoul,
Korea, Republic of
Abstract Body:
Harmful cyanobacterial blooms have become a global issue of concern pertaining to securing water
quality. Due to the massive proliferation of cyanobacteria, the bloom can destroy the aquatic ecosystem
by depleting oxygen in the water. Most importantly, it is also responsible for the production of
hepatotoxin such as microcystin, resulting in liver damage and cancer. Therefore in situ cyanobacterial
detection is imperative for rapid identification and quantification of toxin-producers during a bloom. In
this study, we have developed a pocket-sized electrocoagulator as a pre-concentration technique for
toxic cyanobacterial detection. The electrocoagulator is compatible with NanoGene assay which is well
known for inhibitor resistance as well as high sensitivity and selectivity. The ability of pocket-sized
electrocoagulator was demonstrated using both pure laboratory cyanobacterial culture (Microcystis
aeruginosa) and environmental eutrophic water samples. For comparison, centrifugation was
simultaneously employed as a reference method. Electrocoagulation achieved using aliquots of 3 mL of
sample by operating at 4.5 V of applied voltage for 180 s of operation duration, while centrifugation was
operated at 1224 relative centrifugal force. For laboratory samples, the pre-concentration efficiency of
electrocoagulation achieved ~60%, while that of centrifugation showed ~70% at a range of cell density
from 1.7 × 105 to 4.1 × 106 cells·mL-1. For environmental samples (cell density ranging from 6.5 × 103 to
6.6 × 107 cells·mL-1), electrocoagulation showed the similar pre-concentration efficiency of ~ 60%,
whereas that of centrifugation decreased to below 40%. This means electrocoagulation pre-
concentrates environmental samples more efficiently than centrifugation. The pre-concentrated
samples via electrocoagulator was successfully analyzed with NanoGene assay.
Session Number: 46
Session Number: 46
Abstract Title:
Evaluating the Impact of Changing the Reporter Dye in the Anthrax Lethal Toxin Neutralization Activity
and Toxin Potency Assays
D. A. Boulay, S. Bolcen, L. Cronin, M. H. Jenks, J. Schiffer, H. Li; CDC, Atlanta, GA
Abstract Body:
An anti-anthrax lethal toxin (LTx) neutralization assay (TNA) has been established to quantitatively
measure the toxin neutralizing antibodies (Ab) to LTx (1). In this assay, cell viability is determined
colorimetrically using a tetrazolium salt, 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium
bromide (MTT) as the reporter dye. Although this assay has demonstrated high sensitivity, specificity,
accuracy and precision in both TNA and toxin potency (TP) tests, MTT and other reagents used in this
assay are hazardous chemicals that pose potential health risks to scientists who perform the test. The
assay also requires 2 days to obtain the results. A nonhazardous water-soluble tetrazolium salt, WST-8,
the major component in the Cell Counting Kit-8 (CCK-8), was evaluated to replace MTT in the anthrax
TNA and TP assays. Nine human anti-AVA sera with established Effective Dilution 50% (ED50) values
representing a range from low to high in the TNA assay were selected for evaluation. Assays were
performed as regular TNA except using CCK-8 instead of MTT as the reporter dye with shortened
incubation times for intoxication and color development. Cell intoxication times were evaluated at 2.5,
3.0 and 3.5 hours for the TNA and toxin potency assays. The CCK-8 incubation times of 2 and 2.5 hours
were evaluated for the best reproducibility and matching the ED50 values established from TNA and
Intoxication Concentration 95% killing (IC95) from TP using MTT dye. A minimum of 20 data points were
collected for each sample in the TNA assay or toxin potency assay. Data were analyzed for significant
differences of the ED50/IC95 between those two dyes, inter-operator and intra-operator precisions. At
the condition of 3 hour intoxication and 2.5 hours of incubation after addition of CCK-8, the LTx potency,
expressed as the concentration of PA at IC95 was 52.5ng/ml ± 5.4 calculated from 53 independent data
points, is not significantly different from the established value with the MTT method (51.8ng/ml ± 7.1, P
value = 0.0785). Mean ED50s of the sample panel showed insignificant difference between those two
dyes (P = 0.186). The difference of mean ED50 in each sample between those two dyes was less than
10%. Inter-operator precision (%CV) was less than 30%. Using CCK-8 as the colorimetric dye, the assay
testing period was shortened by more than 16 hours, reducing the assay time from 2 days to 1 day. The
data demonstrates that changing from MTT to CCK-8 is equivalent in overall TNA and TP performance.
CCK-8 is a qualified dye to replace MTT in anthrax TNA.
Session Number: 46
Session Number: 46
Abstract Title:
The Lab. Response Network in Action: A Collaborative Effort to Revise the Original Bioterrorism
Response Guide for Clin. Lab. (Bioterrorism Blue Book)
S. Escott1, E. Bowles2, R. Nickla3, S. Mahlen4; 1MA State Publ. Hlth.Lab., Jamaica Plain, MA, 2WI State
Lab. of Hygiene, Madison, WI, 3OR State Publ. Hlth.Lab., Hillsboro, OR, 4Sanford Hlth.Bismarck,
Bismarck, ND
Abstract Body:
Background: In 2001, the CDC published a clinical laboratory (lab) resource titled, “Bioterrorism
Response Guide for Clinical Laboratories” which was printed in a blue notebook and became known as
“the BT Blue Book”. It served as a primary reference for presumptive and safe recognition of biothreat
agents in a clinical lab. The original version was never updated from CDC, although some State Public
Health Lab (SPHL) BT Training Coordinators (BTCs) and State Training Coordinators (STCs) updated the
guide based on ASM Sentinel Lab Protocol revisions, and for their own State-specific trainings. Methods:
All BTCs and STCs were surveyed to inquire about the status and use of the guide and it was determined
that the sentinel labs still used it and it was in need of a significant update. An initial collaboration was
formed between CDC and APHL to revise the guide with help from the BTCs and STCs, the APHL Sentinel
Lab Training Collaborative Workgroup, and with steering oversight from the APHL Sentinel Lab Partner
and Outreach Subcommittee and ASM. The comprehensive 2 year project began in the fall of 2014, and
in November 2016 an updated guide titled, the “Clinical Laboratory Preparedness and Response Guide”
was released. The revision team was comprised of over 20 SPHL laboratorians, and several staff from
APHL, ASM and CDC participated in designing the layout, writing and editing the content. Results: The
updated guide is over 330 pages containing significant sentinel lab updates to the biothreat organisms,
job aids, checklists, and an expanded all-hazards scope to include chemical and radiological threats.
Many clinical lab regulatory references and requirements for packaging and shipping were also added.
The new guide allows for state specific contact and PH related information to be provided and has
several national resources for general lab preparedness. Biosafety, biosecurity, and risk assessment
content was added. Conclusions: The new “Clinical Laboratory Preparedness and Response Guide” will
serve as a primary reference once again for the next generation of sentinel labs and the SPHL trainings
and outreach efforts. The new guide is available electronically to aid in the search functionality and
increased usage, and is a single resource reference providing a summary of clinical lab preparedness. All
states have access to the same information, ensuring a nationwide, consistent, standardized, and
coordinated emergency response. The new guide will assist the PHL system in preparing and responding
more quickly and efficiently to PH and lab emergencies.
Session Number: 46
Session Number: 46
Abstract Title:
An Unusual Serotype B C. Botulinum Isolate Originating from A Case of Wound Botulism in the United
States
T. Foltz, J. Halpin, J. Dykes, K. Chatham-Stephens, C. Luquez; CDC, Atlanta, GA
Abstract Body:
Clostridium botulinum produces botulinum neurotoxin, which blocks the release of acetylcholine,
leading to neuromuscular paralysis and death if untreated. Wound botulism is an uncommon form of
human botulism; on average 20 cases are reported each year in the US. Over 90% of the cases are
associated with injection drug use (IDU), and most cases are due to toxin type A. Illegal injectable drugs
are the most probable source of infection, but it is unclear if C. botulinum comes from distant or local
sources. The focus of this study was an IDU-associated wound botulism case in Hawaii, due to type B.
We sought to determine the phylogentic relatedness of C. botulinum type B isolated from this wound
botulism case and other environmentally acquired cases in Hawaii. In addition to the wound isolate, we
identified six additional type B infant botulism isolates from Hawaii and a type B infant botulism isolate
from California in our historical strain collection. These were isolated between 1976 and 2015, subjected
to the standard microbiological methods to identify them as toxigenic and added to the CDC strain
collection. Whole genome sequencing was completed on the Ion S5 system and reads were assessed
using FastQC (v. 0.11.5), assembled with SPAdes (v. 3.10.1), and resulting assemblies assessed using
Quast (v. 4.3). We determined the botulinum toxin gene (bont) subtype using CLC Genomics Workbench
and the traditional 7-gene MLST type by querying the PubMLST database with draft genomes. Pairwise
Average Nucleotide Identity (ANI) was used to determine the best reference to use for high quality
single nucleotide polymorphism (hqSNP) analysis. LYVE set (v. 1.1.4f) identified and compared hqSNP
sites and approximated a phylogenetic tree for those isolates harboring bont/B5 genes. Reads were
assembled with an average of 153 contigs (35 - 711) and average coverage of 122x (63.1x - 302.5x). The
California isolate was determined to be subtype bont/B1 and ST-30 while all of the Hawaii isolates were
determined to be subtype bont/B5 and ST-36 except one, which was bont/B5 and a new ST that has
been submitted to PubMLST. Using reference strain CDC67071 LYVE set constructed a phylogenetic tree
and found a high degree of homology between all of the Hawaiian bont/B5 isolates. Although illegal
injectable drugs are the suspected source of infection, it is unclear where and how they become
contaminated with C. botulinum. Our results suggest that the source of contamination for this wound
botulism case was probably local, as the isolates from Hawaii clustered together with few SNP
differences.
Session Number: 46
Session Number: 46
Abstract Title:
Tularemia, As Emerging Disease in Kakheti Region, Georgia
N. Khizanishvili; Natl. Ctr. for Disease Control and Publ. Hlth., Telavi, Georgia
Abstract Body:
Objectives: Tularemia, an Especially Dangerous Pathogen (EDP), is endemic for countries located
between the latitudes 30o - 70o. Georgia is one of them. The last outbreak in Georgia was in 2006. Not
one case of Tularemia was registered in Kakheti Region since 2016. In Telavi, operates the Laboratory
Support Station (LSS) by the National Center for Disease Control and Public Health (NCDC) Georgia. The
US Cooperative Biological Engagement Program (CBEP) in Georgia has built and funds this network of
Laboratories. The LSSs perform BSL 2 lab diagnostic operations as well as disease outbreak investigation,
especially investigation of suspect cases of EDPs. This poster will present the steps taken to safely
respond, confirm and report cases of Tularemia, as emerging disease in Kakheti. Methods and Results: In
18 April 2016, the Telavi LSS received official notification, that one individual from a nearby village was
hospitalized in Tbilisi Infection Disease Center with a Tularemia diagnosis. The Telavi LSS field
investigation team went to investigate the case using EDP biosafety skills and knowledge gained from
CBEP training and CBEP-provided equipment and supplies. Other suspect cases of Tularemia were
registered in 27 April and 17 May at the same place and last case in 30 August at nearby village. The
same investigations were taken, blood samples collected only from diagnosed persons and processed by
Telavi LSS and transported to Lugar Reference Laboratory . Samples were screened for F. tularensis
specific antibodies by using a commercial IgM enzyme linked immunosorbent assay (ELISA). All ELISA
positive samples were confirmed by standard microagglutination test (MAT). All 4 cases were confirmed:
2-oropharyngeal, 1 ulceroglandular and 1 typhoidal forms. All data regarding patient, collection, sample
documentation and diagnostic results were entered into the Electronic Integrated Disease Surveillance
System. So from all 20 confirmed cases in Georgia 2012-2017 Kakheti region has the highest prevalence
Tularemia confirmed cases: 1.25 per 100000 population Kakheti and 0.53 per 100000 population -
Georgia. Conclusions and Significance: This type of response would have been difficult without the
involvement of trained and qualified lab personnel from the NCDC Laboratory network, which are able
to detect, respond and report emerging diseases in Georgia.
Session Number: 46
Session Number: 46
Abstract Title:
Describing A New Foci of Tularemia in the Country of Georgia
L. Malania1, N. Chitadze1, M. Chubinidze1, K. Goginashvili2, M. Donduashvili2, R. J. Arner3, P. Imnadze1;
1Natl. Ctr. for Disease Control and Publ. Hlth., Tbilisi, Georgia, 2Lab. of Ministry of Agriculture, Tbilisi,
Georgia, 3Ryan Arner Sci. Consulting,
Abstract Body:
Background: Tularemia is a zoonotic disease transmitted by direct contact with infected animals or
environmental contamination. Historically, the disease has been considered endemic to east Georgia,
with human cases arising from contaminated rodents, vectors, and water. Herein, we report a new focus
in Kvemo Kartli region of Georgia, previously considered Tularemia-free. Methods: Micro Agglutination
Test (MAT) and ELISA for IgM and IgG was conducted on human and animal samples for F. tularensis
antibodies. Epidemiological investigation of the focus was done through questionnaires to determine
risk factors. In the Kvemo Kartli region, seroprevalence among veterinarians, farmers, and farm animals
was studied. Results: Out of 25 human samples, one veterinarian was detected positive for F. tularensis
antibodies. Of 85 farm animals (55 cattle, 10 sheep, 10 dogs, and 10 goats), a total of 13 animals (11
cattle, 1 goat, and 1 dog) were positive by MAT. Epidemiological investigation revealed a link between
the positive veterinarian and some positive animals, as well as a history of tick bites. The patient
reported prolonged fever and enlarged lymph-nodes. Conclusions: This is first report of human and
animal cases of Tularemia in Kvemo Kartli region. Future studies should track the spread of pathogens to
other regions, better informing policy on control measures.
Session Number: 46
Session Number: 46
Abstract Title:
Environmental Surveillance of Francisella Tularensis Holarctica in the Netherlands
I. Janse, R. van der Plaats, M. van Passel; Natl. Inst. for Publ. Hlth.and the Environment, Bilthoven,
Netherlands
Abstract Body:
Tularemia is an emerging zoonosis with a human, animal and environmental component. The causative
bacterium Francisella tularemia (Ft) is able to infect a range of animal species and infection of humans
may lead to serious disease. Human infections occur through contact with infected animals, ingestion of
food or water, insect bites or exposure to aerosols or water. Ft may persist and replicate in aquatic
reservoirs where the bacterium can be hosted by free-living protozoa. In the Netherland, tularemia was
absent for over 60 years until an endemic patient was diagnosed in 2011, followed by 17 cases since.
Insights into the distribution of Ft may explain the re-emergence of tularemia and address the relevance
of its detection in surface water for infection risks. We performed environmental surveillance of Ft in
surface waters in the Netherlands. Firstly, samples were obtained from routine monitoring -not related
to tularemia- at 127 locations that were visited between 1 and 8 times in 2015 or 2016. Secondly,
sampling efforts were performed after reported tularemia cases (n=8) among hares or humans with an
environmental link in the period 2013-2017. Ft DNA was detected at 22 out of 127 randomly selected
surface water locations (17%) in different parts of the country, included three brackish or saltwater
locations. At most of these positive locations DNA was not detected at each time point and levels were
very low, but at two locations contamination was clearly higher. When sampling followed a tularemia
case, Ft DNA was detected in at least one water sample for 7 out of 8 such instances. By using a protocol
tailored for amplification of low amounts of DNA from environmental samples, 10 gene targets were
sequenced and identified F. tularensis holarctica subclades B1 and B4 at two locations. Environmental
surveillance showed that various surface waters harbor Ft. However, it is not possible to infer or predict
infection chance based on these Ft DNA levels, due to several factors such as limited samples, variable
time periods between cases and sampling and absence of information about viability and infectivity
based on qPCR. Our case-related data support the feasibility of tracking a particular strain as source of
infection. A link between environmental source and case could be substantiated by genotyping, which
was shown to be possible directly on water samples with only low levels of Ft. Applying these techniques
to materials from patients or animals will enable to establish a link between tularemia cases and
environmental samples.
Session Number: 47
Session Number: 47
Session Title: CPHM11 - Laboratory Safety, Security and Biodefense
Abstract Title:
Evaluation of A New Version of Safety Subculture Unit
A. Blair1, P. Reilly2; 1Total Lab. Compliance Experts, LLC, Braintree, MA, 2ITL BioMed., Reston, VA
Abstract Body:
Current methods and tools used to subculture blood culture bottles present a significant risk for
exposure to biohazards and sharps injury. Safety SubCulture Unit 2 (SCU2), is a new subculture device
consisting of a needleless cap that fits on blood culture bottles with an internal plastic piercing tip and
an external plastic dropper intended for dispensing sample drops from a variety of blood culture bottles
for subculture and slide preparation. To evaluate ease of use, a study was performed in a hospital
microbiology laboratory by two experienced laboratory technologists using blood culture bottles
positive for bacteria or yeast. Study metrics included: device stayed on the bottle, initial drop dispensed
≤10 seconds and acceptable drop size (e.g., similar to the original SCU). Supplies used included twenty
(20) SCU2 units, positive blood cultures in Becton Dickinson and bioMerieux aerobic and anaerobic
bottles with and without resins and petri dishes. Positive cultures included categories of Gram Positive
and Negative Bacilli (GPB, GNB), Gram Positive Cocci (GPC), and yeast. Metric results were recorded as
YES/NO. Of the 20 SCU2 devices evaluated: 20 caps stayed on the bottle 19 produced initial drop in
≤10sec. 20 produced drops similar in size to the original SCU Overall performance - SCU2 safely
dispensed drops from blood culture bottles Summary Table<table class="AbstractTable" id="{8D4FE482-
DD85-474C-AB0D-6C8CDF5EB754}"><caption class="AbstractTableCaption"></caption><tr><td
rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1">Blood Culture Bottle
Description</td><td rowspan="1" colspan="4">Organism Categories<br />Evaluated, #</td><td
rowspan="1" colspan="6">Metrics Evaluated</td></tr><tr><td rowspan="1" colspan="1"></td><td
rowspan="1" colspan="4"></td><td rowspan="1" colspan="2">Cap Stays on Bottle,#</td><td
rowspan="1" colspan="2">Initial Drop<br />≤ 10 sec., #</td><td rowspan="1" colspan="2">Drop size
similar to SCU, #</td></tr><tr><td rowspan="1" colspan="1"></td><td rowspan="1"
colspan="1">GPC</td><td rowspan="1" colspan="1">GNB</td><td rowspan="1"
colspan="1">GPB</td><td rowspan="1" colspan="1">Yeast</td><td rowspan="1"
colspan="1">YES</td><td rowspan="1" colspan="1">NO</td><td rowspan="1"
colspan="1">YES</td><td rowspan="1" colspan="1">NO</td><td rowspan="1"
colspan="1">YES</td><td rowspan="1" colspan="1">NO</td></tr><tr><td rowspan="1"
colspan="1">BD Bactec Plus Aerobic w/ Resins</td><td rowspan="1" colspan="1">7</td><td
colspan="1">1</td><td rowspan="1" colspan="1">10</td><td rowspan="1" colspan="1">0</td><td
rowspan="1" colspan="1">9*</td><td rowspan="1" colspan="1">1</td><td rowspan="1"
colspan="1">10</td><td rowspan="1" colspan="1">0</td></tr><tr><td rowspan="1" colspan="1">BD
Bactec Lytic Anaerobic</td><td rowspan="1" colspan="1">4</td><td rowspan="1"
rowspan="1" colspan="1">0</td></tr><tr><td rowspan="1" colspan="1">bM acT/ALERT SA</td><td
colspan="1">0</td></tr><tr><td rowspan="1" colspan="1">bM BacT/ALERT SN</td><td rowspan="1"
rowspan="1" colspan="1">2</td><td rowspan="1" colspan="1">0</td></tr><tr><td rowspan="1"
colspan="1">Sub-Totals</td><td rowspan="1" colspan="1">11</td><td rowspan="1"
rowspan="1" colspan="1">0</td></tr><tr><td rowspan="1" colspan="1">Total</td><td rowspan="1"
rowspan="1" colspan="2">20</td></tr></table> BD: Becton Dickinson; bM: bioMerieux; SA: standard
aerobic, SN: standard anaerobic; GNB: Gram Negative Bacilli; GPC: Gram Positive Cocci; GPB: Gram
Positive Bacilli, *2 clogged SCU2 (repeats successful) *Bottles with resin can potentially clog the piercing
tip and increase the dispensing time. To address this, after agitating the bottle, allow resin to settle then
tilt bottle at a ≤45° angle to allow the resin to gather along the side of the bottle before dispensing.
Safety SubCulture Unit 2 provides an easy to use, efficient method for dispensing samples from blood
culture bottles and improves safety by eliminating the need to use a needle.
Session Number: 47
Session Number: 47
Abstract Title:
Evaluation of Transfer Cap Set with Blood Culture Bottles and Vacuum Tubes
A. Blair1, P. Reilly2; 1Total Lab. Compliance Experts, LLC, Braintree, MA, 2ITL BioMed., Reston, VA
Abstract Body:
Current methods used to obtain samples from blood culture bottles present a significant risk for
exposure to biohazards and sharps injury. ITL BioMedical has developed Transfer Cap Set (TCS),
consisting of a cap with recessed plastic tip, tube holder with multi-sample luer and safety lid. TCS is
intended for safe sample transfer from a blood culture bottle to a vacuum tube for further
testing/processing, e.g. automated streaking. A study was performed at a hospital microbiology
laboratory to evaluate ease of use with multiple manufacturers’ vacuum tubes and blood culture bottles
(with and without resins). Blood culture samples included species of E. coli, S. epidermidis, S. aureus and
categories; Group B Strep, Gram Positive Cocci, Gram Positive and Gram Negative Bacilli, Coagulase
Negative Staph and Yeast. Twenty eight (28) TCS devices were evaluated by two experienced laboratory
technologists. Metrics documented were: Sample Transfer Time recorded as OK or TL (too long) and
Successful Sample Transfer recorded as YES/NO. Additionally, Transfer Time in seconds was measured
on 19 samples. 27 Sample Transfers were successful. 3 from bottles containing resins were initially TL.
Upon repeat with a new TCS, 2 were OK, 1 remained TL. Average Transfer Time was 7.3sec. Overall, TCS
successfully transferred samples from blood culture bottles to vacuum tubes. Summary Table<table
class="AbstractTable" id="{FA051667-80FE-43A3-AF9F-B9ABD577AA87}"><caption
colspan="1"></td></tr><tr><td rowspan="1" colspan="3">Blood Culture Bottle Type</td><td
rowspan="1" colspan="2">Culture Positive</td><td rowspan="1" colspan="2">Resins in Bottle</td><td
rowspan="1" colspan="3">Sample Transfer Time</td><td rowspan="1" colspan="2">Successful Sample
Transfer </td></tr><tr><td rowspan="1" colspan="1">Manufacturer</td><td rowspan="1"
colspan="2">Description</td><td rowspan="1" colspan="1">YES</td><td rowspan="1"
colspan="1">NO</td><td rowspan="1" colspan="1">YES</td><td rowspan="1"
colspan="1">NO</td><td rowspan="1" colspan="1">OK</td><td rowspan="1" colspan="1">TL</td><td
rowspan="1" colspan="1">SEC<br />avg.</td><td rowspan="1" colspan="1">YES</td><td rowspan="1"
colspan="1">NO</td></tr><tr><td rowspan="1" colspan="1">BD</td><td rowspan="1"
colspan="1">BacTec Plus</td><td rowspan="1" colspan="1">Aerobic</td><td rowspan="1"
colspan="1">3*</td><td rowspan="1" colspan="1">13.5</td><td rowspan="1" colspan="1">14</td><td
rowspan="1" colspan="1">1</td></tr><tr><td rowspan="1" colspan="1"></td><td rowspan="1"
colspan="1">BacTec Lytic</td><td rowspan="1" colspan="1">Anaerobic</td><td rowspan="1"
colspan="1">0</td><td rowspan="1" colspan="1">6.5</td><td rowspan="1" colspan="1">10</td><td
rowspan="1" colspan="1">0</td></tr><tr><td rowspan="1" colspan="1">BioMerieux</td><td
rowspan="1" colspan="1">bacT/ALERT SA</td><td rowspan="1" colspan="1">Aerobic</td><td
rowspan="1" colspan="1">0</td><td rowspan="1" colspan="1">ND</td><td rowspan="1"
colspan="1">1</td><td rowspan="1" colspan="1">0</td></tr><tr><td rowspan="1"
colspan="1"></td><td rowspan="1" colspan="1">bacT/ALERT SN</td><td rowspan="1"
colspan="1">Anaerobic</td><td rowspan="1" colspan="1">2</td><td rowspan="1"
colspan="1">2.0</td><td rowspan="1" colspan="1">2</td><td rowspan="1"
colspan="1">0</td></tr><tr><td rowspan="1" colspan="3">SUB-TOTALS</td><td rowspan="1"
colspan="1">3</td><td rowspan="1" colspan="1">7.3</td><td rowspan="1" colspan="1">27</td><td
rowspan="1" colspan="1">1</td></tr><tr><td rowspan="1" colspan="3">TOTALS</td><td rowspan="1"
rowspan="1" colspan="1">7.3</td><td rowspan="1" colspan="2">28</td></tr></table> SA: standard
aerobic, SN: standard anaerobic, TL: too long, *: 2 repeats OK, ND: no data * Use of TCS with bottles
containing resin required angling the bottle so the resin settled along the side preventing it from
obstructing the sample tip and prolonging transfer. TCS provides an easy to use, efficient method for
transferring samples from blood culture bottles into vacuum tubes for further testing/automated
processing. TCS improves safety by eliminating the need to remove the blood culture bottle cap or use a
needle thereby reducing the risks of biohazard exposure and sharps injury.
Session Number: 47
Session Number: 47
Abstract Title:
National Online Cross Sectional Survey on Overview of Biosafety Cabinets Availability, Maintenance and
Safety Operational/Installation Protocols in Pakistan
F. Ahmed1, A. Ikram2, J. Muhammad1, A. Butt1, Z. Rasmussen3, P. Stroot4; 1Pakistan Biological Safety
Association, Islamabad, Pakistan, 2NIH, Islamabad, Pakistan, 3Pakistan Biological Safety Association,
Marryland, MD, 4Pakistan Biological Safety Associat
Abstract Body:
We aimed to have an overview of the biosafety cabinets available, type, safety protocols, manufacturer
details and maintenance in laboratory settings at different academic, research and clinical institutes in
Pakistan. Objectives were to identify the availability of biosafety cabinets in laboratory settings at
different academic, research and clinical institutes in Pakistan. To explore the type and classification of
cabinets being used in Pakistan. In the past two decades many laboratory settings have emerged in
academic, research and clinical settings in Pakistan. With the increase in the number of labs in Pakistan
BSC also periodically increased. For safe and secure use of BSC properly trained staff, safety protocols
and routine annual maintenance and certification are essential. The study design was a cross sectional
survey carried out during February/March 2017. A questionnaire was developed with feedback,
suggestions and guidelines from The Eagleson Institute USA and Fogarty International Center National
Institutes of Health USA. A total of 167 organizations/institutes participated in the survey. Out of the
total sample 71% had a BSC installed and 29% did not have a BSC available. 55% of the BSCs were
installed since less than five years, 31% BSC installed since five to ten years and 14% BSC installed for
more than ten years. Only 43% of the BSC at organizations had laminar flow, 26% BSC had a chemical
fume hood and only 65% had exhaust ducts. 11% of the BSC were self maintained by biomedical
engineer or lab staff at the institute, 31% of BSC were company/manufacturer maintained and 58% of
the BSCs were not maintained routinely. 89% of the respondents also showed interest and agreed to the
need of trainings designed for BSC safe use. Only 37% of the participating institutes had biosafety
committees and biosafety officers. 48% of the institutes had BSC IIA2, 12% had BSC IIB1, 9% had BSC IIB2
and 7% had BSC III. The most common companies of BSC were Thermo Scientific, ESCO, and BioBase.
The survey provides with an overview of the current situation regarding BSC use at a national level.
There was a lack of routine/annual maintenance/certification of cabinets. Safety operations/installation
protocols like having exhaust duct, laminar flow and chemical fume hood were also not available at
most of the institutes. This survey highlights the need to train lab staff in safe BSC use, maintenance,
developing IBCs and having designated BS officers.
Session Number: 47
Session Number: 47
Abstract Title:
Evaluating Bacillus anthracis Inactivation and Dna Extraction Following Heat Stabilization Treatment by
the Denator Stabilizor T1
J. Bugrysheva, P. Michel, P. Juieng, V. Loparev, D. Sue; CDC, Atlanta, GA
Abstract Body:
Transferring biological material from a biosafety level 3 (BSL3) laboratory to BSL2 may be necessary for
downstream analyses such as DNA sequencing and requires efficient inactivation of viable bacteria
without damaging nucleic acids. Heat stabilization treatment using the Stabilizor T1 instrument
(Denator) was previously shown to successfully inactivate Gram-negative non-spore-forming bacterial
pathogens without a negative effect on yield and quality of purified DNA. We evaluated whether heat
stabilization treatment inactivates the Gram-positive, spore-forming pathogen Bacillus anthracis, and
allows for the isolation of high quality DNA in quantities sufficient for whole genome sequencing. The
select agent-excluded, avirulent B. anthracis strain Sterne 34F2 was cultured on Tryptic Soy Agar with
5% Sheep’s Blood (SBA) at 35°C. Suspensions of bacterial cells from SBA colonies were prepared in saline
to a turbidity equivalent to 3.5-4 McFarland and measured using the DensiCHEK Plus (Biomerieux)
instrument. Samples (1ml) were transferred into Maintainor Liquid cards and heat treated at 95°C using
the Denator Stabilizor T1 system. Bacterial inactivation was verified by culturing heat-stabilized cell
suspensions (HSCS) on nutrient media. DNA was isolated using the Epicentre Master Pure Complete DNA
and RNA kit. DNA quantity and quality were determined using the Qubit fluorometer (Invitrogen), a
NanoDrop spectrophotometer (Thermo Fisher Scientific), and by visualization in agarose gel after
electrophoresis. All work was performed in a BSL2 laboratory by trained personnel wearing approved
personal protective equipment. The shortest heat-stabilization treatment time required to consistently
inactivate B. anthracis Sterne suspensions was 180s. This time was sufficient to inactivate Sterne from
colonies grown for 17h but not from older colonies. This is likely due to spore formation. The quantity
and quality of Sterne DNA decreased with increasing durations of heat-stabilization. The amount of
genomic DNA isolated from 1ml of 180s HSCS was ~1µg, which is a 3.5x decrease compared to the
untreated Sterne. The 30kb band for 180s HSCS DNA was visibly present in gel electrophoresis but with
noticeable DNA degradation, and the A260/A280 ratios were lower than 1.80. While a single preparation
of 180s HSCS from colonies grown ≤17h would yield sufficient B. anthracis DNA to prepare short-read
sequencing libraries (Illumina Nextera), multiple aliquots and re-purification may be needed if larger
quantities of high quality DNA are required.
Session Number: 47
Session Number: 47
Abstract Title:
Biological Indicator Incubation Time for Vhp Decontamination
S. Blount1, S. Ziegler2, S. Orr1; 1Southern Res., Birmingham, AL, 2Texas BioMed. Res. Inst., San Antonio,
TX
Abstract Body:
Chemical and biological indicators are used to ensure successful decontamination of spaces and
equipment when using Vaporized Hydrogen Peroxide (VHP). While chemical indicators show immediate
results upon exposure, biological indicators coated with Geobacillus stearothermophilus spores require
incubation for at least 7 days per manufacturer’s instructions. Furthermore, a seven day hold of
equipment or rooms until confirmation of zero spore growth may restrict the ability to perform
operations in those spaces. However, experience has shown that positive biological indicator results
indicating incomplete spore kill appear well before day seven. We ran a series of validation experiments
to demonstrate the effectiveness of a shortened incubation time for biological indicators. Prior to the
experiment, VHP cycle parameters were established that would achieve a target 50% ±20% kill of
biological indicators to allow for positive samples for verification of incubation times needed for spore
growth. This target also increases potential incubation times by reducing the amount of viable spores
necessary to create sufficient metabolic activity for the incubated media to indicate growth. The
experiment included 4 test runs of chemical and biological indicator pairs placed in a sealed box test
chamber and exposed to the 50% ±20% kill sterilization cycle. Upon cycle completion, chemical indicator
results were immediately recorded and biological indicators were processed by dropping the spore-
coated disks into media and incubated at 55-60ºC for a total of 7 days with recording intervals at 12, 24,
48, and 72 hours and on the 7th day. 52.3% (69 of 132) of biological indicators recorded as positive for
growth. Of those, 66 of 69 (95.6%) indicated positive after 12±3 hours of incubation and 100% indicated
positive at or before 48±2 hours of incubation. All chemical indicators exhibited some color change due
to VHP exposure but none matched the full color change from purple to the passing yellow control
color. The validation experiment showed an incubation period of 48 hours is sufficient for remaining
viable spores to indicate positive for growth. A shortened incubation period increases the flexibility
without compromising safety for continuing operations with equipment and spaces following VHP
decontamination.
Session Number: 47
Session Number: 47
Abstract Title:
Stories Behind the Spectra: Improving Lab Safety, Outbreak Response and Strain Characterization Using
Maldi-Tof Ms
J. Gartin, J. Pryor, R. Welsh, K. Silberman, J. Ortiz Santos, J. McQuiston; CDC, Atlanta, GA
Abstract Body:
Introduction: MALDI-TOF is a leading tool in microbial diagnostics; however, the full scope of its
capabilities is unclear. Experiences in CDC’s Special Bacteriology Reference Lab (SBRL), Zoonotic Select
Agents Lab (ZSAL), and Mycotic Disease Lab (MDB) advocate for non-traditional applications of MALDI-
TOF that address critical needs in laboratory safety, outbreak response, and strain classification.
Methods: Spectra are acquired with extended direct transfer or full protein extraction protocol. Spectral
evaluations are performed with MBT Compass (4.0) and BioNumerics (v7) software. Main Spectral
Profiles (MSPs) require 20 spectra with peak frequencies >70%, as well as corroborating sequence-based
confirmations. Results: MicrobeNet included representation (20) of Brucella spp., resulting in high
confidence scores for Brucella samples. Compass 4.0 cannot differentiate Brucella spp.; however,
analysis using BioNumerics identified Brucella spp. to the species level. Using MicrobeNet, internal and
external evaluations find Elizabethkingia anophelis to be significantly more prevalent than expected.
SBRL has classified over 300 clinical isolates and aided hospital and public health labs in over 100
Elizabethkingia identifications. Over 70% of historical E. meningoseptica were reclassified as E.
anophelis. MicrobeNet provides supplementary representation of Candida auris. Over 700 isolates from
CDC and public health labs have tested positive using MicrobeNet. Analysis of C. auris strains using
BioNumerics demonstrated clustering matches distinctive geographic origins determined by whole
genome sequence (WGS) analysis. Discussion: With early detection diagnostics returning incorrect or
inclusive results, Brucella exposures remain a concern in clinical labs. With MicrobeNet’s added
representation, possible Brucella can be flagged in the early stages of diagnostic workflow. BioNumerics
clustering can reliably classify Brucella species. For both E. anophelis and C. auris outbreaks, database
expansion of MALDI-TOF decidedly improved triaging processes vital to outbreak response. The 2016
Elizabethkingia outbreak and corresponding representation in MicrobeNet enlightened scientists to a
long misunderstanding of the genus. While further investigation is necessary, the associations found
between WGS and spectral clustering for E. anophelis and C. auris indicate capabilities beyond species
identification and that MALDI-TOF could potentially determine clinically- or epidemiologically-relevant
patterns.
Session Number: 47
Session Number: 47
Abstract Title:
Evaluation of the Biosafety Risk for the Bd Max™ Mdr-Tb Assay on the Bdmax™ System
K. E. McMurdie1, E. Stonebraker1, C. C. Whiteford1, J. Leitch1, K. Bryan-McNeal1, K. J. Andrews1, J. D.
Balarashti2, R. S. Tuttle2, M. Porter1; 1Becton, Dickinson and Company, Sparks, MD, 2Aerosol Res. and
Engineering Labs, Olathe, KS
Abstract Body:
The BD MAX™ MDR-TB assay* is a new molecular test that detects Mycobacterium tuberculosis
Complex (MTBC) DNA and genetic mutations associated with rifampin and isoniazid resistance, directly
from sputum specimens. We assessed the biohazard risk for the BD MAX MDR-TB assay* by examining
MTBC inactivation by the Sample Treatment Reagent (STR) and by assessing MTBC viability and aerosol
production during automated sample processing on the BD MAX instrument. When negative sputa
spiked with MTB strain H37Rv or multidrug-resistant MTB isolates were treated with STR at a 2:1 STR-
sputum ratio, a minimum of a 7-log reduction in MTBC viability was observed. No viable MTBC were
recovered from the wash buffers or eluates collected at various steps of the BD MAX process. Cross-
contamination studies demonstrated no aerosol contamination occurred during automated processing.
Sputa spiked with high titers of Bacillus globigii (Bg) and M. bovis (BCG) were processed and no growth
was observed for either organism on solid culture media. Collectively, these findings support that pre-
treatment of sputa with STR and performing the MDR-TB assay* on the BD MAX inactivate MTBC
organisms. *Product under development. Not available for sale or use.
Session Number: 47
Session Number: 47
Abstract Title:
Rapid and Label-Free Optical Screening of Anthrax Spores Using Quantitative Phase Imaging and Deep
Neural Network
Y. Park, Y. Jo; KAIST, Daejeon, Korea, Republic of
Abstract Body:
Background: The spore-forming bacterium Bacillus anthracis is the causative agent of the disease
anthrax, which has been abused for biological warfare and terrorism. Although it is crucial to establish
early warning systems for anthrax attacks, most of the conventional methods for screening of anthrax
spores have suffered from limited sensitivity and/or specificity to cope with realistic settings such as
aerosolized spores. Methods: We developed an optical method for rapidly screening anthrax spores
with single-spore sensitivity and sub-genus specificity. The proposed method is based on holographic
microscopy or quantitative phase imaging, which exploits the sample’s endogenous refractive index
distribution as the imaging contrast, for label-free imaging that is ideal for ultrahigh-throughput analysis.
The measured holographic images of individual spores from several Bacillus species are utilized by a
deep learning algorithm, which is adopted to holographic microscopy for the first time, to extract the
fingerprints of anthrax spores encoded in the images. Results: We present a new optical method for
rapid screening of anthrax spores with single-spore sensitivity and sub-genus specificity. In contrast to
conventional machine learning techniques, the new deep learning framework enables end-to-end
training of species classifiers due to representation learning capability. HoloConvNet automatically
recognized and exploited the inter-species dry mass difference between the Bacillus species, which is
consistent with the structural characteristics of the microbes, from the raw phase images. The network
outperformed all previous methods based on conventional optics and/or machine learning, achieving
the test accuracy higher than 95% for certain anthrax screening tasks. Conclusions: We demonstrated
that the label-free detection of the pathogen Bacillus anthracis spores within seconds. The proposed
method combines holographic microscopy with deep learning, for the first time to our knowledge, and is
sufficiently broad and general to be applied to various single-cell identification problems. We
demonstrated sub-genus diagnosis of the pathogen Listeria monocytogenes with no modification of
HoloConvNet. We believe that our demonstration would facilitate novel approaches at the interface
between quantitative phase imaging and deep learning.
Session Number: 109
Session Number: 109
Session Title: Candida auris: The World is on the Lookout!
Session Start Date Time: 6/8/2018 12:15:00 PM
Abstract Title:
Viability of Candida Auris and Other Candida Species after Three Maldi-Tof Preparation Protocols
A. Bateman, A. Sterkel, A. Valley, D. Warshauer; Wisconsin State Lab. of Hygiene, Madison, WI
Abstract Body:
Background: Candida auris is an emerging, often multidrug-resistant, yeast of public health concern. This
organism can persist in the environment, which complicates efforts to prevent transmission in health
care settings. Commercially-available identification techniques can mis-identify this species. Matrix-
assisted, laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry is one of the most
accurate methods for identifying this species. However, there is a lack of data about C. auris viability
after various preparation protocols for MALDI-TOF testing. Methods: We tested 10 C. auris isolates and
20 isolates of various other Candida species with three different MALDI-TOF preparation protocols: on-
plate extraction with formic acid (extended direct method), CDC 1:1 ethanol:water extraction, and
Bruker tube extraction with formic acid and acetonitrile. Viability was assessed after various steps in the
Bruker tube extraction by removing a small aliquot after each step. After spotting the Bruker MALDI-TOF
target plate in duplicate with each preparation and overlaying with matrix, a flocked swab dampened
with RPMI broth was used to remove the dried spots, and growth in RPMI broth and on Sabouraud
Dextrose plates was assessed. Positive controls consisted of spotting yeast on the MALDI target plate,
followed by removing with a dampened flocked swab and assessing growth. Results: No growth in RPMI
broth or on Sabouraud Dextrose plates was observed after 48 hours with any Candida species treated
with the 1:1 ethanol:water extraction or after any step in the Bruker tube extraction. The extended
direct method greatly decreased the viability; with C. auris, there were no colonies on any of the 10
isolates, and only 5/10 RPMI broths showed growth. With the extended direct method for C. albicans,
every RPMI broth showed growth and colonies were present on Sabourad Dextrose plates, but the
number of colonies was <1% of the positive control. Conclusions: These data indicate that the CDC 1:1
ethanol:water and Bruker tube extraction methods are sufficient to kill Candida species, including C.
auris, prior to MALDI-TOF analysis. This information can help inform risk assessments at clinical and
public health microbiology laboratories performing MALDI-TOF yeast identification.
Session Number: 109
Session Number: 109
Abstract Title:
Rapid, Accurate Identification of Candida Auris by Using A Novel Maldi-Tof Database (Library)
J. Bao1, R. Master1, K. Azad2, D. Schwab2, R. Clark1, R. Jones1, E. Moore1, K. Shier1; 1Quest
Diagnostics, chantilly, VA, 2Quest Diagnostics, San Juan Capistrano, CA
Abstract Body:
Background: The newly emerging multidrug-resistant yeast Candida auris can cause serious infections.
Accurate identification has been hampered by misidentification when using biochemical-based
identification systems and low identification rate when using existing research-use only (RUO) libraries
from matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) platforms. We developed a
novel database, CMdb, to quickly and easily identify C. auris on the Biotyper MALDI-TOF system (Bruker
Baltonic). Methods: Strains from the internationally-collected CDC C. auris panel (10 C. auris and 10
other closely related yeast species) and one in-house clinical C. auris isolate were selected to create the
mean spectrum projections (MSPs) for the new database. A rapid direct on-plate extraction method was
used to create the new database and afterwards to identify the clinical isolates on the biotyper system
(Bruker). The CMdb was evaluated on 23 clinical C. auris isolates, the 20 CDC strains, 52 isolates of 10
other yeast species and 28 isolates of 16 bacterial species. Saboraud Dextrose Agar (SDA) was the
primary media for yeast culture. Results: The new database CMdb was composed of 22 MSPs from 5
yeast species (C. auris and 4 other closely related yeast species, C. haemulonii, C. duobushaemulonii, C.
krusei, and Kodameae ohmeri). When using the CMdb, all the 23 clinical isolates, plus the 10 CDC
strains, of C. auris were correctly identified (100%). The identification log score was consistently greater
than 2.40 with an average of 2.50, In comparison, when using the RUO database, 13 (39%) C. auris
isolates were identified with average log score of 1.76 (p<0.001, R-project.org). The 4 closely related
non-C. auris species were correctly identified by the same CMdb database. No misidentification was
observed when CMdb was used to test other yeasts and bacterial isolates. When counting the
identification per spectrum produced, all the spectra produced from C. auris and the 4 other species had
the correct identification with the CMdb (100%), while 32% of spectra had identifications with Bruker’s
RUO database. Sensitivity tests using YeastOne (Sensititre, OH) for the clinical isolates indicated that all
23 clinical isolates (100%) were highly resistant to fluconazole (>256 µg/mL) and two isolates expressed
multidrug resistance. Conclusions: The identification of C. auris using this novel CMdb is an accurate
(100%), easy to use with an on-plate extraction method and has significantly higher identification log
scores.
Session Number: 109
Session Number: 109
Abstract Title:
Outstanding Abstract Award: Rapid Identification of Routine Clin. Candida Species and Discrimination
between Fluconazole-Resistant and Fluconazole-Susceptible Candida Auris by Reagent-Free Atr-Ftir
Spectroscopy
L. Lam1, P. J. Dufresne2, J. Sedman1, A. A. Ismail1; 1McGill Univ., Ste-Anne-de-Bellevue, QC, Canada,
2Laboratoire de santé publique du Québec, Ste-Anne-de-Bellevue, QC, Canada
Abstract Body:
Candida auris and C. haemulonii are phylogenetically related, leading to frequent clinical
misidentification with routine biochemical identification systems. With Candida auris emerging as a
multidrug-resistant (MDR) yeast, rapid identification is critical for appropriate patient care. Recent
studies have shown that Fourier transform infrared (FTIR) spectroscopy is a promising method for
identifying medically important yeasts, and subspecies-level discrimination has been demonstrated for
several yeast species. When coupled with the attenuated total reflectance (ATR) mode of spectral
acquisition, FTIR spectroscopy is a low-cost, reagent-free technique that provides results within minutes
after initial culture. In previous work by our group, an ATR-FTIR spectral database for use in the
identification of medically important yeasts was developed and validated. In the present study, this
spectral database was expanded to include MDR C. auris (n = 10; 7 are fluconazole resistant), C.
haemulonii (n = 2), and C. duobushaemulonii (n = 3) and was employed to identify daily routine Candida
samples received from local hospitals over a two-month period. Frozen MDR samples, obtained from
the CDC antimicrobial resistance bank, were cultured on Sabouraud dextrose agar (SAB) at 30°C for 48 h
immediately prior to ATR-FTIR spectral acquisition. Daily Candida samples were provided for ATR-FTIR
analysis on SAB after incubation at 35°C for 24 h. ATR-FTIR spectra were acquired in triplicate by direct
transfer of a single isolated colony from the sample culture plate onto the sampling surface of a portable
ATR-FTIR spectrometer; spectral acquisition time was ~1 min/spectrum. Species-level identification of
50 daily routine Candida samples by matching their spectra with those in the previously created spectral
database yielded 94% concordance with the MALDI-TOF MS reference method. Hierarchical cluster
analysis of the spectra of the MDR Candida samples together with the spectra of 9 Candida species in
the spectral database yielded 100% correct classification at the species level, confirming that C. auris
and C. haemulonii can be discriminated by ATR-FTIR spectroscopy. Discrimination between fluconazole-
resistant and fluconazole-susceptible C. auris samples was also achieved by HCA of their spectra. Thus,
in addition to providing a new rapid method for routine clinical identification of yeasts, ATR-FTIR
spectroscopy could contribute to timely detection of life-threatening strains for proper patient care,
diagnosis and treatment.
Session Number: 109
Session Number: 109
Abstract Title:
Characterisation of Candida Auris on Chromid® Candida Agar Medium
L. Dévigne1, A. Foray1, J. Roche2, S. Orenga1, S. Ghirardi1; 1bioMérieux, La Balme Les Grottes, France,
2bioMérieux, Marcy l'Etoile, France
Abstract Body:
Background: Candida auris is an emerging multidrug-resistant fungus that causes a wide range of
infections sometimes associated with high mortality rates. Its early detection allows to take appropriate
infection control measures and to adapt antimicrobial therapy. The aim of this study is to identify the
biochemical profile of C. auris thanks to VITEK®2 YST cards and to characterize C. auris on chromogenic
agar culture media. A second step allows to screen enzymatic substrates which could discriminate
specifically C. auris. Methods: Twenty (20) strains of C. auris and 60 strains form other species of
Candida coming from bioMérieux (bMx) and ATCC collections were characterized by VITEK®2 YST cards
and color of colonies on chromID®Candida (CIDCan) (bMx), CHROMagarTMCandida (CMACan) (Becton
Dickinson) and BrillianceTMCandida (BrilCan) (Oxoid) media. Subsequently, synthetic enzymatic
substrates targeting the most relevant activities were incorporated in variations of the CIDCan medium.
The screening of these substrates was performed with 17 strains of Candida, including 4 C. auris using
calibrated suspensions. Reading was performed after 24 and 48 h incubation. Results: On CIDCan, the
isolated colonies of C. auris became pink after 48 h incubation. Conversely, this species was colorless on
CMACan even at 48 h. On BrilCan, the coloration of colonies was brown since 24 h of incubation. Based
on VITEK®2 biochemical profiles and literature data analysis it appears that pink and brown color of C.
auris colonies for CIDCan and BrilCan, were respectively due to β-glucosidase and phosphatase activities.
Data found in literature and VITEK®2 YST results showed that C. auris is able to hydrolyse D-Raffinose, as
well as few other Candida species. The screening of enzymatic substrates has shown that some
phosphatase enzymatic substrates combined with the β‑glucosidase and hexosaminidase activities
could allow the characterization and pre-identification of C. auris by a specific coloration of colonies.
Conclusions: This study highlights metabolic activities of interest to differentiate C. auris. These are β-
glucosidase, phosphatase activities and D-raffinose hydrolysis. This biochemical profile allowed to
screen some enzymatic substrates and to define interesting combinations of substrates which could
allow the pre-identification of C. auris.
Session Number: 109
Session Number: 109
Abstract Title:
Validation of A Real-Time Pcr Assay for the Detection of Candida Auris In Urine Matrix and Analysis of
Surveillance Samples
L. Leach, H. Schwab, S. Chaturvedi; Wadsworth Ctr., NYSDOH, Albany, NY
Abstract Body:
Background: Candida auris is an emerging multidrug-resistant yeast, which is causing invasive
healthcare-associated infection with multiple fatalities in the New York. Approximately 50% of the
clinical cases in the NY have been identified from blood culture, and other cases were identified from
various body sites including approximately 25% from urine. The precise mechanism leading to presence
of C. auris in urine is currently not clear but possible colonization might pose serious threat of
transmission in the healthcare-facilities. We have recently developed a real-time PCR assay for the
surveillance samples (J. Clin. Microbiol. doi: 10.1128/JCM.01223-17). In the present study, we have
extended this study to urine matrix for rapid identification. Method: A real-time PCR assay targeting the
ITS2 sequence of C. auris was validated in a urine matrix. DNA was extracted using an in-house
developed freeze/heat/bead beating protocol. The assay was first tested using a spiked urine matrix to
determine the assay’s performance. Following validation of the assay, 27 surveillance urine samples
were screened for C. auris. Results: The limit of detection of the real-time PCR for urine samples was 1 C.
auris CFU/PCR reaction. Real-Time PCR yielded positive results from 7 urine samples with 100% and 95%
clinical sensitivity and specificity when compared to culture results. C. auris DNA was detected in one
urine sample with negative culture results indicating the presence of dead or culture defective C. auris.
Conclusion: We report the validation of a real-time PCR assay for the detection of the emerging fungal
pathogen C. auris in a urine matrix. The results indicated that the assay is a powerful tool for the direct
detection of C. auris from urine. It demonstrated high analytical accuracy and precision. The accurate
and rapid screening of C. auris from urine of patients and colonized individuals would allow for effective
control and prevention of this multidrug-resistant deadly fungal pathogen in healthcare-facilities.
Session Number: 117
Session Type: Rapid Fire
Session Number: 117
Session Title: Microbe Trek, The Next GENEration: Boldly Testing as No One has Done Before
Abstract Title:
Outstanding Abstract Award: Application of Next-Generation Sequencing in Identification of Pathogens
During Clin. Infectious Diseases, A Prospective Study
J-W. Ai, P. Cui, Y. Zhu, Y. Qian, Y. Qian, X. Zhou, Y. Ying, W. Zhang; Huashan Hosp. affiliated to Fudan
Univ., Shanghai, China
Abstract Body:
Background: The implementation of the NGS enabled a fast microbiological diagnostic method without
requiring a predefined range of suspicious pathogens [1-2]. This study aims to evaluate the diagnostic
efficacy of NGS during clinical approach to patients with suspected infections. Methods: Patients with
suspected infections were enrolled and high-throughput sequencing was performed on the collected
samples using BGISEQ-100 platform. Data was processed and mapped to the Microbial Genome
Databases after filtering low quality data and human reads. Results: We prospectively enrolled 1048
patients, including 234 suspected blood stream infections, 284 suspected respiratory infections, 312
suspected central nervous infections and 218 focal infections. The overall NGS sensitivity is 46.5%, while
blood stream, respiratory, focal, and central nervous system infection had a sensitivity of
78.72%,89.4%,96.4% and 19.4% respectively. Among which, NGS had a significantly higher sensitivity
than traditional laboratory method (46.5% vs. 32.4%) and the diagnostic sensitivity of CNS infection is
significantly lower than others (P<0.05). When combined with traditional laboratory methods, the
overall diagnostic sensitivity further increased to 54.3%. Among different pathogens, the diagnostic
sensitivity of protozoa and virus were the highest (100%, 11/11), followed by virus (69.5%, 168/242) and
bacteria( 74.29%, 351/473), while fungi had a lowest sensitivity of 33.3% (P value<0.05). Furthermore, in
blood stream infection, antibiotics application was found to associate with a decreased accumulative
sensitivity in both NGS and traditional culture (16.1% vs 15.2%, P>0.05)(Figure 1). Conclusions: This
study highlights the promising potential of NGS in rapid etiological diagnosis during clinical approach of
suspected infectious diseases.<br /><p><a
href="http://files.abstractsonline.com/CTRL/a2/5/a1d/ff0/a19/473/1a2/401/320/1d7/e57/49/g6817_1.
src="http://files.abstractsonline.com/CTRL/a2/5/a1d/ff0/a19/473/1a2/401/320/1d7/e57/49/g6817_1.j
Session Number: 117
Session Number: 117
Abstract Title:
Evaluation of Sample Transportation Solutions and 16s Rrna Gene Regions for Microbial Study
Z. Chen, M. Hui, P. Hui, P. Wong, P. Chan; The Chinese Univ. of Hong Kong, Hong Kong, Hong Kong
Abstract Body:
Background: The amplicon profiling of 16S ribosomal RNA (rRNA) gene is widely used to investigate the
microbial diversity. One of fundamental challenge has been the choice of sample collection methods and
16S rRNA gene primers for profiling accuracy of microbial community. Methods: We compared six DNA
preservation solutions without cold chain for stool sample collection, and compared 16S rRNA gene
regions of V1-V2 and V3-V4 for gut and oral microbial communities. The stool samples in different DNA
preservation solutions were cultured for bacteria growth in aerobic and anaerobic conditions. Results:
Different to the stool samples in PBS and the culture medium, all collections in six DNA preservation
solutions showed high identical microbial communities when compared to the freshly frozen samples,
with three most common taxa of Bacteria (mean abundance of 24%), Faecalibacterium (18%) and
Escherichia-Shigella (14%). The microbial community was inter-individual diversified (R2=0.80, p<0.001)
irrespective of preservations (R2=0.10, p=1.000). Both aerobic and anaerobic cultures indicated four
commercial available preservations could inhibit the growth of bacteria within minutes. When 16S rRNA
gene regions of V1-V2 and V3-V4 were compared, the weighted UniFrac distances showed similar
clustering for stool samples (mantel test 0.9676, p<0.001), oral rinse samples (mantel test 0.9047,
p<0.001), and the head and neck squamous cell carcinoma tissues (mantel test 0.9419, p<0.001).
However, the 16S V1-V2 primer pair failed to amplify Bifidobacteriales due to in part the high rate of
mismatch of 27f primer to bifidobacteria. Conclusions: We provide solutions for stool sample collection
without cold chain and the promising 16S rRNA gene primers. The data may be useful as a guideline to
obtain microbial samples with stable bacteria DNA and reduce PCR-based bias in oral and gut microbial
diversity studies.
Session Number: 117
Session Number: 117
Abstract Title:
Metagenomic Detection of Viral, Bacterial, and Parasitic Infections from Dried Blood Spots
L. M. Filkins1, K. Edes1, M. R. Couturier2, D. T. Leung1, R. Schlaberg2; 1Univ. of Utah, Salt Lake City, UT,
2ARUP Lab., Salt Lake City, UT
Abstract Body:
Background: Febrile illness in travelers and patients in remote areas may be due to diverse pathogens,
often with overlapping clinical presentation, requiring timely diagnoses and pathogen-specific
treatments. Medical care is frequently delayed by days or weeks in this population due to limited access
to quality health care facilities abroad, further challenging infectious disease diagnoses. Next generation
RNA sequencing (RNA-seq) from peripheral blood dried blood spots (DBS) enables hypothesis-free,
broad-spectrum testing for diverse pathogens from a single, low volume sample collected during the
acute stage of illness when sensitivity is generally greatest. We evaluated the analytic performance of
metagenomic RNA-seq for the detection of viral, parasitic, and bacterial infections from DBS.
Materials/Methods: Cultured Zika virus, P. falciparum, and Salmonella enterica were spiked into venous
blood from healthy human donors, serially diluted, spotted on Whatman 903 filter paper, and stored for
up to 10 weeks under dry conditions and room temperature. DBS were disrupted by bead beating and
total RNA extracted using the miRNeasy Micro Kit (Qiagen). cDNA libraries were prepared using KAPA
Stranded RNA-Seq Library Preparation Kit and sequenced on an Illumina NextSeq 500 instrument (5-14
million sequences/sample). Sequences were mapped to pathogen and human reference sequences
using Geneious software (Biomatters). Limit of detection was defined as the pathogen load required for
detection of at least one pathogen-derived read. Results: Equivalent pathogen detection was achieved
from DBS stored in ambient, dry temperatures for 3 days and 10 weeks demonstrating stability of total
RNA in these conditions. Zika virus, P. falciparum, and S. enterica were detected at a wide range of
concentrations from spiked DBS samples using RNA-seq. Preliminary limits of detection were
determined to be 2670 copies/ml for Zika virus, 4-110 copies/ml for P. falciparum, and 30-170 colony-
forming units/ml for S. enterica. Conclusions: DBS can be used for long-term preservation of RNA in dry,
ambient conditions, enabling simple patient self-collection and sample storage while traveling for
metagenomic pathogen detection. The impact of storage in heat and humidity on DBS stability is being
evaluated. These analytic performance studies demonstrate detection of representative parasitic and
viral pathogens at or below expected loads during acute illness. Additional optimization of library
preparation and sequencing may improve pathogen sensitivity.
Session Number: 117
Session Number: 117
Abstract Title:
Development and Evaluation of Ngs Standards for Virome Research
B. Benton, J. Lopera, S. King, D. Mittar, R. Shabman; ATCC, Manassas, VA
Abstract Body:
Compared to the abundance of studies, applications, and publications on the human bacterial
microbiome, there are limited reagents and publications focused on the “virome.” Next-generation
sequencing (NGS) has enabled virus genome sequencing on a large scale at an affordable cost. However,
the complexities involved in the NGS methodology, and the diversity of viral genomes pose a significant
challenge to NGS assay standardization. Therefore, there is a critical need for standardized reference
materials across the research and diagnostics communities to serve as controls in assay development.
To support this need, we have developed a viral panel comprising quantified genomic nucleic acids
prepared from diverse viral RNA and DNA. We included members of the Orthomyxoviridae, Flaviviridae,
Picornaviridae, Reoviridae, and Herpesviridae families, since these families differ in their genomic
organization, envelope properties, and genome size. We normalized input viral nucleic acid copy
numbers by droplet digital PCR to a concentration of 2 x 104 genome copies per virus, and subjected the
panel to library construction and NGS on the MiSeq® (Illumina) sequencing platform. The data from both
the RNA and DNA virus library construction generated approximately 24 million paired-end reads with
only 7-8% virus-specific reads. DNA sequencing demonstrated an abundance of 94.84% of herpesvirus
and RNA sequencing efficiently recovered Flavivirus (80% abundance), Herpesvirus (16% abundance),
and Orthomyxovirus (3.8% abundance). While we normalized input genomic copies by ddPCR,
abundance by NGS varied significantly and Reovirus and Enterovirus were not detected. This finding may
be attributed to the difference in their genome organization, thereby warranting library preparation and
sequencing assay optimization. This proof-of-concept study highlights the need for, and utility of, such
virome standards to allow researchers to optimize their sequencing methods to recover sequences from
diverse viral families.
Session Number: 117
Session Number: 117
Abstract Title:
Ethics in Clin. Metagenomics Data Sharing: High Correlation of Sample Collection Date and Patient
Admission Date in Microbiological Testing
A. Greninger, R. Shean; Univ. of Washington, Seattle, WA
Abstract Body:
Background: Infectious pathogens are known for their rapid evolutionary rates with new mutations
arising over days to weeks. The ability to rapidly recover whole genome sequences and analyze the
spread and evolution of viral pathogens using metagenomics and sample collection dates has lead to
interest in real-time tracking of infectious transmission and outbreaks. However, the level of temporal
resolution afforded by these analyses may conflict with definitions of what constitutes protected health
information (PHI) and privacy requirements for de-identification for publication and sharing of such
data. In the United States, dates and locations associated with patient care that provide greater
resolution than year or the first three digits of the zip code are generally considered patient identifiers;
admission and discharge dates are specifically named as identifiers in Department of Health and Human
Services guidance. Methods: To understand the degree to which one can impute admission dates from
specimen collection dates, we examined sample collection dates and patient admission dates associated
with more than 270,000 unique microbiological results from the University of Washington Laboratory
Medicine Department between 2010 and 2017. Cumulative distribution curves were plotted and
compared using two-sample Kolmogorov-Smirnov tests. Results: Across all positive microbiological tests,
the sample collection date exactly matched the patient admission date in 68.8% of tests. Collection
dates and admission dates were identical from emergency department and outpatient testing 86.7%
and 96.5% of the time, respectively, with more than 99% of tests collected within one day from the
patient admission date. Samples from female patients were significantly more likely to be collected
closer to admission date that those from male patients. Conclusions: We show that PHI-associated dates
such as admission date can confidently be imputed from deposited collection date. We suggest that
publicly depositing microbiological collection dates at greater resolution than the year may not meet
routine Safe Harbor-based requirements of patient de-identification. At the UW Virology lab, we have
made a decision to deposit only the collection year for the more than 700 viral genomes deposited in
NCBI to date. We recommend the use of Expert Determination to determine definitions of PHI for a
given study and/or direct patient consent if clinical laboratories or phylodynamic practitioners desire to
make higher resolution data available.
Session Number: 117
Session Number: 117
Abstract Title:
Silver Nanoparticles Inhibit Virulent Hydrolytic Enzymes Of Staphylococcus aureus and Serratia
Marcescens
J. Vanterpool1, E. Stuffle2, E. Vanterpool2; 1Oakwood Adventist Academy, Huntsville, AL, 2Oakwood
Univ., Huntsville, AL
Abstract Body:
Background: Staphylococcus aureus and Serratia marcescens are opportunistic pathogens that infect
over one million people in the United States each year. Because of superbug development and natural
drug resistance in some microbes, antibiotic treatment is becoming less effective. It is imperative to
identify alternative ways of reducing the virulence potential of these pathogens. Proteases of S. aureus
and Methicillin-Resistant Staphylococcus aureus (MRSA) have been shown to destroy host tissues. Some
proteolytic enzymes include the staphopains (ScpA, SspB), metalloproteases, and serine proteases. S.
marcescens produces proteases such as serralysin that can also contribute to the destruction of host
tissue. Reducing the function of the toxins and/or proteolytic enzymes of these pathogens may be a
potential strategy of reducing pathogenicity. Silver has been used as an antimicrobial agent for years,
but its effect on S. aureus, MRSA and S. marcescens proteolytic enzyme function has not been
evaluated. The hypothesis of this study is that silver ions and/or nanoparticles can inhibit the proteolytic
enzyme function of S. aureus, MRSA and S. marcescens through interfering with the enzymatic
structure. Methods: To test the hypothesis, experimental procedures including synthesis of silver
nanoparticles, spectrometry, and peptidase-, collagenase- and gelatinase assays were done. Results:
Results show that silver ions and nanoparticles can reduce bacterial proteolytic activities by up to 70% in
S. aureus and S. marcescens. Silver ions and nanoparticles show little inhibitory effect on S. aureus or S.
marcescens gelatinase/collagenase activities but are shown to reduce the collagenase/gelatinase activity
levels of MRSA by approximately 40%. Conclusions: In conclusion, the findings of this study support the
hypothesis that silver nanoparticles can inhibit bacterial enzyme function. Silver may be a potential
protease inhibitor treatment option that can help treat and prevent complications caused by these
pathogens.
Session Number: 117
Session Number: 117
Abstract Title:
Fully Integrated Automation of Ngs Library Preparation for Foodborne Pathogens
H. Zhu1, B. Kim1, R. Yasmin1, G. Kastanis2, M. Allard2, R. A. Montagna1; 1Rheonix, Inc., Ithaca, NY, 2Ctr.
for Food Safety & Nutrition/FDA, College Park, MD
Abstract Body:
Background: The identification of foodborne pathogens is important to prevent and track major
outbreaks and to identify sources of contamination in food processing plants. Next Generation
Sequencing (NGS) is a powerful tool to identify such organisms, but considerable effort is required to
isolate DNA and prepare the DNA libraries. To simplify the process, DNA isolation and library prep were
integrated on a single instrument with sequencing performed on MiSeq instruments. Methods: FDA’s
Center for Food Science & Nutrition (CFSAN) provided 12 strains of Salmonella as well as its proficiency
panel (PP) consisting of 4 strains of Salmonella and 2 strains of E. coli O157:H7 used to evaluate the
ability of various labs to correctly identify these pathogens using NGS. Nextera DNA Preparation
reagents were used on the Rheonix Encompass workstation to prepare the NGS libraries. In addition
sequencing results from six strains of Salmonella and six strains of E. coli were evaluated after combining
DNA isolation and library preparation on the same instrument. Results: Purified, well-characterized DNA
was initially used to optimize the library preparation portion of the fully automated process. Sequence-
ready DNA libraries were prepared from these DNA samples on the Encompass workstation within
approximately 3 hours, which included about 30 minutes of hands on time. Sequencing of these libraries
by CFSAN on MiSeq instruments yielded excellent quality metrics. In the case of the 12 strains of
Salmonella received from CFSAN, quality scores exceeded 30 with depth of coverage exceeding 112X.
For the PP samples, the quality scores for both forward and reverse reads exceeded Q30 with the depth
of reads in the range of 30X - 40X. In addition, 95-98% of the reads could be mapped to the reference
genomes. Then, additional studies were performed starting with cultured Salmonella and E. coli
microbes to optimize the combined DNA isolation and library preparation on a single instrument.
Sequencing of these libraries on MiSeq instruments yielded similar results and quality scores.
Conclusions: The ability to reduce the hands-on time and improve quality metrics using automated DNA
isolation and subsequent NGS library preparation will help to reduce costs, improve turn-around time,
and provide consistently reliable data. Automation of the entire process on a single instrument will also
significantly reduce the amount of training required as well as the capital equipment costs and required
laboratory space as compared to manual processes using multiple pieces of equipment.
Session Number: 117
Session Number: 117
Abstract Title:
Comparison of Microbial Dna Enrichment and Extraction Kits Preparatory to Metagenomic Shotgun
Sequencing of Blood
P. Vijayvargiya, K. Greenwood Quaintance, S. Cunningham, M. Thoendel, Z. Esquer, A. Tande, R. Patel;
Mayo Clinic, Rochester, MN
Abstract Body:
Shotgun metagenomic sequencing is an agnostic approach for detection of microbial nucleic acids (NAs)
in clinical specimens. When NAs are extracted from human whole blood, the majority of sequencing
reads are of human origin, even in infected specimens. Human reads can be removed bioinformatically;
however, this requires deep sequencing to find microbial reads, and thereby engenders cost. Here, we
compared four commercial NA extraction and two microbial NA enrichment kits, selected based on their
compatibility with downstream next-generation sequencing, to determine which would provide the
highest yield of microbial NAs from human blood. MolYsis Complete (MC), AllPrep PowerViral DNA/RNA
(AP), ZymoBIOMICS DNA/RNA Mini (ZD) and Qiagen UCP Pathogen (QU) nucleic acid extraction kits were
tested. MC includes a built-in protocol for removal of human NAs. The other three kits were used in
combination with MolYsis Basic (MB) or NEBNext Microbiome DNA Enrichment (NN) kits for removal of
human NAs. Pseudomonas aeruginosa and Candida albicans were spiked together into 15ml EDTA blood
to final concentrations of 105 CFU/ml each. Aliquots of spiked and unspiked blood were stored at -80°C.
NAs were extracted using various combinations of extraction and enrichment kits (table).The quantity of
total extracted NAs was determined by Qubit HS DNA assay. Microbial NAs were quantitated using
quantitative PCR (qPCR), targeting the 16S rRNA gene for P. aeruginosa and ITS for C. albicans. PCR was
performed on a Roche LightCycler™ 1.0. Overall, MC, which provides both DNA extraction and
enrichment, provided the highest yield of microbial NAs of the seven extraction/enrichment protocols
studied for both organism-types (table).<table class="AbstractTable" id="{32D36E96-84F7-4F5B-A413-
8A10E47B764B}"><caption class="AbstractTableCaption"></caption><tr><td rowspan="1"
rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1">Kits used</td><td rowspan="1"
colspan="1">16S rDNA qPCR (crossing point)</td><td rowspan="1" colspan="1">ITS qPCR<br
/>(crossing point)</td><td rowspan="1" colspan="1">Qubit HS DNA amount (ng/mL)</td></tr><tr><td
rowspan="1" colspan="1">MC</td><td rowspan="1" colspan="1">16.76</td><td rowspan="1"
colspan="1">15.47</td><td rowspan="1" colspan="1">1,968</td></tr><tr><td rowspan="1"
colspan="1">AP/MB</td><td rowspan="1" colspan="1">28.75</td><td rowspan="1"
colspan="1">20.25</td><td rowspan="1" colspan="1">Not detected</td></tr><tr><td rowspan="1"
colspan="1">ZD/MC</td><td rowspan="1" colspan="1">27.01</td><td rowspan="1"
colspan="1">16.71</td><td rowspan="1" colspan="1">148</td></tr><tr><td rowspan="1"
colspan="1">QU/MC</td><td rowspan="1" colspan="1">28.93</td><td rowspan="1"
colspan="1">AP/NN</td><td rowspan="1" colspan="1">18.28</td><td rowspan="1"
colspan="1">ZD/NN</td><td rowspan="1" colspan="1">19.57</td><td rowspan="1"
colspan="1">QU/NN</td><td rowspan="1" colspan="1">17.55</td><td rowspan="1"
colspan="1">18.75</td><td rowspan="1" colspan="1">12,580</td></tr></table>
Session Number: 117
Session Number: 117
Abstract Title:
Moderator
Shangxin Yang; TriCore Reference Lab., Albuquerque, NM
Abstract Body:
Session Number: 173
Session Number: 173
Session Title: What's Up With Total Lab Automation?
Abstract Title:
Comparison of Streaking Patterns, Plating Consistency, and Isolation of the Wasp and Inoqula Benchtop
on Blood Agar, Macconkey Agar
M. L. Faron, H. Samra, B. Mesich, B. W. Buchan, N. A. Ledeboer; The Med. Coll. of Wisconsin,
Milwaukee, WI
Abstract Body:
Numerous publications have demonstrated that automatic plating creates more consistent streaking
patterns and isolates more bacteria than manual plating; however, it is unclear what streaking patterns,
systems, and plates are optimal for culture. In this study, we compared the WASP and benchtop InoqulA
for their ability to reproducibly inoculate plates and isolate colonies. To test precision of inoculum,
sterile saline was inoculated at concentrations ranging from 103 to 108 CFU/mL using 4 pathogens. Each
concentration was plated to blood and MacConkey agar, using the WASP 1uL and 10uL loop and a
calibrated pipette at 10uL on the InoqulA in triplicate. Plates were assessed for isolation of >5 colonies,
which is indicative enough organism to complete ID and AST. Urine cultures were then plated in singlet
following the above methodology. A technologist reviewed each plate recording preliminary results.
Significant growth was based on clean-catch urine culture guidelines. Furthermore, two technologists
were blinded to the streaking methods and ranked quality of streaking patterns from 1-3 to evaluate
technologist preference to streaking quality with 3 being the highest. Evaluation of plates for the need
to subculture demonstrated that >5 colonies were accessible for testing for all streaking methods when
plating between 104 -107CFU/mL. At 108 CFU/mL the inoqulA required subculture for 18% of specimens
tested. Out of 149 urine specimens, 8 differences were observed. Of these 8, 5 specimens had the same
pathogens, but workup differed because the organisms were near the significance threshold (10K/mL).
For 2 specimens 50% of the plates reported likely contamination, while the other 50% required workup
based on laboratory policy. The final specimen did not recover gram positive cocci using the WASP 10uL,
likely due to containing >100k of gram negative organisms. Overall head to head comparisons were
concordant in >97% of specimens tested. The need to subculture was similar for full plates ranging from
4-6% of specimens plated. Technologist preferred the WASP 1uL loop with an average score of 2.4
followed by the WASP 10uL loop (2.1), and InoqulA (1.9). These data demonstrated that in most cultures
the automated system will not affect results; however, use of 1uL for plating may improve sensitivity as
all pathogens were detected from this method. Use of 10uL for inoculation may decrease sensitivity and
increase turnaround time as these data demonstrated that over inoculation can reduce isolation and
inhibit detection of other pathogens.
Session Number: 173
Session Number: 173
Abstract Title:
Comparison of Copan Wasp versus Bd Kiestra Inoqula in Isolating Colonies from Positive Urine Culture
Specimens
M. Noorbakhsh1, S. Novak_Weekley2; 1Sutter Hlth.Shared Lab., Livermore, CA, 2Copan Diagnostics,
Inc., Murrieta, CA
Abstract Body:
Background: Automated plating instruments become available in the last decade to reduce labor
associated with specimen inoculation and improve the quality of plating. We used the two most
common instruments on the market today, WASP® (Copan Diagnostics), and BD KiestraTM InoqulA.
Crucial to these instruments is the ability for the plater to produce enough isolated colonies so that
work up off the primary plates can be performed. In this study the ability of each instrument to produce
well isolated colonies (at least 3 or more colonies), so that culture work up could proceed without sub-
culturing and time delay. Methods: Total of 294 turbid urine specimens submitted for routine culture at
Sutter Shared Laboratory were plated on Blood and MacConkey agar plates by the WASP with 1µL loop
on whole plate (WW1), WASP with dual 1µL loop and on bi-plates (WB1) and by the InoqulA pipetting
(KW10). All plates were transferred to the WASPLab™ for incubation in CO2 and images were taken at
18 hours. Images from each set of plates were examined for the quality of the streaking through
different parameters presented in a questionnaire. Results: Out of the 294 urine samples, 211 were
found positive for WB1, 213 for WW1, and 207 for KW10. A concordance of 96% was calculated among
the clinical outcome of the 3 conditions analyzed. WB1 positive samples shown 272 isolates and 234 had
enough colonies for work-up (86%), for WW1 282 isolates were present and 250 had enough colonies
for work-up (89%), for KW10 270 isolates were present and 224 had enough colonies to work-up (83%).
To estimate the difference in colony isolation ability, a score was given (1colony= 1 score, >6 colony= 7
score): no difference was found between WW1 vs KW10 (p= 0.404) and in WB1 vs KW10 (p= 0.402) for
isolates with colony counts between ≥10,000 CFU/mL and <100.000 CFU/mL. A significant difference
was found in samples with a concentration ≥100,000 CFU/mL: the number of isolated colonies was
higher in WW1 in comparison with KW10 (p= 0.001). No statistical difference was found between KW10
and WB1 (p= 0.921). Conclusions: The ability of the instrument to isolate colonies for immediate work
up without subculture can impact when the preliminary or final results are given to the provider, while
poor isolation of colonies results in sub-optimal patient care and delay in turn-around-time. Based on
this study both the WASP and Kiestra InoqulA systems were comparable in terms of isolating colonies in
a positive urine specimen, however WASP performs slightly better (89% versus 83%) for providing more
colonies for further workup.
Session Number: 173
Session Number: 173
Abstract Title:
Validation of Group B Streptococcus Screening Cultures Using Total Lab. Automation
A. Crozier1, W. Lainhart2, C-A. D. Burnham2; 1Barnes-Jewish Hosp., Saint Louis, MO, 2Washington Univ.
Sch. of Med. in St. Louis, Saint Louis, MO
Abstract Body:
Background: Total laboratory automation (TLA) has the potential to standardize culture processing,
incubation, and work up of Group B Streptococcus (GBS) screening cultures. The objective of this study
was to optimize and evaluate the performance characteristics of TLA for GBS screening cultures.
Methods: For initial evaluation of TLA for GBS cultures, various sample inoculation volumes and
streaking patterns were evaluated. Patient specimens (n=67), collected using the BD Eswab, were
cultured using bioMérieux CHROMID® StreptoB agar and LIM broth for both manual and TLA (BD
Kiestra) processing/plating and culture methods. For each paired culture, the following metrics were
evaluated during this process validation: 1) accuracy of specimen label reading, and media selection and
labeling, 2) routing of media to the correct incubators, 3) streaking of the media, 4) imaging of media at
the correct incubation times, and 5) correlation of the results between the methods. To assess
reproducibility, 12 contrived specimens (3 Streptococcus agalactiae, 3 S. pyogenes, 3 S. porcinus, and 3
negative for Streptococcus) were each tested over 3 days. Results: Optimal TLA processing conditions
for GBS screening cultures included the use of 50 µL of specimen inoculated onto the solid (streaked
using pattern 19) and liquid media. The cultures were incubated at 35°C in air. The CHROMID® plate was
imaged at 18 hours. LIM broths of cultures without GBS detected on solid medium were subcultured (50
µL) after 24 hours of incubation using TLA Second Inoculation to a 5% sheep's blood agar plate. This
subculture was incubated at 35°C in 5% CO2 and imaged at 18 and 35 hours. Cultures were read only on
day shift (0700-1530). The TLA system performed well (both patient specimens and reproducibility
cultures) with 100% accuracy of specimen label reading, media selection, media labeling, routing of
media to the correct incubators, streaking of media, and imaging of media at the correct times. Twenty-
four (35.8%) cultures were positive, with 100% positive and negative agreement between the methods.
In reproducibility studies, all 36 cultures had comparable results (positivity and organism abundance).
Conclusions: Optimization of TLA for GBS screening cultures includes batched sample processing. TLA for
GBS screening is accurate compared to conventional methods, improves standardization of specimen
inoculation and reading, and reduces hands-on time for sample processing.
Session Number: 173
Session Number: 173
Abstract Title:
Impact of Lab. Automation on Microbiology Specimen Processing and Culture Reporting in A Large
Integrated Healthcare Organization
N. Z. Khoury1, K. M. Ellyson2, A. R. Borowick2, R. L. Bergsbaken2, C. A. Janita2, D. A. Olson1;
1Hlth.Partners and Park Nicollet Med. Groups, Bloomington, MN, 2Regions Hosp., St. Paul, MN
Abstract Body:
The consolidated microbiology laboratory for the HealthPartners Medical Group, located at Regions
Hospital (RH) in St. Paul, MN, serves more than 55 primary care clinics, 22 urgent care locations, 55
medical and surgical specialties, nursing homes, and 6 hospitals spanning eastern Minnesota and
western Wisconsin. High culture processing volumes (>40,000 cultures per month) and a need for rapid
and efficient specimen processing, drove the microbiology laboratory decision to implement the BD
Kiestra™ Work Cell (WCA) system. This platform enables automated specimen processing, plate
incubation and digital imaging. Automated plating occurs 24/7 and culture interpretation with reporting
occurs from 5AM -11PM, daily. The WCA was validated for urine cultures, beta-cervical cultures, and
MRSA screens in September, 2016. Blood cultures were validated 12 months later (Sept 1, 2017). We
conducted a review of pre- and post-Kiestra go-live to evaluate volume/FTE and turnaround time
efficiencies earned, utilizing the WCA automation line. Volume data (2014-2017) demonstrate an
increase in specimens processed (Figure 1). However, the additional volume did not negatively impact
the critically important positive blood culture turnaround time. Despite off-site blood culture processing
and 4 hour courier delivery routes to RH, the turnaround time to final result for positive blood cultures
remained consistent (range 2-11 days), pre- and post-Kiestra go-live, without accruing significant
overtime and staying under budgeted FTE.<br />
Session Number: 173
Session Number: 173
Abstract Title:
Comparative Evaluation of Puritan and Copan Sputum Solution for Recovery of Respiratory Pathogens
Using Automated Plating
M. George; Albany Med. Ctr., Albany, NY
Abstract Body:
Liquefaction and thorough mixing of sputum specimens ensures a representative sample for culture.
Buffered phosphate solutions of dithiothreitol (DTT) are often used for this purpose. Tubes with DTT
that are adaptable to automated plating systems are now available from Puritan Medical Products (P)
and Copan Diagnostics (C). P (1.0 mL/tube) and C (0.5 mL/tube) sputum solutions (SS) were inoculated
with 100 uL of approx. 107 cfu/mL of ATCC and clinical strains of H. influenza (HI), M. catarrhalis (MC), S.
pneumoniae (SPN), S. pyogenes (SPY), P. aeruginosa (PA), S. aureus (SA), C. albicans (CA), K. pneumoniae
(KP), B. cepacia (BC), and S. maltophilia (SM). Tubes were plated using a BD Kiestra InoqulA delivering 10
uL of sample onto SBA or CHOC with zigzag streaking. Tubes were plated at 0, 2, 4, and 6 h. Changes in
colony counts of 2 log10 or more as compared to time 0 h were considered significant. Sputum samples
were also tested in SS. Using a transfer pipet, 100 uL of sputum was added to P and C SS and vortexed.
Tubes were monitored for liquefaction every 15 minutes. Once liquefied, samples were plated onto SBA,
CHOC and MAC using 30 uL with 4 quadrant streaking. Culture growth was graded 1+, 2+, 3+ and 4+ and
compared to non-liquefied, manually plated cultures. Differences in growth of 2 grades or more were
considered significant. Viability of SPY, PA, SA, CA, KP, BC and SM did not significantly change after
exposure to P and C SS for up to 6 h. However, for HI and SPN there were no viable organisms remaining
at 6 h when exposed to C with significant decreases starting at 2 h for some strains of HI and at 4 h for
some strains of SPN. There were also significant decreases in colony counts for MC exposed to C for 6 h.
For sputum samples, the time required for adequate liquefication ranged from 15 – 45 minutes. C
treated samples remained more viscous than with P after the same exposure time with fibrin clot
equipment errors. P and C treated samples yielded equivalent results as compared to nontreated
manually plated samples and compared to each other. C and P cultures often yielded higher colony
counts as compared to manually plated specimens. P and C SS tubes hold the potential of providing an
efficient mechanism for liquefaction and automated plating of sputum samples. Care must be exercised
with the length of time that samples are exposed to SS especially when using C. Sputum samples treated
with P and C were comparable to each other and to non-liquefied manually plated samples. The lower
volume of SS in C may cause mechanical issues with some automated plating systems due to higher
viscosity as compared to P.
Session Number: 173
Session Number: 173
Abstract Title:
Moderator
Karissa Culbreath; The Univ. of New Mexico, Albuquerque, NM
Abstract Body:
Session Number: 203
Session Number: 203
Session Title: Outbreak Detection in the 21st Century
Abstract Title:
Outbreak of Listeria Monocytogenes in South Africa: Whole-Genome Sequencing Analysis of Isolates
A. M. Smith, K. H. Keddy, N. P. Tau, S. L. Smouse, S. T. Duze, N. Ramalwa, A. Ismail, M. Allam, N.
Govender, K. M. McCarthy, J. Thomas; Natl. Inst. for Communicable Diseases, Sandringham, South
Africa
Abstract Body:
Background: Globally, there is growing concern about the increasing prevalence of Listeria
monocytogenes associated with foodborne outbreaks. Data concerning the prevalence and
epidemiology of L. monocytogenes in South Africa are lacking. A marked increase in listeriosis cases in
South Africa was noted in mid-2017. As of 05 January 2018, a total of 727 laboratory-confirmed clinical
listeriosis cases was documented for 2017. We describe whole-genome sequencing (WGS) data for L.
monocytogenes isolates associated with this outbreak. Methods: Standard microbiological techniques
were used to confirm identification of L. monocytogenes. For WGS analysis, raw sequencing data
generated on Illumina MiSeq equipment were analyzed using tools available in the CLC Genomics
Workbench Software; trimmed reads were assembled using the ‘De novo Assembly Tool’. Assembled
WGS data were analyzed using bioinformatics tools and on-line analysis pipelines available at the Center
for Genomic Epidemiology (CGE), Technical University of Denmark (http://cge.cbs.dtu.dk/services/).
Results: For the 727 laboratory-confirmed clinical listeriosis cases documented throughout the country
in 2017, most were from the Gauteng (61%, 442/727), Western Cape (13%, 92/727) and KwaZulu-Natal
(7%, 51/727) provinces. Isolates were mostly recovered from blood culture (70%, 507/727) and
cerebrospinal fluid (24%, 176/727). WGS data were available for 296 of these clinical (human) isolates;
data were also available for an additional 107 non-human (food/environmental) isolates. Among human
isolates, 15 multi-locus sequence typing (MLST) subtypes (STs) were identified; 89% (264/296) of isolates
belonged to a single ST (ST6). Isolates in this ST6 cluster were highly related, showing <20 single
nucleotide polymorphism differences following phylogenetic analysis. Among non-human isolates, 20
STs were identified; no non-human ST6 isolate has yet been confirmed. Conclusions: The majority (89%)
of the outbreak isolates are highly related and associated with a single MLST subtype ST6, a subtype
associated with unfavourable outcomes in patients with meningitis. This suggests that most cases in this
outbreak were exposed to a single contaminated food or multiple foods contaminated at a single
source. Presently, the source of the outbreak is unknown, so it is uncertain which food/s may be
implicated. Investigation into listeriosis cases will continue with trace back and further investigation of
any Listeria-positive food/environmental samples.
Session Number: 203
Session Number: 203
Abstract Title:
Use of Whole Genome Sequencing During A Multistate Outbreak of Multidrug Resistance
Campylobacter jejuni Linked to Pet Store Puppies
L. A. JOSEPH1, L. FRANCOIS WATKINS1, J. CHEN2, K. TAGG2, C. BENNETT2, H. CAIDI1, J. FOLSTER1, M.
LAUGHLIN1, L. KOSKI3, R. SILVER3, L. STEVENSON4, S. ROBERTSON1, J. PRUCKLER1, M. NICHOLS1, H.
POUSEELE5, H. A. CARLETON1, C. BASLER1, C. FRIEDMAN1, A. GEISSLE
Abstract Body:
Background: Campylobacter jejuni is a leading cause of bacterial foodborne and zoonotic illness in the
United States. Pulsed-field gel electrophoresis (PFGE) has historically been used for Campylobacter
surveillance and outbreak investigation; however, PulseNet is implementing whole genome sequencing
(WGS) for surveillance/outbreak investigation. In 2017, CDC and several state health departments
investigated a multistate outbreak of campylobacteriosis linked to pet store puppies. In this
investigation, we used whole genome multi-locus sequence typing (wgMLST) to link outbreak-associated
human and canine C. jejuni isolates. We also used the WGS data to predict the antimicrobial
susceptibility of these isolates. Methods: We sequenced human and canine C. jejuni isolates using the
Illumina MiSeq. Sequences were analyzed using the Campylobacter wgMLST v.5 allele database in
BioNumerics 7.6, developed in collaboration with domestic and international partners. WGS analysis,
using an in-house antimicrobial resistance workflow (based on ResFinder 3.0), was used to determine
predictive resistance. Antimicrobial susceptibility testing (AST) was performed by the National
Antimicrobial Resistance Monitoring Team using the broth microdilution method. We performed PFGE
on a subset of C. jejuni isolates using the PulseNet Campylobacter protocol. PFGE data were analyzed in
BioNumerics 6.6.10 and named using PulseNet nomenclature guidelines. Results: The wgMLST analysis
differentiated epidemiologically-linked C. jejuni isolates from PFGE-indistinguishable sporadic isolates
and separated the outbreak isolates into three clades (59-282 alleles different). Clade 1 contained four
clinical isolates from four states (4–30 alleles different). Clade 2 contained 11 clinical isolates from six
states (1–21 alleles different). Clade 3 contained 18 clinical isolates from 11 states and nine canine
isolates from two states (0–32 alleles different). Five PFGE patterns were produced by isolates in clades
two and three. All isolates were multidrug resistant using WGS data which was confirmed by AST.
Conclusions: Our study showed that wgMLST analysis provided greater resolution and epidemiological
concordance compared with PFGE during this outbreak investigation. Predicted resistance results were
comparable to AST and were available in real-time. This study demonstrated the power of WGS data for
linking outbreak-associated C. jejuni isolates and determining the antimicrobial resistance profiles of
these isolates in a single workflow.
Session Number: 203
Session Number: 203
Abstract Title:
The Worlds Largest Waterborne Campylobacteriosis Outbreak
B. Gilpin; Insitute of Environmental Sci. & Res., Christchurch, New Zealand
Abstract Body:
Background: It is estimated that over 5500 cases of campylobacteriosis occurred in Havelock North, New
Zealand in August 2016 as a consequence of the consumption of untreated drinking water contaminated
with sheep faeces. Methods: Multiplex Binary Typing (MBiT) and whole genome sequencing (WGS) was
undertaken on Campylobacter jejuni isolates from 284 notified cases of campylobacteriosis from the
outbreak region. Genotypes were compared with isolates from reticulated water, the supply bore and
sheep faeces. Results: 208 cases with at least 10 different genotypes of C. jejuni were linked to the
outbreak (Figure 1). MBiT analysis confirmed a link between cases and the water 1 day after receipt of
primary isolation plates, could be performed on non-viable isolates, and allowed screening of almost
500 isolates, of which WGS was performed on a subset. WGS was completed on the first set of isolates
within 4 days of isolation. Comparisons between the two methods suggested that MBiT genotypes
differing by 1 or 2 alleles should be considered as potentially indistinguishable. WGS using multilocus
sequence typing (MLST) or SNP analysis produced equivalent results. Six of the genotypes had less than
2 wgMLST differences from isolated recovered from the water and/or sheep faeces confirming the
source of contamination. Conclusions: Combined use of epidemiology and genotyping was better able to
define the exposure period, with genotyping helping exclude cases with other causes of
campylobacteriosis. There was no evidence of significant secondary transmission beyond initial outbreak
period. Genotyping of campylobacter isolates was essential for refining case definition, confirming
exposure period and connecting cases with reticulated water and with the contamination sources.
Investigating and genotyping geographically separated cases with limited exposure periods contributes
strongly to determination of exposure period and sources.<br /><p><a
href="http://files.abstractsonline.com/CTRL/6b/f/447/fbb/d22/4c4/5a3/678/e2f/a36/d9d/bc/g7010_1.j
pg" target='_blank' address=no ><img
src="http://files.abstractsonline.com/CTRL/6b/f/447/fbb/d22/4c4/5a3/678/e2f/a36/d9d/bc/g7010_1.jp
g" alt="" border="0" width="600" height="392" /></a></p>
Session Number: 203
Session Number: 203
Abstract Title:
Identification of Clostridium Botulinum B1 Okra Isolates from A Foodborne Outbreak in China, 2016
Y. Lin1, Y. Jiang1, Z. Gong2, Y. Wang3, Y. Qiu1, M. Jiang1, Q. Chen1, Q. Hu1, X. Shi1; 1Shenzhen Ctr. for
Disease Control and Prevention, Shenzhen, China, 2Southern Med. Univ., Coll. of Publ. Hlth., Guangzhou,
China, 3The Univ. of Shenzhen-Hongkong Hosp.
Abstract Body:
Background: Botulinum neurotoxins (BoNTs) of seven serotypes from A to G is the reason for genetically
diverse of C. Botulinum. Among the species, including A, B, E and F subtypes are prevalently identified
worldwide which cause deadly diseases to the infected. The infection of C. botulinum is commonly
related to foodborne poisoning, with the typical symptoms of dysphagia, dysarthria, ophthalmoplegia
and weakness of limbs. In the study, we review a food poisoning of Clostridium botulinum in Shenzhen,
China. Two poisoning patients involved in the incident had recovered and discharged for the reason of
timely treatment and accurate detection. Methods: In the processing of detection, serum, enema and
suspected food samples were assayed for botulinum neurotoxins by the methods of Detection Kit for
Botulinum A/B/E, detection of morphology, real-time PCR. Enema and suspected food samples were
cultured and isolated for C. botulinum. Mouse bioassay had also been done for detecting enema
samples. Botulinum neurotoxins were identified by specific antitoxins of BoNTs/A, B, E. MALDI-TOF MS
detection was used for confirming, while Gene sequencing technique was used for typing. Results: Cases
of patient’s enema specimens were confirmed for the existence of C. botulinum type B by the methods
of real-time PCR, mouse bioassay and MALDI-TOF MS. The virulence of neurotoxins from the food
poisoning was approximately estimated to 10000 MLD/ml-100000 MLD/ml. Based on phylogenetic
analysis, the isolation of C. botulinum was typed into C. botulinum B1 okra which is far different from C.
botulinum E3 str. Alaska identified in Shenzhen, 2015. Conclusions: It is confirmed that the isolates of C.
botulinum from the food poisoning in Shenzhen, China was typed into C. botulinum B1 okra, with the
virulence of neurotoxins estimated to 10000 MLD/ml-100000 MLD/ml.
Session Number: 218
Session Number: 218
Session Title: SATURDAY - CPHM Late-breakers
Poster Board Number: SATURDAY - CPHM LB1
Abstract Title:
Seroepidemiologic Survey on Severe Fever with Thrombocytopenia Syndrome in Farmers on the Jeju
Island
S. Heo, J. Yoo; Jeju Natl. Univ. Hosp., Jeju-si, Korea, Republic of
Abstract Body:
Background: Severe fever with thrombocytopenia syndrome (SFTS) is an endemic zoonotic disease, its
overall mortality was about 22.0% between 2013 and 2016 in South Korea. The incidence of SFTS was
increasing annually, its incidence of Jeju Island was 8.66/100,000 person (1.19/100,000 people in
mainland) in South Korea. The present study was designed to determine the seroprevalence of SFTS
virus (SFTSV) in the healthy population on Jeju Island. Methods: We conducted the cross-sectional study
for antibody to SFTSV using enzyme-linked immunosorbent assays in the healthy agricultural and
livestock population between 2015 and 2017 in Jeju Island in South Korea. We collected a data with
sociodemographic variables, medical history, type of occupation, and history of tick bite. Results: A total
254 person were included during the study periods in the total rural 16 area. Total 421 samples
subjected to ELISA for SFTSV, each sample was collected the 142 samples in 2015, 170 samples in 2016,
and 109 samples in 2017. 72 persons consecutibly collected during 3 years. The mean age 59.9 years
(range, 27-84), and 68.9% were male. The distribution of occupation was as follows: grain, 13 samples;
vegetable 120 samples; fruits, 125 samples; livestock, 27 samples; others, 8 samples (Figure 1.
geographic distribution of 421 samples). The overall seroprevalence of SFTSV-IgG was 2.4% in examined
specific population. Six patients and eight samples were positive for SFTSV-IgG. SFTSV-IgG was
associated with type of occupation variable, it was a fruit farmer. They have no a history of tick bite and
contact for SFTS patient. Conclusions: This result suggests that SFTSV has circulated on Jeju Island, a few
proportion of infected individuals develop disease. The incidence of SFTS would be much more than we
expected in Jeju Island. Further study would be needed a study for correlation between ecology of
vectors and prevalence of SFTSV IgG of fruit growing farmers.
Session Number: 218
Session Number: 218
Abstract Title:
Development of a Scoring System to Target Molecular Diagnostic Tests for Skin and Soft Tissue
Infections
K. Balasubramanian1, L. Ryckebusch1, Y. Li1, R. B. Schechter2, T. A. Moreno1, A. Del Tredici1;
1CogenDx, San Diego, CA, 2Palomar Hlth.Wound Care Ctr., San Marcos, CA
Abstract Body:
Skin and soft tissue infections (SSTIs) are common and can lead to complications such as amputation and
increased mortality. Successful treatment of SSTIs is dependent on timely pathogen identification and
initiation of appropriate antimicrobial therapy. Molecular diagnostics can aid in the therapeutic process
due to their capacity for rapid and sensitive detection of multiple targets. We developed a
pathogenicity-based scoring system to identify microbial targets for a PCR-based SSTI diagnostic tool.
We curated public health documents, clinical and microbiology practice guidelines, epidemiological
surveys and published clinical studies to develop the scoring system. Pathogenicity was ranked on a
scale of 1 to 5, where 1 is high priority for inclusion and 5 is low priority. Organisms were assigned the
highest applicable score. See Table. To evaluate the scoring system, we retrospectively analyzed culture
reports of wound specimens from 388 patients treated at two outpatient wound care clinics from
December 2016 to February 2018 and assessed the frequency of the curated organisms. We identified
and curated 70 bacterial and fungal species as candidates for inclusion in the panel. Organisms with
pathogenicity scores of 1 included Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus
agalactiae and Enterococcus faecalis. Analysis of 445 speciated culture results identified these bacteria
as the most frequently detected pathogens, accounting for 35%, 11%, 7.4% and 6.5% of the results
respectively. In contrast, Clostridium perfringens, which was assigned a score of 2, was only detected
once in culture, demonstrating that the pathogenicity score can aid in the curation of fastidious and less
frequently detected organisms. By applying the scoring system, we prioritized over 40 organisms for
inclusion in the panel, which accounted for 84% of the organisms detected in the culture reports. Using
a scoring system, we ranked microorganisms that are frequently reported in culture reports for SSTIs
and selected candidates for a PCR-based SSTI panel. Continued application of this scoring system will aid
in the targeting of PCR-based diagnostics of microorganisms, an accurate and precise method that may
have clinical utility for the management of SSTIs.<br /><table border="1" cellpadding="1"
class="DisplayTable" id="{8F5C5401-2E3D-4E33-ADC1-39DF859A7031}"><caption>Pathogenicity Score
Criteria</caption><tr><td rowspan="1" colspan="1">Score</td><td rowspan="1" colspan="1">SSTI
Pathogenicity</td><td rowspan="1" colspan="1">Number of Organisms</td></tr><tr><td rowspan="1"
colspan="1">1</td><td rowspan="1" colspan="1">WHO1, IDSA2 or CDC3 Public Health Threat</td><td
rowspan="1" colspan="1">21</td></tr><tr><td rowspan="1" colspan="1">2</td><td rowspan="1"
colspan="1">CDC High Virulence SSTI4</td><td rowspan="1" colspan="1">3</td></tr><tr><td
rowspan="1" colspan="1">3</td><td rowspan="1" colspan="1">ASM Microbiology Lab SSTI
SOP5</td><td rowspan="1" colspan="1">35</td></tr><tr><td rowspan="1" colspan="1">4</td><td
rowspan="1" colspan="1">Other SSTI Pathogens</td><td rowspan="1"
colspan="1">11</td></tr><tr><td rowspan="1" colspan="1">5</td><td rowspan="1" colspan="1">Not
of Major Importance</td><td rowspan="1" colspan="1">0</td></tr></table>
Session Number: 218
Session Number: 218
Abstract Title:
Application of Phage Display to Identify Chemotherapeutic Treated Colon Cancer
D. McAtee1, D. Bedi2, K. Vig3; 1Southern Univ. Shreveport Louisiana, Shrevport, LA, 2Tuskegee Univ.,
Tuskegee, AL, 3Alabama State Univ., Montgomery, AL
Abstract Body:
Colon Cancer (CC) is a carcinoma which originates in the cells of the large intestine, and often
metastasizes to the liver. Currently, the therapeutic treatments available are not capable of specifically
identifying malignant cells and often are cytotoxic to healthy benign tissue. Bacteriophages (BPs), or
“phage”, are nanosized viruses which specifically bind to and infect bacterium and can only proliferate
inside the host cell. The same mechanism of targeting and rapidly infecting the host cell is being
genetically manipulated and bio-medically applied in nanomedicine to CC cells for diagnostic and
therapeutic approaches. In the present study, 16 BPs were tested for their specificity binding to SW620
cells using an Enzyme Linked Immunosorbent Assay (ELISA). The in vitro assay was conducted using
SW620 cells showing ≥90% viability. Cells were treated for 24-hours with Camptothecin (CPT), a
chemotherapeutic treatment that inhibits DNA topoisomerase I for 24-hours followed by washing with
washing buffer. ELISA was performed using M13 antibody to determine its affinity to phage. The
diagnostic technique showed the 3 phages L2, E2, and L47 had the highest selectively, identifying and
binding to CPT treated SW620 cells. To determine the concentration of selected phage binding to
SW620 cells, a phage titration assay was conducted on L2, E2, and L47 phages. Further studies are being
carried out to demonstrate that these phages can be used as theranostic biomarkers or antineoplastic
on targeted SW620 cells.
Session Number: 218
Session Number: 218
Abstract Title:
Initial Optimization of Metagenomic Next-Generation Sequencing from Cerebral Spinal Fluid
H. B. Miller1, P. Thielen2, S. Salzberg3, F. Brietwieser3, K. C. Carroll1, P. J. Simner1; 1Johns Hopkins Sch.
of Med., Baltimore, MD, 2Applied Physics Lab, Johns Hopkins Univ., Baltimore, MD, 3Ctr. for
Computational Biology, Johns Hopkins Sch. of Med., B
Abstract Body:
Introduction: Numerous reports show the potential of metagenomic next-generation sequencing
(mNGS) to detect pathogens in clinical samples, but the complexity of testing, lack of guidelines and cost
creates significant hurdles to the implementation in clinical microbiology labs. The purpose of this study
was to optimize a mNGS assay using CSF. Methods: Optimization was performed by spiking pooled,
pathogen-negative, remnant CSF with various microbes (bacteria, viruses, fungi and AFB). Both DNASeq
and RNASeq were performed on the Illumina MiSeq using Nextera XT library preparation chemistry. We
evaluated multiple methods for each aspect of testing including: 1. Sample processing (storage
conditions, bead-beating [BB], host depletion [HD] methods, type of extraction (manual vs automated),
whole transcriptome/whole genome amplification [WTA/WGA] and the use of internal controls [IC]); 2.
Library preparation (nucleic acid [NA] inputs and DNA fragment size-selection); 3. NGS (sequencing cycle
number [150, 600], sequencing depth/multiplexing);4. Bioinformatics (online software tools [Illumina
BaseSpace Apps] vs manual pipeline with curated database [Kraken, Pavian]). Results/Discussion: 1.We
found sample storage conditions affected RNA virus detection. BB increased detection of hard to lyse
microbes (fungi, mycobacteria), but negatively impacted gram-negative bacteria and some viruses.
Saponin/DNase provided the best balance between HD/microbial DNA preservation. ICs resulted in
confounding E. coli reads, but allowed assessment of analytical sensitivity. The manual magnetic bead
extraction kit provided the best downstream detection rates. 2: cDNA from WTA and DNA with WGA run
together provide best coverage and help address amplification bias between methods. Normalization
method based on DNA fragment size and sample concentration led to a 10X increase in reads/flow cell.
3: For NGS, the best balance (TAT, carryover) is a 150 cycle flow cell with minimal multiplexing. 4: Online
tools are easier to learn and require fewer resources, however in-house bioinformatics pipelines provide
more in-depth analysis and allow for curated/tailored databases. Conclusions: Each step in an NGS
workflow introduces bias and has the potential to improve pathogen recovery. Careful assessment of
each step of mNGS has allowed us to validate approaches for detection of diverse pathogens from CSF.
Session Number: 218
Session Number: 218
Abstract Title:
Detection of Yersinia enterocolitica in Stool Specimens by Multiplex PCR: False Positive or Viable
Pathogen?
L. Westblade1, R. Marrero2, A. Brudie2, K. Callan2, A. Robertson2, M. La Spina2, A. Chen2, A. Low2, J.
Hargrave2, S. Jenkins1; 1Weill Cornell Med., New York, NY, 2New York-Presbyterian Hosp.-Weill Cornell
Med. Ctr., New York, NY
Abstract Body:
Background: Molecular assays for diagnosis of gastrointestinal (GI) infections present new challenges
and opportunities: challenges in recovery of detected organisms for subsequent testing; opportunities
for greater understanding of the prevalence and pathobiology of these agents. Since implementation of
the FilmArray® GI panel (BioFire Diagnostics, LLC) at our Medical Center in May 2016, we have detected
Yersinia enterocolitica (Yent), a foodborne pathogen, in 141 stool specimens. Prior to adoption of the GI
panel, Yent was recovered once between Jan 2011 - Apr 2016 in stool culture. Thus, we were unsure if
this increase in positivity reflected detection of Yent or was due to false positive GI panel results.
Therefore, our aim was to validate GI panel Yent positive results using conventional and cold enrichment
culture methods. Methods: Twenty stool specimens collected between Sept 2017 and Jan 2018 in Para-
Pak C&S media (PAK, Meridian Bioscience, Inc) positive for Yent using the GI panel were simultaneously
evaluated using conventional culture and cold enrichment. Conventional culture involved inoculating
~50 µL of PAK preserved stool onto MacConkey (MAC) and Cefsulodin-Irgasan-Novobiocin (CIN) agars
(Becton, Dickinson and Company [BD]) and incubating at 25°C in ambient air for up to 48 h. For cold
enrichment, 1 mL of PAK preserved stool was transferred to phosphate buffered saline (PBS, BD) and
incubated at 4°C for up to 21 days. At defined points (3, 7, 14, and 21 days), ~50 µL aliquots of PBS were
subcultured onto MAC and CIN agars and incubated at 25°C in ambient air for up to 48 h. Isolates were
identified using MALDI-TOF MS (Bruker Daltonics, Inc). Results: Yent was isolated from 45% (9/20) of
specimens using conventional culture. An additional 7 isolates were recovered after cold enrichment,
resulting in an overall recovery of 80% (16/20) and an increase in recovery of 43.8% (7/16) compared to
conventional culture. Upon inspection of cold enrichment data, 4 isolates were recovered after 3 days, 2
after 7 days, and 1 after 14 days. Isolates were further identified at our Public Health Laboratory: 14
identified as Yent biogroup 1A, 1 as Yent biogroup 4, and 1 as Yersinia frederiksenii. Conclusion:
Increased detection of Yent by a molecular assay in our Medical Center is the result of detection of Yent
rather than false positive results. Cold enrichment permitted the recovery of additional isolates
compared to conventional culture, supporting the use of this tool for recovery of Yent for subsequent
analyses.
Session Number: 218
Session Number: 218
Abstract Title:
Evaluation of FASTinov® Kit for Antimicrobial Susceptibility Testing on Gram-Negative Bacilli Directly
from Urine
J. Oliveira1, S. Costa-de-Oliveira2, R. Teixeira-Santos2, A. Silva-Dias2, I. Martins-Oliveira2, A. Rodrigues1,
C. Pina-Vaz2; 1Faculty of Med., Univ. of Porto, Porto, Portugal, 2FASTinov, SA, Porto, Portugal
Abstract Body:
Background: Urinary tract infections are the most common infections in both hospitalized and
community patients. The emergence of resistant bacteria is aggravated by incorrect or inadequate use
of antimicrobial drugs. Quicker antimicrobial susceptibility tests are urgently need. FASTinov® kits allow
the determination of antimicrobial susceptibility profile directly from biological products (TTR 2h). The
present study intends to evaluate the performance of FASTinov® gramneg kit for AST on gram-negative
bacilli directly from urine. Methods: A total of 8 quality control strains (E.coli ATCC 35218, ATCC 25922,
ATCC 8739; K. pneumomia BAA 1705, BAA 1706, ATCC 700603 and OXA-48 and E. aerogenes ATCC
13048) were included in this study. Those selected bacteria were inoculated into 5 mL Muller-Hinton
Ca2+ broth and incubated overnight (37°C; shaking at 130 rpm). Urine from heathy donors was collected
and filtered (0.45 µM pore size). Cell culture was diluted 1:40 to a total of 20 mL urine and incubated for
±120 min. Bacteria was extracted from urine according to IFU, inoculated in a FASTinov® gramneg kit
and incubated for 1 h. Ampicillin, amoxicillin-clavulanic acid, cefotaxime, ceftazidime, cefoxitin,
ceftalozane-tazobactam, piperacillin-tazobactam, ciprofloxacin, gentamicin, amikacin, meropenem,
imipenem and colistin were studied; additionally screenings for detection of enzymatic mechanisms of
resistance was provided; afterwards, cells were analyzed BD AccuriTM C6 Flow Cytometer. EUCAST and
CLSI protocols were selected on the FASTinov using a dedicated software. Results obtained from FC and
microdilution broth reference method were compared. All experiments were performed twice. Results:
The overall agreement between FASTinov® gramneg kit and broth microdilution reference method was
96% for both EUCAST and CLSI protocols. One major discrepancy was detected for gentamicin and
ciprofloxacin, and one minor discrepancy was observed for amikacin and imipenem; there were no very
major discrepancies. Regarding the screening for detection of enzymatic resistance mechanisms, ESBL
and carbapenemases enzymes were correctly detected. Excellent reproducibility was obtained.
Conclusions: We hereby presented a fast flow cytometric protocol which revealed to be an excellent
tool for AST on gram-negative bacilli directly from positive urine in a time-to-result of 2 hours versus 48h
of the routine method, with high agreement with broth microdilution reference method.
Session Number: 218
Session Number: 218
Abstract Title:
Two-Site Evaluation of the Colistin Broth Disk-Elution Screen to Determine In Vitro Activity of Colistin
P. J. Simner1, Y. Bergman1, M. Trejo2, A. A. Roberts1, R. Marayan1, T. Tekle1, S. Campeau3, P. D.
Tamma1, J. Hindler2, R. Humphries3; 1Johns Hopkins Univ. Sch. of Medine, Baltimore, MD, 2Univ. of
California, Los Angeles, Los Angeles, CA, 3Accelerate Diag
Abstract Body:
Background: Colistin antimicrobial susceptibility testing (AST) can only be performed by reference broth
microdilution (rBMD), a method few laboratories have access to. A practical method for clinical
microbiology labs to identify colistin minimum inhibitory concentrations (MICs) is essential; especially
with the increasing prevalence of infections caused by multidrug-resistant gram-negative bacilli (GNB).
The purpose of this study was to evaluate the colistin broth disk-elution (CBDE) test to perform colistin
AST. Methods: CBDE was compared to colistin rBMD for a collection of GNB at two laboratories. Isolates
tested included 121 retrospective clinical isolates, 45 prospective clinical isolates, and 7 mcr-1 positive
Escherichia coli. CBDE is performed with 4, 10 ml cation-adjusted Muller Hinton broth (Remel) tubes per
isolate, to which 0, 1, 2, and 4 colistin disks (10 µg; BD) are added, generating a final concentration of 0
(growth control), 1, 2 and 4 µg/mL. Tubes are incubated at room temperature for 30 min, after which a
50 µL aliquot of a 0.5 McFarland inoculum suspension is added. Colistin MIC values are read visually,
after 18-20 h incubation at 35C. MICs were interpreted using CLSI breakpoints.<br />Results:<br />Table
1: Summary of CBDE Results Compared to rBMD<table class="AbstractTable" id="{F5DA4E5C-23B9-
404D-89CE-8B9753C1BAD8}"><caption class="AbstractTableCaption"></caption><tr><td rowspan="1"
rowspan="1" colspan="1">Isolates </td><td rowspan="1" colspan="1">N</td><td rowspan="1"
colspan="2">BMD Results</td><td rowspan="1" colspan="1">CA<br />(%)</td><td rowspan="1"
colspan="1">EA<br />(%)</td><td rowspan="1" colspan="1">VME<br />(%)</td><td rowspan="1"
colspan="1">ME<br />(%)</td></tr><tr><td rowspan="1" colspan="1"></td><td rowspan="1"
colspan="1"></td><td rowspan="1" colspan="1">S or WT</td><td rowspan="1" colspan="1">R or
NWT</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1"
colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1">Site
1</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1"
colspan="1">A. baumannii</td><td rowspan="1" colspan="1">12</td><td rowspan="1"
colspan="1">0</td></tr><tr><td rowspan="1" colspan="1">P. aeruginosa</td><td rowspan="1"
colspan="1">Enterobacteriaceae</td><td rowspan="1" colspan="1">24</td><td rowspan="1"
colspan="1">0</td></tr><tr><td rowspan="1" colspan="1">Site 2</td><td rowspan="1"
colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1"
colspan="1">Retrospective CRE</td><td rowspan="1" colspan="1">65</td><td rowspan="1"
colspan="1">0</td></tr><tr><td rowspan="1" colspan="1">A. baumannii</td><td rowspan="1"
colspan="1">0</td><td rowspan="1" colspan="1">0</td></tr><tr><td rowspan="1" colspan="1">P.
aeruginosa</td><td rowspan="1" colspan="1">14</td><td rowspan="1" colspan="1">14</td><td
colspan="1">97a</td><td rowspan="1" colspan="1">0</td><td rowspan="1"
colspan="1">0</td></tr><tr><td rowspan="1" colspan="1">Enterobacteriaceae</td><td rowspan="1"
colspan="1">0</td><td rowspan="1" colspan="1">0</td></tr><tr><td rowspan="1" colspan="1">Both
Sites</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1"
colspan="1">mcr-1 E. colib </td><td rowspan="1" colspan="1">7</td><td rowspan="1"
colspan="1">0</td></tr><tr><td rowspan="1" colspan="1">Overall</td><td rowspan="1"
colspan="1">GNB</td><td rowspan="1" colspan="1">173</td><td rowspan="1"
colspan="1">0</td></tr></table><br />S: Susceptible, R: Resistant, WT: wild-type, NWT: non-wild-type;
CA: categorical agreement; EA: essential agreement; VME: very major error; ME: major error<br />a 1 P.
aeruginosa with a MIC of 0.5 µg/mL had an MIC of 2 µg/mL by CBDE<br />b 3 mcr-1 E. coli had MICs of 2
µg/mL by CBDE and 4 µg/mL by BMD. These results were reproduced at the 2 sites.<br />Conclusions:
CBDE has CA and EA of 98% and 99% compared to colistin rBMD. CBDE is an easy and practical method
to perform colistin AST. Mcr-1 isolates yielded MICs of 2 ug/mL by CBDE and 4ug/mL by rBMD; as such
isolates with colistin MICs of 2 µg/mL should be confirmed by rBMD and those with MIC ≥ 2 µg/mL
should be tested for mcr genes.
Session Number: 218
Session Number: 218
Abstract Title:
Alternative Faster MALDI-TOF Based Procedure to Detect KPC-2 Carbapenemase Directly in Clinical
Isolates
R. Figueroa1, D. Cejas1, V. Rumi2, C. Barberis3, C. Vay3, G. Gutkind1, J. Di Conza1; 1Facultad de
Farmacia y Bioquímica, Buenos Aires, Argentina, 2Facultad de Veterinaria, Buenos Aires, Argentina,
3Facultad de Farmacia y Bioquímica, Hosp. de Clínicas, Bu
Abstract Body:
Background: Fast and accurate detection of carbapenemases is critical in clinical microbiology. The KPC
is the most prevalent class A carbapenemase in Enterobacterales, worldwide. Rapid methods for KPC
detection are needed for guiding antibiotic therapy as early as possible and prevent further
dissemination of this resistance marker. MALDI-TOF mass spectrometry (MS) is currently used for
species identification. ß-lactamases detection, using the analysis of ß-lactam hydrolysis products, by
MALDI-TOF is reliable but time and labour consuming. Detection of KPC has also been inferred by
MALDI-TOF after detection of an accompanying protein in KPC-producing microorganisms. The aim of
this work was to detect KPC-2 in different enterobacterial isolates applying an easy to perform, fast and
direct MALDI-TOF MS procedure. Materials/Methods: 117 clinical isolates of K. pneumoniae (65), S.
marcescens (24), E. coli (20) and E. cloacae (8) were included in this study. Susceptibility tests were
performed according to CLSI. Isolates were analyzed by PCR and grouped according to the presence of ß-
lactamases genes. Detection of KPC-2 ß-lactamase was carried out by MALDI-TOF (Bruker) after protein
extraction with organic solvents. MALDI-TOF spectra were recorded by MALDI Biotyper system and
analyzed using ClinProTool software (Bruker). A KPC-2 producing E. coli XL1 blue transformant was used
as a positive control. Those isolates negative for blaKPC (ESBL producers or susceptible strains) and E.
coli XL1 blue were included as negative controls. Results: Forty one clinical strains (33 K. pneumoniae, 4
S. marcescens, 3 E. coli and 1 E. cloacae) were categorized as KPC-2 producers. A distinct peak at m/z
~28,477 Da, in agreement with the theoretical MW of the mature protein of KPC-2 was detected in the
E. coli XL1 transformant when the instrument operated at a mass range of 17,000 to 50,000 Da. This
distinctive peak was present in all KPC-2 producing clinical isolates spectra, and consistently absent in
the negative control group (n = 76). Conclusions: Even if our results are preliminary, we here
demonstrate that MALDI-TOF MS has the potential to detect the most clinically relevant acquired class A
carbapenemase, the KPC-2-enzyme. To the best of our knowledge, this is the first report where mature
KPC-2 enzyme was detected by MALDI-TOF in a timely way, substantially less than that needed for ß-
lactam hydrolysis. This procedure shows an improvement in the lab abilities due to an easy, fast and
direct detection of KPC-2 with extensive clinical implications.
Session Number: 218
Session Number: 218
Abstract Title:
Evaluation of T2 Magnetic Resonance Candida auris Panel as a Rapid Diagnostic for this Emerging
Multidrug Resistant Yeast in Clinical Skin-Swab Samples
J. Sexton1, M. Bentz1, R. Welsh1, B. Manning2, R. Shivers3, A. Litvintseva1; 1Ctr. for Disease Control,
Atlanta, GA, 2T2 Biosystems, Lexington, MA, 3T2 Biosystems, Atlanta, GA
Abstract Body:
Background: Candida auris is a multidrug-resistant pathogenic yeast whose recent emergence is of
increasing public-health concern. C. auris is capable of colonizing patients’ skin and can survive for
weeks in the healthcare environment, facilitating patient-to-patient transmission and fueling healthcare-
associated outbreaks. Rapid and accurate detection of C. auris on skin swabs of asymptomatically
colonized patients is essential for implementation of infection control measures to prevent continued
spread within healthcare facilities. Currently, skin swabs are processed using a culture-based method
that is highly sensitive and specific, however, testing requires up to 14 days. This delay prevents timely
isolation and decontamination of patients and highlights the need for an alternative method for species
identification from patient samples. Methods: The culture-independent T2 Magnetic Resonance (T2MR)
system is a rapid diagnostic platform shown to detect target pathogens of interest from unprocessed
blood samples in <5 hours. In this study we evaluated the performance of a new C. auris-specific T2
assay in the unique context of patient skin swabs. The evaluation of the C. auris panel was performed in
two stages, first using contrived samples prepared in a representative skin swab matrix, and second in a
direct comparison of clinical patient samples. To create a matrix representative of a typical skin swab, 40
patient skin swabs previously demonstrated to be negative for C. auris were pooled together at equal
ratios. We then compared the ability of the T2 assay to detect specified concentrations of C. auris added
to this representative skin swab matrix relative to a phosphate buffered saline (PBS) reference
condition. Results: The C. auris-specific T2 assay was able to detect C. auris as low as 5 CFU/mL in both a
PBS and swab matrix control. Inclusivity testing determined that the T2 C. auris assay can detect all 4
phylogenetically distinct clades of C. auris. Finally, we compared the identification of C. auris from 89
clinical patient skin-swabs with the T2 C. auris assay and the culture-based diagnostic as a reference
method to determine sensitivity (>90%) and specificity (>95%) for the T2 C. auris assay. Conclusions:
Overall, our data show that the T2 C. auris assay performed well as a rapid alternative to culture that
could help expedite the detection of C. auris in patient skin swabs and facilitate a more timely response
to the existing and future outbreaks.
Session Number: 218
Session Number: 218
Abstract Title:
Gastrointestinal Yersiniosis in Nebraska: A Laboratory and Epidemiological Analysis Following the
Introduction of the BioFire FilmArray® Gastrointestinal Panel
A. Craney1, R. Fowler1, A. Bartling2, K. Flaherty2, P. Iwen2; 1Univ. of Nebraska Med. Ctr., Omaha, NE,
2Nebraska Publ. Hlth.Lab., Omaha, NE
Abstract Body:
Background: Yersinia enterocolitica (YE), an important but often overlooked food-borne pathogen, can
be difficult to detect in stool by routine culture, likely resulting in under reporting of yersiniosis. Since
the introduction of the BioFire FilmArray Gastrointestinal (GI) panel in 2015 to multiple laboratories in
Nebraska, a >4-fold increase in yersiniosis has been reported in the state when compared to prior years,
with 16 cases reported in 2015, 47 cases in 2016 and 17 cases in 2017. This study was done to evaluate
culture methods for recovery of YE from stools positive by the GI panel and subsequently to evaluate
these isolates further using laboratory epidemiological methods for outbreak investigating. Methods:
Stools preserved in maintenance media from laboratories that had YE detected by the GI panel were
either directly cultured to CIN agar plates incubated at room temperature (RT) for up to 5 days or 500 μl
placed into sterile PBS (10 ml) and incubated at 4°C (cold enrichment) for up to 2 weeks with
incremental subcultures to CIN agar. Isolates detected from these stools were subsequently tested by
pulsed-field gel electrophoresis (PFGE) and whole genome sequencing (WGS) (Illumina MiSeq). Cluster
analysis of PFGE profiles and WGS single nucleotide polymorphism (SNP) analysis were performed using
BioNumerics 7.6. Results: 26 stools positive by the GI panel were cultured for YE. YE was recovered from
14 stools (53.8%) following RT incubation and a total of 21 stools (80.1%) following a cold enrichment
incubation. These isolates were further evaluated using PFGE and WGS. Cluster analysis of PFGE
patterns revealed high diversity among isolates; however, four clusters of indistinguishable PFGE
patterns, were observed. SNP analysis of the WGS of these isolates mirrored the PFGE patterns with
most isolates found to be unrelated. Two of the 4 potential clusters from PFGE had WGS with ~180 SNPs
identified between isolates, suggesting that the isolates were unrelated. However, the isolates within
the other 2 clusters showed no SNPs between them, suggesting that the isolates within each cluster
were clonal. Conclusion: This study showed that the addition of the cold enrichment method to routine
culture improved recovery of YE. The enhanced discriminatory power of WGS did show that 2 clusters of
yersiniosis may have occurred in 2016 although a majority of the isolates were unrelated. Additional
epidemiological studies and further bioinformatic analysis of the WGS to evaluate these YE cases and
future cases are ongoing.
Session Number: 218
Session Number: 218
Abstract Title:
Prevalence Of Salmonella Enterica Serovar Typhimurium, Salmonella Derby And Salmonella Serotype
4,[5],12:i:- (a Monophasic Variant of S. Typhimurium) from Swine Herds in Lombardia, Italy.
A. Grassi1, S. Giovannini2, C. Salogni2, E. Giacomini2, V. Baldo2, M. D'Incau2, P. Pasquali3, G. Alborali2;
1I-VET, Brescia, Italy, 2IZSLER, Brescia, Italy, 3Istituto Superiore Sanità, Rome, Italy
Abstract Body:
Background: Salmonella represents the second most common food-borne pathogens in Europe.
Salmonella infection in pigs may cause fever, diarrhoea, prostration and mortality; however, most
infected pigs remain healthy carriers, and those infected at the end of the fattening period could pose a
threat to human health. Salmonella enterica serovar Typhimurium (Salmonella Typhimurium), its
monophasic variant (Salmonella 4,[5],12:i:-) and Salmonella Derby are the major cause of gastroenteritis
in both humans and animals and have been responsible for an increased number of foodborne
infections in humans in Europe in recent years. The aim of this study was to estimate the prevalence of
Salmonella Typhimurium, Salmonella Derby and Salmonella serotype 4,[5],12:i:- in fecal samples from
swine herds in the highest density region of swine farms of Italy. Methods: 10 pens for each farm were
sampled resulting in a total of 2039 pens (851 weaning, 1048 growing, 215 fatteners) from 192 out of
527 pig farms of the area were included in this study. 150 g of fecal material were collected from each
pen. Samples were analyzed using the ISO 6579: 2002/ Amendment 1: 2007 protocol. Suspect
Salmonella colonies were subjected to biochemical identification and serological identification using
Salmonella group-specific antisera. For the statistical analysis, the animals were classed according to the
weight: <30kg (weaning), 30-89 kg (growing) and >90 kg (fatteners). Results: 373/2067 (18%) samples
were positive for Salmonella spp, 18% in weaning, 17,64% in growing and 22% in fatteners pig. 37/2067
(1,7%) samples were positive for S. Typhimurium, 2,23% in weaning, 1,54% in growing, and 1,39 in
fatteners pig. 205/2067 (9,9%) samples were positive for S. Typhimurium monophasic variant, 10,57% in
weaning, 10,52% in growing and 6,04% in fatteners pig. 51/20167 (2,46%) samples were positive for S.
Derby, 0,82% in weaning, 2,68% in growing, 3,25% in fatteners pig. Conclusions: Salmonella carriage in
pigs is a significant food safety issue. S. Typhimurium, S. Typhimurium monophasic variant and S. Derby
represents 78,5% of total Salmonella strains isolated in this study, these data confirm the dominance of
this emergent serovars in pig herds
Session Number: 219
Session Number: 219
Session Title: CPHM02 - Antimicrobial Susceptibility Testing: Gram-positive and Fastidious Bacteria
Poster Board Number: SATURDAY - 205
Abstract Title:
An Nosocomial Infection of Enterococcus faecalis In A Neonatal Intensive Care Unit
C. Liu, H-l. Sun; Peking Union Med. Coll., Beijing, China
Abstract Body:
Background: The immune function of premature is not mature and enterococcus has increasingly
become one of the most common pathogenic bacteria of hospital infection in neonatal intensive care
unit. The aim of this study is to investigate a suspected nosocomial infection of Enterococcus faecalis in
a neonatal intensive care unit and discussed the causes, prevention and control measures. Methods:
There were 5 cases of nosocomia infection of Enterococcus faecalis in the neonatal intensive care unit in
June, collected the object samples and probiotics samples which most of the prematures have taken.
Identified them by MALDI-TOF Mass Spectrometry. Mltilocus sequence typing, PFGE and drug sensitive
test were done to analysis the relationship of them. Results: The gestational age of 5 nosocomia
infection cases were <32 weeks. 6 strais were isolated from 5 cases. 3 strains were ST25, the same with
the cultrued strain of trigeminy viable organism powder. two strains were ST624, the same with the
strain isolated from Object Surface. Toxin gene detection showed asa + 100%, cyt + 5/6, esp +
83.3%(5/6) , gel + 100 %, hyln + 0. Ampicillin, linezolid, vancomycin, left oxygen and chloromycetin
antibiotic sensitive rates were 100%, Quinupristin - dalfopristin antibiotic resistance rate was 100%.
Mltilocus sequence typing ang PFGE have the similar results. Conclusions: This nosocomial infection
outbreak may caused by object samples and probiotics. It can be controlled by standardized medical
behavior, which can decline the nosocomial infection incidence and mortality of preterm infants in
NICU.<br /><p><a
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" alt="" border="0" /></a></p>
Session Number: 219
Session Number: 219
Abstract Title:
Occurrence and Evaluation of Antimicrobial Susceptibility of Staphylococcus aureus Isolated from
Chicken Eggs, Eastern Ethiopia
J. Kemal1, W. Beji1, G. G. Tesfamariam2; 1Haramaya Univ., Dire Dawa, Ethiopia, 2Jigjiga Univ., Jigjiga,
Ethiopia
Abstract Body:
Background: Staphylococcus aureus is responsible for a variety of infections in humans and animals that
particularly causes staphylococcal food poisoning when it present in foods. This study was aimed to
isolate Staphylococcus aureus present on the shell surfaces and in the contents of chicken eggs, and
determine antimicrobial susceptibility patterns. Methods: A total of 335 egg samples were obtained
from open market (n = 174) and poultry farm (n = 161). A sterile cotton swab was used to sample the
surface of eggs. After sterilizing the shells, the egg contents were sampled. Isolation of Staphylococcus
aureus was done based on culture characteristics, Gram staining and biochemical tests. The isolates
were subjected to antimicrobial susceptibility testing using disc diffusion method following the
procedure of the Clinical and Laboratory Standards Institute. Results: Out of the total 335 eggs sample
examined, 93(27.8%) samples yielded S. aureus. Out of these, 28(17.4%) were from poultry farm while
65(37.4%) were obtained from open market. Similarly, 63(18.8%) were from the shell while 30(8.9%)
were from the content. The occurrence of S. aureus in the egg shell collected from open market was
significantly higher than egg shell obtained from poultry farm (CI=0.2904 - 0.9078; p = 0.021). The level
of S. aureus in egg contents was also significantly higher in the open market (CI=0.0962- 0.6085; p =
0.003). All 76 S. aureus isolates were resistant to at least one of the antimicrobials tested with overall
3.9-92.0% level of resistance pattern showing higher resistant to penicillin (92%), ampicillin (89.5%) and
amoxicillin (55.3%). A lower level of resistance was observed to chloramphenicol, gentamycin and
ciprofloxacin with complete susceptibility to vancomycin. Multiple drug resistance to more than two
antimicrobial agents was detected in 86.8% of the total S. aureus isolates. Conclusions: The study
showed high level of S. aureus with considerable antimicrobial resistant pattern. Further study is needed
to better define bacterial resistance to antimicrobial agents with emphasis on surveillance of multiple
drug resistant.
Session Number: 219
Session Number: 219
Abstract Title:
High Rates of False-Resistance of S. Aureus to Trimethoprim-Sulfamethoxazole Observed on the Vitek 2
Gp-75 Antimicrobial Susceptibility Testing Card
P. M. Luethy1, P. J. Williams2, J. K. Johnson1; 1Univ. of Maryland Sch. of Med., Baltimore, MD, 2Univ. of
Maryland Med. Ctr., Baltimore, MD
Abstract Body:
Background: Trimethoprim-sulfamethoxazole (SXT), an antibiotic that inhibits folate synthesis, can be
used to treat a variety infections, including those caused by Staphylococcus aureus. Typical susceptibility
rates of S. aureus to SXT (as monitored by yearly antibiogram) at our institution typically exceed 90% for
both methicillin-sensitive (MSSA) and methicillin-resistant (MRSA) S. aureus clinical isolates.
Interestingly, SXT susceptibility rates for MRSA decreased from 91% in 2015 to 83% in 2016 as
determined by Vitek 2 GP-75 card results. Methods: S. aureus clinical isolates that tested resistant to
SXT as determined by Vitek 2 GP-75 cards were retested against SXT E-test strips to confirm
susceptibility testing results. A small subset of isolates was chosen for strain typing by the DiversiLab®
System and was performed by bioMérieux. Results: Preliminary data from approximately 4.5 months of
testing found that 134 of 912 clinical S. aureus isolates were resistant to SXT by the Vitek 2 GP-75 card.
Of the potentially resistant isolates, only 26 (19.4%) were MSSA. Confirmatory testing with SXT E-test
strips found that 130 (97.0%) of the isolates were actually sensitive to SXT, resulting in a Major Error
rate of 14.3%. Six of the falsely resistant isolates were strain typed, with five found to be likely clonal in
nature. Conclusions: Due to the high Major Error rate associated with SXT and S. aureus on the Vitek 2
GP-75 card, confirmation testing by another method should be performed to rule-out false SXT
resistance. Although five of the six isolates that were typed were identical, further typing is required to
determine if these Major Errors are strain specific. In addition, testing of previous years’ isolates may
reveal that previously observed minor decreases SXT susceptibility were a result of false-resistance.
Session Number: 219
Session Number: 219
Abstract Title:
Rapid Phenotypic Detection of Vancomycin-Resistant Enterococci from Positive Blood Cultures Using
Vancomycin-Impregnated Disks
M. C. Dobler1, B. D. McCollister2, N. Madinger2, M. Bandali3; 1UCHlth.Univ. of Colorado Hosp., Aurora,
CO, 2Univ. of Colorado Anschutz Med. Campus, Aurora, CO, 3Univ. of Colorado Hosp., Aurora, CO
Abstract Body:
Background: Delay of appropriate therapy to patients with bloodstream infection due to vancomycin-
resistant enterococci (VRE) has been associated with poor outcomes and excess mortality. Rapid
detection methods to determine the presence of vancomycin resistance are available, but many are
prohibitively expensive. We investigated the use of vancomycin antibiotic disks on initial subculture
from positive blood culture bottles as a cost-effective approach to rapidly detect vancomycin resistance
in enterococcal isolates. Methods: Positive blood culture bottles that demonstrated Gram-positive cocci
in chains by Gram stain were subcultured onto sheep blood agar plates and were overlaid with a 30μg
vancomycin-impregnated antibiotic disk in the first quadrant. After overnight incubation, bacterial
isolates that were identified as Enterococcus faecalis or Enterococcus faecium were secondarily
evaluated by the vancomycin direct disk test. Isolates that demonstrated bacterial growth up to the disk
were considered vancomycin resistant, while those demonstrating any zone of inhibition were
considered vancomycin sensitive.The sensitivity results obtained by the direct vancomycin disk method
were compared to antibiotic susceptibility testing performed by automated BD Phoenix System or Kirby
Bauer assays, and time to detection was compared between methods for 10% of the isolates. Results: A
total of 100 isolates were tested. None of the 56 E. faecalis isolates were found to be resistant to
vancomycin by direct vancomycin disk testing from positive blood cultures. Of 44 E. faecium isolates, 32
(72.7%) had growth up to the vancomycin disk and were considered resistant. All of the sensitivity
results obtained by the direct vancomycin disk method matched formal sensitivity testing, revealing
100% sensitivity and 100% specificity for determining vancomycin resistance by the direct vancomycin
disk method. Time to determine vancomycin susceptibility averaged 24.46 hours for the direct
vancomycin disk method compared to 48.85 hours for traditional antimicrobial susceptibility testing,
which was statistically significant. Conclusion: Use of a vancomycin disk on initial subculture of positive
blood cultures is a rapid and cost effective strategy for determining vancomycin resistance in
Enterococcus faecium and Enterococcus faecalis isolates and can significantly decrease time to detection
and notification of providers and infection control.
Session Number: 219
Session Number: 219
Abstract Title:
A Multi-Site Study Comparing A Commercially Prepared Dried Mic Susceptibility Sys. to the Clsi Broth
Microdilution Method for Eravacycline Using Fastidious Organisms
N. M. Holliday1, S. M. Andrus1, C. C. Knapp1, S. B. Killian1, J. M. Lindley2, J. M. Streit2, B. J. Olson3, T. R.
Fritsche3, K. Becker4, E. A. Idelevich4, E. Scopes5, A. M. Leonte5, C. Fyfe6; 1Thermo Fisher Scientific,
Oakwood Village, OH, 2JMI Lab., Nort
Abstract Body:
Background: Eravacycline (ERV) (Tetraphase Pharmaceuticals, Watertown, MA) is a novel, fully-synthetic
fluorocycline displaying broad spectrum activity against a variety of organisms. A 4-site study was
performed to determine the accuracy and reproducibility of ERV susceptibility testing against
Streptococcus spp. and Haemophilus influenzae using the Thermo Scientific™ Sensititre® dried MIC
susceptibility system (Thermo Fisher Scientific, Cleveland, OH) compared with the CLSI (M07) reference
broth microdilution method (BMD). Methods: ERV (0.002-16µg/mL) was tested against 537 recent
clinical isolates, 118 challenge isolates, and 15 reproducibility isolates of Streptococcus spp. These
isolates consisted of S. pneumoniae (253), S. pyogenes (121), S. agalactiae (130), S. mitis group (62), S.
salivarius (35), S. anginosus group (69). ERV (0.001-16µg/mL) was also tested against 393 recent clinical
isolates, 50 challenge isolates, and 10 reproducibility isolates of H. influenzae (beta lactamase positive
and negative). The Sensititre MIC susceptibility system was inoculated per manufacturers’ instructions,
and the BMD method was performed per CLSI guidelines. Quality control organisms were tested daily
and were within acceptable ranges. Results: Comparison of Streptococcus spp. MIC results on the
Sensititre system to the CLSI BMD method for automated and manual reads resulted in 99.1% and 99.4%
essential agreement (EA, +/- 1 log2 dilution) for ERV, respectively. Overall the essential agreements for
reproducibility (+/- 1 log2 dilution of the modal MIC) using automated and manual reads were 98.5%
and 98.9%. Comparison of H. influenzae MIC results on the Sensititre system to the CLSI BMD method
for manual read methodology resulted in 98.6% essential agreement (EA, +/- 1 log2 dilution) for ERV.
Overall agreements for reproducibility (+/- 1 log2 dilution of the modal MIC) using manual read
methodology was 100%. Conclusion: The Sensititre susceptibility system demonstrates an equivalent
level of performance compared to the CLSI BMD method when testing ERV against fastidious organisms,
specifically Streptococcus spp., and H. influenzae. The high level of agreement obtained by the Sensititre
susceptibility system and the CLSI BMD method suggests that this is an acceptable method for
susceptibility testing of ERV.
Session Number: 219
Session Number: 219
Abstract Title:
A Multi-Site Study Comparing A Commercially Prepared Dried Mic Susceptibility Sys. to the Clsi Broth
Microdilution Method for Fda Approved Delafloxacin (Baxdela) Using Fastidious Organisms
Fritsche3, K. Becker4, E. A. Idelevich4, E. Scopes5, A. M. Leonte5, S. McCurdy6; 1Thermo Fisher
Scientific, Oakwood Village, OH, 2JMI Lab., N
Abstract Body:
Background: Delafloxacin (DLX) (BAXDELATM) (Melinta Therapeutics, New Haven, CT), is a
fluoroquinolone antibiotic that has been FDA approved for the treatment of acute bacterial skin and skin
structure infections (ABSSSI) caused by gram positive and gram negative organisms. A 4-site evaluation
was designed to determine the accuracy and reproducibility of DLX susceptibility testing against
Streptococcus spp., using the Sensititre dried MIC susceptibility system compared with the CLSI (M07)
reference broth microdilution method (BMD). Both auto and manual read methodologies were
employed. Materials and Methods: DLX (0.0005-8 µg/ml) was tested against 258 recent clinical isolates,
59 challenge isolates and 10 reproducibility isolates. Microorganisms tested included 124 Streptococcus
pyogenes, 131 Streptococcus agalactiae, 67 Streptococcus anginosus Group and 5 other Streptococcus
spp. (reproducibility). The Sensititre dried MIC susceptibility system was inoculated per manufacturers’
instructions. BMD was performed per CLSI guidelines. Recommended CLSI quality control (QC)
organisms were tested daily and all results were within the published QC ranges. Results: Comparisons
of the indicated Streptococcus spp. MIC results on the FDA cleared Sensititre dried MIC susceptibility
system to the CLSI BMD for automated and manual reads resulted in 99.1% and 99.7% essential
agreement (EA; +/- 1 log2 dilution) and 99.1% and 100% categorical agreements respectively. Overall
agreement for the reproducibility (+/- 1 log2 dilution of the modal MIC) using automated and manual
reads was 99.6% and 97.9%. Conclusions: The results of this DLX study on the Sensititre dried MIC
susceptibility system (both automated and manual read), as per the FDA guidance, demonstrated an
equivalent level of performance compared to the CLSI BMD when testing DLX against Streptococcus spp.
The FDA has determined that DLX is substantially equivalent for the indications of Streptococcus
pyogenes, Streptococcus agalactiae, and Streptococcus anginosus Group on the Sensititre dried MIC
susceptibility system and has been cleared for in vitro diagnostic use.
Session Number: 219
Session Number: 219
Abstract Title:
Comparison of Methods for Broth Microdilution Antimicrobial Susceptibility Testing of Lipophilic
Corynebacterium Spp
K. Prasidthrathsint1, M. Fisher2; 1Univ. of Iowa Carver Coll. of Med., Iowa City, IA, 2Associated Regional
and Univ. Pathologists, Univ. of Utah Sch. of Med., Salt Lake City, UT
Abstract Body:
Historically, antimicrobial susceptibility testing (AST) was not routinely performed on Corynebacterium
spp. as they were often considered contaminants. When performed, AST was not standardized, and was
particularly challenging for lipophilic species. Despite standardization by the Clinical and Laboratory
Standards Institute (CLSI, M45, 3rd ed.), current methods may not support growth of some lipophilic
Corynebacterium spp. for AST. Tween 80 may improve the growth of such isolates, but its effect on
broth microdilution results compared to those using CLSI-recommended CAMHB with lysed horse blood
(CAMHB-LHB) media are unclear. Therefore, we performed AST on 41 recent clinical isolates of lipophilic
corynebacteria in both CAMHB-LHB and CAMHB with Tween 80 (CAMHB-T) to evaluate the impact of
Tween. Isolates tested (C. accolens [3], C. bovis [3], C. jeikeium [8], C. tuberculostearicum [13], C.
urealyticum [8], C. glucuronolyticum [1], C. kroppenstedtii [3], C. macginleyi [1], and C.
pyruviciproducens [1]) were identified by MALDI-TOF or 16S rRNA gene sequencing. AST was performed
concurrently in custom Sensititre broth microdilution panels using CAMHB-LHB, and CAMHB with 0.5%
Tween 80. Daptomycin wells were supplemented with calcium to 50 µg/ml. Panels were incubated at 35
°C, ambient atmosphere and read at 24 h (48 h for beta-lactams susceptible at 24 h). Quality control was
performed using Streptococcus pneumoniae ATCC 49619, Staphylococcus aureus ATCC 29213, and
Enterococcus faecalis ATCC 23212. The results were reported only if QC values were in range. Minimal
inhibitory concentrations (MICs) and interpretations were compared between methods. CAMHB-T had
minimal effects on most results (penicillin, ceftriaxone, meropenem, vancomycin, gentamicin,
levofloxacin, doxycycline, clindamycin, TMP-SMX, rifampin, quinupristin/dalfopristin, and linezolid), with
≥ 90% essential agreement (EA) versus CAMHB-LHB. Erythromycin had slightly lower EA (88%), but 22%
of daptomycin MICs were >2x higher when tested in Tween 80. All drugs had acceptable CA except
daptomycin (85%). Overall, testing lipophilic corynebacteria in CAMHB-T gives accurate results for most
drugs, but further testing may be required to establish the reliability of erythromycin results.
Unfortunately, daptomycin MICs are often elevated and cautious interpretation is needed due to risk of
false-nonsusceptibility.
Session Number: 219
Session Number: 219
Abstract Title:
Comparison of Five Commonly Used Automated Susceptibility Testing Methods for Accuracy Evaluation
in China Antimicrobial Resistance Surveillance Sys. (Carss) Hospitals
M. Zhou1, H. Sun2, Y. Xu3; 1Peking Union Med. Coll. Hosp., B, China, 2Peking Union Med. Coll. Hosp.,
Bei, China, 3Peking Union Med. Coll. Hosp., Beijing, China
Abstract Body:
Background: To evaluate the performance of five automated antimicrobial susceptibility testing (AST)
systems in China (Vitek 2, Phoenix, Microscan, TDR and DL), commonly used in the China Antimicrobial
Resistance Surveillance System (CARSS) program. Methods: Two “unknown” isolates, S1 (ESBL-producing
Escherichia coli) and S2 (KPC-producing Klebsiella pneumoniae), were concurrently sent to 886 hospitals
in China for identification and AST. Using the gold standard of broth microdilution method (BMD), the
performance of 5 automated AST systems was evaluated. Essential agreement (EA), categorical
agreement (CA), and major and minor error rates were calculated for each method. Results: The
majority (851/ 886, 96%) of the hospitals returned timely feedback with most (392, 46.1%) using Vitek 2,
and about 16% each using Phoenix, Microscan, and DL systems. The remaining 50 (5.9%) used the TDR
system. Minimum inhibitory concentrations (MICs) of 22 antimicrobials were evaluated for 2 study
isolates plus 3 ATCC strains. All hospitals correctly identified the 2 unknown isolates. In total, 1581
susceptibility results for 3 ATCC strains were submitted by 780 (91.2%) hospitals, either with one (121,
14.2%), two (517, 60.5%) or three (142, 16.6%) ATCC strain results. For the two study isolates, TDR and
DL systems performed the worst in MIC determination and susceptibility categorization of cefazolin and
cefepime, while the Microscan system had difficulties in categorising the isolates as resistant or
susceptible, for aztreonam and ertapenem. CAs were >90% for most of the antimicrobials tested for
both isolates, among which no EAs with BMD was noted for ampicillin, piperacillin, cefazolin,
cefuroxime, ceftriaxone, and trimethoprim/sulfamethoxazole. In terms of VMEs and MEs, all four
systems except Vitek 2 showed unacceptable VMEs for cefazolin (S1 and S2) and MEs for ceftazidime,
cefepime and aztreonam (isolate S1), while Vitek 2 demonstrated a high VME rate for cefepime (10.0%)
and meropenem (6.2%) for isolate S2. Conclusions: None of the five automated systems met the criteria
for acceptable AST performance, but Vitek 2 provided a relatively accurate and conservative
performance for most of the antimicrobials, save for cefepime and meropenem.
Session Number: 219
Session Number: 219
Abstract Title:
Impact of Inter-Species Interaction on the Staphylococcus aureus Antibiogram
M. J. Lee, S. McHale, N. Singh; Wadsworth Ctr. - NYSDOH, Albany, NY
Abstract Body:
Background: Polymicrobial infections (PMI) are infections that involve more than one microbe. One of
the biggest challenges in tackling PMI is the lack of an antimicrobial susceptibility test. To bridge this gap
in methodology, we developed a polymicrobial susceptibility test (PMST). The PMST is specific for
contact-independent, non-biofilm testing conditions. As a proof-of-concept, we performed PMST on
Staphylococcus aureus in the presence of Pseudomonas aeruginosa or Klebsiella pneumoniae culture
supernatants (S/N). Methods: Briefly, for the PMST method, 1x107 CFU from an overnight culture is
inoculated in a flask with 100 mL of cation-adjusted Muller-Hinton broth, incubated for 16-18 hours at
200 rpm, 35°C, spun down to collect the S/N, membrane filtered, and stored at -80°C. About 1-5x104
CFU/50 μL of test isolate (different species than the S/N) is added to the thawed S/N, 50 μL of this is
inoculated into a 96 well plate containing 50 μL per well of media with drug dilutions, and incubated
overnight at 35°C. The MIC is read the next morning per CLSI guidelines. To test for meropenem (MER)
resistance, MER impregnated disks were incubated for 4 hours in the respective S/N, then placed on a
lawn of S. aureus, incubated overnight, and zone of inhibition measured. To test for presence of folate in
the S/N, a commercial ELISA kit was used (MyBiosource, Inc) and data interpreted per manufacturer’s
instructions. Results: Performing PMST on S. aureus with carbapenem-resistant K. pneumoniae (KP-CRE)
S/N showed an increase in minimum inhibitory concentration (MIC) for oxacillin (OXA) from 0.12 to ≥ 64
μg/mL, and MER from 0.5 to 2 μg/mL. MER disk test was performed to determine if beta-lactamases (BL)
were present in the KP-CRE S/N. While disks incubated in S. aureus or ESBL-K. pneumoniae S/N showed
large zone sizes, disks incubated in KP-CRE S/N showed no zone of inhibition suggesting that BL is
present in KP-CRE S/N and likely responsible for the increased MER MIC. Similarly, S. aureus incubated in
P. aeruginosa S/N showed an increase in the bactrim MIC from 0.03 to 8 μg/mL. An ELISA was performed
to determine if different folic acid levels in S/N can explain the change in MIC. However, no differences
were found, suggesting that a secreted factor by P. aeruginosa is likely responsible. Conclusion:
Collectively, our preliminary data show that inter-species interactions can alter susceptibility profiles
and caution should be exercised in prescribing oxacillin or bactrim to cover S. aureus in PMI. Additional
gene expression and biochemical studies will be needed.
Session Number: 219
Session Number: 219
Abstract Title:
Correlation between Broth Microdilution and Disk Diffusion Methods Results When Testing Ceftaroline
against Methicillin-Resistant Staphylococcus aureus(Mrsa), and Molecular Characterization of
Ceftaroline Nonsusceptible Isolates
H. S. Sader, P. R. Rhomberg, T. B. Doyle, R. K. Flamm, R. E. Mendes; JMI Lab., North Liberty, IA
Abstract Body:
Background: Discrepancy rates between MIC and disk zones may vary according to the percentage of
isolates with MIC values in the range of +/- 1 doubling dilution of the breakpoints. The prevalence of
MRSA isolates with ceftaroline (CPT) MICs of 1-4 μg/mL varies widely by geographic region. We
evaluated the disk-MIC correlation when testing CPT against MRSA collections with various CPT
susceptibility (S) rates. Methods: 158 MRSA isolates were evaluated, including 106 randomly selected
isolates collected outside the United States and 52 CPT-non-S isolates. Four subsets with CPT
intermediate (I)/resistant (R) rates of 5%/0%, 10%/2%, 15%/4%, and 20%/8% were also analyzed.
Isolates were tested by CLSI broth microdilution (BMD) and disk diffusion (DD) methods. DD was
performed with 30 μg disks and Mueller-Hinton agar (MHA) from 2 manufacturers each; thus, there
were 4 DD results for each MIC result. CLSI/US FDA BMD and DD breakpoints were applied. Selected
isolates (n=51) were characterized by whole genome sequencing. Results: The randomly selected
collection had CPT S/R rates of 86.8%/0.0%, and the very major (VM), major (Ma) and minor (Mi) error
rates were 0.0%, 0.0% and 11.1%, respectively. In contrast, the collection enriched for CPT-non-S
isolates had CPT S/R rates of 62.0%/15.8%, and VM, Ma, and Mi errors of 3.8%, 0.0%, and 23.1%,
respectively. Error rates for the collections with increased CPT non-S (NS) rates are shown in the Table,
with VM and Mi error rates increasing as the %I and %R increases. Mean values for zone diameters
varied nearly 2 mm between the 2 disks (p<0.001 [t-test]) independent of the MHA. No mutation in the
SCCmec was observed in 5 of 15 isolates with CPT MIC of 2 μg/mL; whereas 3 of 11 isolates with CPT
MIC of 1 μg/mL exhibited mutations in the penicillin-binding domain (PBD; 1 isolate) or in the non-PBD
(2 isolates). Isolates with CPT MIC of ≥4 μg/mL showed ≥1 mutation in the PBD and/or non-PBD.
Conclusions: A direct correlation existed between disk-MIC discrepancy rates and the proportion of CPT-
NS isolates, and error rates became unacceptable (>10% Mi and/or >1% VM) when the collection had
>10-15% CPT-NS isolates. Current DD breakpoints (≥24 mm / ≤20 mm for S/R) appeared appropriate, but
error rates varied substantially according to the disk reagent used.<br /><table border="1"
cellpadding="1" class="DisplayTable" id="{57D2EEAE-80AE-4FD9-B0B7-
CBAF7C998626}"><caption></caption><tr><td rowspan="2" colspan="1">Ceftaroline
susceptibility</td><td rowspan="1" colspan="3">Error rates (%)</td></tr><tr><td rowspan="1"
colspan="1">VM</td><td rowspan="1" colspan="1">Ma</td><td rowspan="1"
colspan="1">Mi</td></tr><tr><td rowspan="1" colspan="1">5% I / 0% R</td><td rowspan="1"
colspan="1">0.0</td><td rowspan="1" colspan="1">0.0</td><td rowspan="1"
colspan="1">5.6</td></tr><tr><td rowspan="1" colspan="1">10% I / 2% R</td><td rowspan="1"
colspan="1">23.1</td></tr></table>
Session Number: 219
Session Number: 219
Abstract Title:
Evaluation of Three Testing Methods for Telavancin Activity against S. Aureus with Reduced
Susceptibility
L. Saravolatz, J. Pawlak; St John Hosp. and Med. Ctr., Grosse Pointe Woods, MI
Abstract Body:
Telavancin (TLV) is a lipoglycopeptide antibiotic that exhibits concentration dependent bactericidal
activity via a dual mechanism of action involving inhibition of bacterial cell wall synthesis as well as
disruption of cell membranes. In January, 2014, the Clinical and Laboratory Standards Institute (CLSI)
revised the broth microdilution susceptibility testing method for telavancin. The purpose of this study
was to assess the activity of telavancin against a collection of S.aureus isolates using three testing
methods. We tested 74 isolates of S.aureus with reduced susceptibility to other antimicrobial agents
against TLV. The three methods included Etest strips, two approved broth microdilution methods
including microtiter plates prepared in house and lyophilized dry format Sensititre Custom plates. The
revised method followed CLSI guidelines using dimethyl sulfoxide as the solvent and diluent for
antibiotic preparation. This method also includes the addition of polysorbate–80 at 0.002% to mitigate
against lipoglycopeptides binding to plastics. The TLV results are categorized based upon
vancomycin(VAN) and daptomycin(DAP) susceptibility(SUS). The percent susceptible was the same when
comparing the three different methods for three of the five groups listed below. The major source of
variation was for VISA-daptomycin non-susceptible (NONSUS) isolates and occurred in 9 cases. All
differences were within one doubling dilution. The TLV MIC ranges are provided in µg/ml in the table
below.<table class="AbstractTable" id="{DDB7CE6D-B2C2-4A65-A7EF-2F7208381648}"><caption
rowspan="1" colspan="1"></td><td rowspan="1" colspan="2">Broth Microtiter</td><td rowspan="1"
colspan="2">Sensititre Plates</td><td rowspan="1" colspan="2">Etest Strips</td></tr><tr><td
rowspan="1" colspan="1">VAN</td><td rowspan="1" colspan="1">DAP</td><td rowspan="1"
colspan="1">Range</td><td rowspan="1" colspan="1">%SUS</td><td rowspan="1"
colspan="1">Range</td><td rowspan="1" colspan="1">%SUS</td><td rowspan="1"
colspan="1">Range</td><td rowspan="1" colspan="1">%SUS</td></tr><tr><td rowspan="1"
colspan="1">SUS</td><td rowspan="1" colspan="1">SUS</td><td rowspan="1" colspan="1">0.06-
0.12</td><td rowspan="1" colspan="1">100%</td><td rowspan="1" colspan="1">0.03-0.12</td><td
rowspan="1" colspan="1">100%</td><td rowspan="1" colspan="1">0.032-0.125</td><td rowspan="1"
colspan="1">100%</td></tr><tr><td rowspan="1" colspan="1">SUS</td><td rowspan="1"
colspan="1">NON SUS</td><td rowspan="1" colspan="1">0.06-0.12</td><td rowspan="1"
colspan="1">100%</td><td rowspan="1" colspan="1">0.06-0.12</td><td rowspan="1"
colspan="1">100%</td></tr><tr><td rowspan="1" colspan="1">VISA</td><td rowspan="1"
colspan="1">SUS</td><td rowspan="1" colspan="1">0.03-0.25</td><td rowspan="1"
colspan="1">88%</td></tr><tr><td rowspan="1" colspan="1">VISA</td><td rowspan="1"
colspan="1">NON SUS</td><td rowspan="1" colspan="1">0.06-0.5</td><td rowspan="1"
colspan="1">38%</td></tr><tr><td rowspan="1" colspan="1">VRSA</td><td rowspan="1"
colspan="1">SUS</td><td rowspan="1" colspan="1">0.25-4</td><td rowspan="1"
colspan="1">0%</td><td rowspan="1" colspan="1">0.5-4</td><td rowspan="1"
colspan="1">0%</td><td rowspan="1" colspan="1">0.25-4</td><td rowspan="1"
colspan="1">0%</td></tr></table> Overall, all three testing methods showed similar results. For most
of the strains the percent susceptibility ranged from 88% to 100%. VRSA were resistant to TLV by all
three methods. The greatest discordance was in the VISA-Daptomycin non-susceptible group where the
% susceptibility ranged from 38% to 54%. Clinicians should be aware that all three methods will
generally provide similar results for Telavancin susceptibility testing except for VISA-daptomycin non-
susceptible strains. For these isolates repeat susceptibility testing should be considered.
Session Number: 219
Session Number: 219
Abstract Title:
Performance of Etest Telavancin for Antimicrobial Susceptibility Testing of Staphylococcus aureus and
Enterococcus faecalis
T. Armstrong1, H. Dwivedi1, C-A. Burnham2, W. Lainhart2, S. Campeau3, R. Humphries3, G. W. Procop4,
M. Tuohy4, D. Wilson4, V. Sauvonnet5, D. Halimi5, G. Zambardi5; 1bioMérieux, Inc, Hazelwood, MO,
2Washington Univ. Sch. of Med., St. Louis, MO, 3Univ. of
Abstract Body:
Background: Telavancin is a lipoglycopeptide antibacterial drug with activity against select Gram-positive
bacterial species. In this study, the performance of ETEST® Telavancin (TLA), an in vitro technique for
determining the antimicrobial susceptibility of Enterococcus faecalis and Staphylococcus aureus, was
evaluated against the Clinical and Laboratory Standards Institute (CLSI) broth microdilution reference
method (BMD). Method: A total of 666 isolates (289 E. faecalis and 377 S. aureus) were tested by
ETEST® TLA and BMD methods. Isolates were subcultured on tryptic soy agar plates supplemented with
5% sheep blood. After overnight incubation 0.5 McFarland suspensions were prepared to inoculate
ETEST® TLA and BMD. Results were read after 16 - 20 hours incubation. Results were analyzed for
essential agreement (EA), category agreement (CA), major and very major error rates and compared to
the FDA performance criteria, EA and CA (≥ 90%), major error rate (≤ 3.0% ) and very major error rate (≤
1.5%) using the FDA breakpoints for Telavancin (S. aureus susceptible S <u><</u> 0.125 µg/mL and E.
faecalis S <u><</u> 0.25 µg/mL). Results: ETEST® TLA results including the rate of Telavancin non-
susceptible results and the performance against Enterococcus faecalis and Staphylococcus aureus are
summarized in table 1 and 2 respectively. Overall, ETEST® TLA met the FDA performance acceptance
criteria for EA and CA (>90%), major error rate (<3%) and very major error rate (<1.5%) Conclusion:
ETEST® TLA performance for E. faecalis and S. aureus met the FDA performance criteria. When
compared to the broth microdilution reference method, ETEST® TLA proved to be a suitable test for
susceptibility testing of E. faecalis and S. aureus. Table 1 Telavancin Non-Susceptible Result Rate<table
class="AbstractTable" id="{8F950786-F0EC-42FF-81D4-704365156B98}"><caption
colspan="1"></td></tr><tr><td rowspan="1" colspan="1">Species</td><td rowspan="1"
colspan="1">Percentage Non-Susceptible Results</td></tr><tr><td rowspan="1"
colspan="1">Vancomycin susceptible Enterococcus faecalis </td><td rowspan="1" colspan="1">0.0%
(0/0)</td></tr><tr><td rowspan="1" colspan="1">Vancomycin susceptible and resistant Enterococcus
faecalis </td><td rowspan="1" colspan="1">13.1% (38/289)</td></tr><tr><td rowspan="1"
colspan="1">Staphylococcus aureus</td><td rowspan="1" colspan="1">1.3% (5/377)</td></tr></table>
Table 2 ETEST® TLA Performance for Enterococcus faecalis and Staphylococcus aureus <table
class="AbstractTable" id="{6869AC40-304C-4F49-8240-0957B9B42E5E}"><caption
rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1">Species</td><td rowspan="1"
colspan="1">EA</td><td rowspan="1" colspan="1">CA</td><td rowspan="1" colspan="1">Major Error
Rate</td><td rowspan="1" colspan="1">Very Major Error Rate</td></tr><tr><td rowspan="1"
colspan="1">Vancomycin susceptible Enterococcus faecalis </td><td rowspan="1" colspan="1">91.6%
(230/251)</td><td rowspan="1" colspan="1">97.6% (245/251)</td><td rowspan="1" colspan="1">2.4%
(6/251)</td><td rowspan="1" colspan="1">0.0% (0/0)</td></tr><tr><td rowspan="1"
colspan="1">Vancomycin susceptible and resistant Enterococcus faecalis </td><td rowspan="1"
(0/38)</td></tr><tr><td rowspan="1" colspan="1">Staphylococcus aureus</td><td rowspan="1"
(0/5)</td></tr></table>
Session Number: 219
Session Number: 219
Abstract Title:
Characterization of Linezolid Non-Susceptible Enterococcus faecium from Missouri and Pakistan
M. A. Wallace1, R. F. Potter1, A. W. D'Souza1, S. Andleeb2, G. Dantas1, C-A. D. Burnham1; 1Washington
Univ. Sch. of Med., St. Louis, MO, 2Natl. Univ. of Sci. and Technology, Islamabad, Pakistan
Abstract Body:
Objective: The objective of our study was to characterize linezolid (LZD) non-susceptible isolates of E.
faecium from two distant geographical regions. Methods: Thirty-one (31) LZD non-susceptible E. faecium
isolates from Missouri (USA) and 48 isolates from Islamabad (Pakistan) were evaluated. Linezolid and
daptomycin were analyzed using gradient diffusion (Etest, bioMerieux) and interpreted according to CLSI
guidelines. Tedizolid and dalbavancin were evaluated using gradient diffusion (Liofilchem) according to
the manufacturer's specifications using E. faecalis breakpoints as a surrogate for interpretation. Disk
diffusion was performed with vancomycin, ampicillin, doxycycline (Hardy Diagnostics), and
quinupristin/dalfopristin (BD BBL). DNA was extracted from each isolate and PCR for cfr was performed.
Results: 100% (31/31) of the LZD non-susceptible USA isolates were also tedizolid non-susceptible.
93.8% (45/48) of the Pakistan isolates were tedizolid non-susceptible. Minimum inhibitory concentration
ranges for linezolid and tedizolid are represented in Table 1. 29% (9/31) of USA isolates and 0% (0/48) of
Pakistan isolates were susceptible to dalbavancin. 100% (48/48) of Pakistan isolates and 90.3% (28/31)
of USA isolates tested susceptible to daptomycin. All tested isolates (79/79) were resistant to ampicillin.
74.2% (23/31) (USA) and 100% (48/48) from Pakistan were vancomycin resistant. 77.4% (24/31) (USA)
and 95.8% (46/48) (Pakistan) isolates were doxycycline non-susceptible. 80.6% (25/31) of USA isolates
and 77.1% (37/48)of Pakistan isolate were susceptible to synercid. None of the Pakistan and 9.7% (3/31)
USA isolates were found to harbor the plasmid-mediated linezolid resistance determinant cfr.
Conclusion: Most isolates of LZD non-susceptible E. faecium evaluated were not susceptible to tedizolid
or dalbavancin. The plasmid-mediated linezolid resistance gene cfr was rare in this cohort of LZD non-
susceptible E. faecium.<br /><p><a
href="http://files.abstractsonline.com/CTRL/08/b/8a0/196/690/4a0/fae/d3d/0b5/ba2/70e/6a/g4713_1
.png" target='_blank' address=no ><img
src="http://files.abstractsonline.com/CTRL/08/b/8a0/196/690/4a0/fae/d3d/0b5/ba2/70e/6a/g4713_1.
png" alt="" border="0" width="600" height="159" /></a></p>
Session Number: 220
Session Number: 220
Session Title: CPHM03 - Diagnostic Bacteriology: Consider the Source
Abstract Title:
Comparative Study of Genepoc™ Gbs Lb Assay and Genexpert® Gbs Lb Assay for the Detection of Group
B Streptococcus in Prenatal Screening Sample
T. Choera, B. Jung-Hynes, D. J. Chen; Univ. of Wisconsin- Madison, Madison, WI
Abstract Body:
Streptococcus agalactiae, often referred to as Group B Streptococcus (GBS), is a gram-positive bacterium
found in the rectum and vagina of approximately 25% of pregnant women. As GBS infections in the
United States are a leading cause of meningitis and sepsis in newborns, the CDC recommends GBS
screening for all pregnant women at 35-37 weeks of gestation and administration of intrapartum
prophylaxis (in those that tested positive) as an effective means of controlling disease transmission.
Several FDA approved molecular diagnostic tests are available for rapid and accurate detection of GBS in
antepartum women. Here, we report a clinical comparison of two: our existing clinical test, Xpert® GBS
LB and a novel recently FDA-cleared test, GenePOC™ GBS LB. Samples from women undergoing prenatal
screening were submitted to the University of Wisconsin’s clinical microbiology laboratory for GBS
testing. Vaginal-rectal swabs were enriched in Lim broth for 18-24 hours; post-enrichment, each sample
was tested by GeneXpert® and revogene™ within 72 hours. Results from both tests were compared for
accuracy. Concordant results between the assays were recorded, and additional tests were not
performed. Discrepancies between the initial tests were resolved in two ways: repeating the tests on
both platforms and culturing the enriched sample to check for GBS growth. Results from these
secondary tests were compared against two definitions of true positivity: 3 of 4 molecular tests yielding
a positive result and GBS growth in bacterial culture. These definitions are deemed as standard 1 and 2,
respectively. A total of 95 samples were tested on both instruments with 94% concordant results
between the two assays. When standard 1 was applied to define true positives for the detections of
GBS, GeneXpert demonstrated a sensitivity and specificity of 82% and 100%, respectively, while
GenePOC demonstrated a sensitivity and specificity of 100% and 96%, respectively. Three of six samples
initially identified as negative for GBS by the GeneXpert were correctly identified as positive by the
GenePOC GBS LB when using standard 1 for comparison. When standard 2 was applied, the GeneXpert
demonstrated a sensitivity and specificity of 100%, while the GenePOC demonstrated a sensitivity and
specificity of 100% and 93%, respectively. Both tests were comparable in assay run time as well as
technical instrument use. Comparatively, we observed higher sensitivity for the GenePOC GBS LB assay,
and higher specificity for the GeneXpert GBS LB assay when applying both standards for comparisons.
Session Number: 220
Session Number: 220
Abstract Title:
Evaluation of the Solana Gbs Assay for Analytic Performance and Workflow Efficiency
G. Capraro; Carolinas Pathology, Charlotte, NC
Abstract Body:
Background: Streptococcus agalactiae (GBS) can cause serious infections in newborns and adults,
including bacteremia, pneumonia, meningitis, and skin/soft tissue infections. Neonatal mortality due to
early-onset GBS disease has decreased over the past two decades, primarily because of national
guidelines recommending universal prenatal screening. Conventional lab methods to support this
activity include culturing vagino-rectal specimens for phenotypic identification of GBS; however,
molecular GBS assays are becoming more widely used in clinical labs. The purpose of this study was to
determine the analytical performance and workflow efficiency of a recently FDA-cleared molecular test
for detection of GBS colonization in pregnant patients, compared to colorimetric enrichment broth
culture. Methods: A total of 200 prospective swabs submitted for routine prenatal care were tested by
conventional culture. Briefly, specimens were inoculated into Carrot Broth (CB) and observed for a
positive orange color change within 24 h of incubation. Broth specimens with no color change were
subcultured to GBS Detect Agar (GDA). Residual CB was tested at 24 h using the Solana GBS (SG) assay
according to manufacturer instructions, and results were compared to those obtained by culture.
Results: Of 40 CB-positive specimens, 40 (100%) were positive by SG. Of 160 CB-negative specimens, 145
(91%) were negative by SG and 15 (9%) were positive by SG. Of note, 1 CB- and SG-negative specimen
grew an isolate that was identified as GBS by MALDI-ToF, and 4 specimens were positive by SG only. This
study demonstrated a GBS prevalence of 26% and an overall agreement of 97.5%. Analytic sensitivity,
specificity, positive- and negative-predictive values were calculated to be 98.1%, 97.3%, 92.7%, and
99.3%, respectively. Workflow analysis based on a full run of 12 specimens revealed an average hands-
on time of 1 minute, 23 seconds per specimen, and an average time for personnel to interface with the
instrument of 12 seconds per specimen. Based on this analysis and an assay run time of 29 minutes, the
theoretical maximum number of specimens that can be tested with this assay in an 8-hour shift was
calculated at 198. Conclusions: The SG assay demonstrated excellent analytical performance and a
relatively short hands-on time, and lends itself to efficient small batch processing. For 11 (21%) of 52
GBS-colonized patients in this study, molecular results were available approximately 24 h prior to
culture results, demonstrating improved turnaround time over conventional methods.
Session Number: 220
Session Number: 220
Abstract Title:
High Prevalence of β-Hemolysing Streptococci, Non-Serologic Group A Precludes Molecular Testing for
Streptococci Pyogenes Only
E. A. Clark, K. A. Heath, M. Oethinger; Providence St. Joseph's Hlth., Portland, OR
Abstract Body:
Background: Pharyngitis caused by Streptococcus pyogenes (β-hemolyzing Streptococci serologic group
A), “BSA”) is usually treated to prevent sequelae such as rheumatic fever or glomerular nephritis,
especially in children. Other β-hemolyzing, non-Group A Streptococci (Streptococci of serologic group
C/G/F) can cause clinically almost identical disease but without these sequelae. The purpose of this
study was to understand how frequently β-hemolyzing, non-Group A Streptococci (“BS Non-A”) are
identified and reported by our clinical microbiology laboratory. Materials/Methods: The study analyzed
data from calendar year 2016 that were gathered at eight hospitals and multiple physician offices
throughout Oregon. First-line of diagnosis of BSA was a rapid antigen test (OSOM Streptococcus A) on
throat swabs. Swabs from children with negative results were sent to the regional Clinical Microbiology
Laboratory, and for adults only if clinically indicated. Throat swabs were cultured on blood agar,
incubated at 35 ± 2 C in 5% CO2 and read after 24 and 48 hours. Arcanobacterium haemolyticum was
ruled out by Gram stain. Agglutionation tests (Streptex) were performed on colonies of beta-hemolytic
Gram-positive cocci. Cultures were reported semiquantitatively as “Streptococcus pyogenes (group A)
present“, “beta-hemolytic Streptococcus (non-Group A) present”, or “No Streptococci present”. Results:
In 12 months, 5246 rapid Strep A antigen tests were performed of which 847 (16.1%) were positive (35%
in children <= 16 yrs., 65% in adults > 16 yrs.) A total of 4691 throat swabs were cultured of which more
than half were positive for beta-hemolytic streptococci: 968 (22%) for BSA and 1488 (34%) for BS Non-A.
Given a sensitivity of our Strep A antigen test of almost 90% it appears that BS Non-A play a role in
almost as many cases as BSA. Conclusion: Antigen or molecular methods that target only Group A
Streptococci may miss a large proportion of bacterial pharyngitis cases that are caused by beta-
hemolytic Streptococci other than Group A (Group C/G/F Streptococci). Even though the latter
infections have not been associated with the same sequelae as the former, treatment with a beta-
lactam antibiotic rapidly decreases morbidity. This calls for molecular tests of the future that target and
call out all beta-hemolytic Streptococci.
Session Number: 220
Session Number: 220
Abstract Title:
Evaluation of A New Chromogenic Group B Streptococcus Agar and Use of Digital Analysis for Detection
of Group B Streptococcus In Vaginal/Rectal Swabs
M. Pham1, J. Cumpio2, K. Van Horn1; 1Southern California Permanente Med. Group, Chino Hills, CA,
2Southern California Permanente Med. Group, North Hollywood, CA
Abstract Body:
Detection of Group B Streptococcus (GBS) colonization during pregnancy aids in the prevention of early
onset GBS disease in newborns. A new Group B chromogenic culture medium combined with
segregation software (SSW) developed for the WASPLabTM system (Copan Diagnostics) may aid in the
accurate detection of GBS colonization as well as enhanced laboratory workflow management. We
evaluated the LIM broth/chromID® Strepto B agar (STRB, bioMerieux) for detection of GBS along with
the WASPLab SSW for automated digital analysis for separation of negative and positive culture results.
The comparative method was Carrot broth/GBS Detect medium (Hardy Diagnostics). Vaginal/rectal
swabs were processed by the WASPLab system for both methods with initial inoculation into the
enhancement broths which were incubated off-line at 35-37oC overnight, followed by WASP inoculation
of the plates and incubation at 35-37oC for 20 h (Detect) and 24 h (STRB). WASPLab digital images of
GBS Detect (beta hemolysis) and STRB (pink to red colonies considered positive) were visually examined.
STRB also had digital images analyzed by the SSW to automatically segregate negatives from positives.
All positives were confirmed as GBS or not GBS by phenotypic methods. There were 245 samples
acceptable for comparison with 152 negative by Detect and STRB and 87 positive by both methods.
There were 4 cultures positive by Detect that were negative by STRB and 2 cultures that were negative
by Detect and positive by STRB. Sensitivity was 96% and specificity 99%, however the adjusted specificity
was 100% with the 2 STRB positive/ Detect negative cultures considered as true positives. The 245 STRB
plate images were analyzed by SSW after initial visual examination. There were 89 STRB visually
examined as positive with 100% detected as positive by SSW with no false negatives. Of the remaining
156 STRB negative plates, 124 (79%) were negative by SSW. There were 32 positives indicated by SSW
that were determined to be negative for GBS by additional phenotypic testing. These were determined
to be SSW false positive most likely due to blue/purple colonies with a pink hue in the medium. The
STRB is equivalent to the Detect for detection of GBS. The SSW is a powerful tool for automatic
separation of positive and negative cultures to help laboratory workflow.
Session Number: 220
Session Number: 220
Abstract Title:
CmpTmGbs Transcultswab and Bi-Plate Detects and Identifies Group B Streptococcus During
Antepartum Screening
L. Wu1, Feiling Wang, Fang Ming, Fan Jiang, Rui Dong, Xiaoyue Liu, Jianhua Zou, Xuri Chen,Yuanfang Zhu,
Y. Tang2; 1Baoan Maternal and Child Hlth.Hosp., Jinan Univ., Shenzhen, China, 2Mem. Sloan Kettering
Cancer Ctr. and Weill Med. Coll. of Cornell Univ.,
Abstract Body:
Background: Culture from LIM broth-enriched vaginal-rectal swabs (VRS) is considered the gold standard
for screening maternal carriage of Group B Streptococcus (GBS); however, successful screening depends
on the timing of specimen collection, process and enrichment as well as the subsequent culture
methods. We performed a clinical validation of CMPTM GBS TransCultSwab and Bi-Plate (Creative Co,
Suzhou, China) for detection and identification of GBS in pregnant women.Methods: From August 15 to
September 20, 2017, three VRS were collected simultaneously and randomly from each enrolled 35-37
week pregnant woman using the TransCultSwab, a Copan (Brescia, Italy) and a Yangpu (Guangzhou,
China) swab. The TransCultSwab was incubated/transported for 6-24 h before being plated on blood
agar plate (BAP) or Bi-Plate. Isolates were identified as GBS by using matrix-assisted laser
desorption/lionization-time of flight mass spectrometry. Results: A total of 1,440 pregnant women were
recruited during the study period. The Copan-LIM-BAP reference method detected 194 GBS resulting in
a positive rate of 13.5%. Sensitivities, specificities, positive and negative predictive values were 90.9%,
91.0%, 93.9% and 98.5% for the TransCultSwab-BAP, 90.2%, 99.2%, 94.6% and 98.5% for the
TransCultSwab-Bi-plate, and 94.8%, 98.6%, 91.5% and 99.2% for the routinely used Yangpu-Dijing-BAP
methods, respectively. Hands-on time was 17.1, 25.4, 32.8 and 31.9 s for the TransCultSwab-BAP, the
TransCultSwab-Bi-plate, the Yangpu-Dijing-BAP, and the Copan-LIM-BAP standard. Test turnaround time
was 34.7 to 35.0 h when the TransCultSwab was used, which was more than 10 hours shorter than the
broth-culture methods (46.4-46.5 h, Exact Fisher p<0.0001).Conclusions: CMPTMGBS TransCultSwab
possesses equivalent accuracy for GBS screening with significantly shortened hands-on time and test
turnaround time.
Session Number: 220
Session Number: 220
Abstract Title:
Evaluation of A Multiplex Real Time Pcr Assay for the Detection of Mycoplasma And Ureaplasma Species
At A Large Tertiary Care Facility
S. Chandrasekaran, H. K. Huse, O. B. Garner; Univ. of California, Los Angeles, Los Angeles, CA
Abstract Body:
Background: Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma urealyticum, and Ureaplasma
parvum are small, fastidious bacteria that lack cell walls. These species are often isolated from the
genital tracts of sexually active adults leading to a variety of clinical manifestations ranging from
asymptomatic carriage to adverse pregnancy outcomes including miscarriage and preterm labor.
Colonization of infants can occur through passage of the birth canal or in utero, leading to pneumonia,
bacteremia, and death in very low birth weight infants. These bacteria represents a unique group of
organisms which are often ignored by laboratories due to their fastidious growth requirements.
Recently, molecular tests have been developed for the detection of Mycoplasma and Ureaplasma spp.
from urine and genital swabs using real-time PCR (RT-PCR). This study evaluated lyophilized RT-PCR
reagents (custom manufactured by BioGX for UCLA) for use with the BD MAX™ to detect Mycoplasma
hominis, Mycoplasma genitalium, Ureaplasma urealyticum, and Ureaplasma parvum. Methods:
Remnant urine samples from previous Mycoplasma and Ureaplasma culture requests were processed on
the BD MAX™ using the BD MAX™ ExK™ DNA-1 extraction kit. The lyphophilized reagents were used for
PCR analysis. Additionally, purified DNA from these organisms were used to spike culture negative urine
samples. A total of 154 urine samples were evaluated and compared to the initial culture results.
Results: Compared to culture, M. hominis (N=25) had a sensitivity of 96% and specificity of 98% with 1
false negative and 1 false positive. For Ureaplasma spp. (N=42), PCR had a sensitivity of 100% and
specificity of 91% with 6 false positives and 0 false negatives. In total, PCR testing of 25 specimens
resulted in discordant results. After repeat testing with BioGX reagents, 18 discrepant results were
resolved. Specimens with remaining discrepant results were sent to Mayo Medical Laboratories for PCR
analysis, and results for 3 more specimens were resolved. Conclusions: The custom manufactured
reagents targeting Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma urealyticum, and
Ureaplasma parvum, when utilized with the BD MAX™ platform, provided sensitive and specific
identification of these species from urine. Future studies include validation using genital swabs as a
specimen source.
Session Number: 220
Session Number: 220
Abstract Title:
A Three-Assay Comparator for Validation of Ivd Naats for Mycoplasma Genitalium
B. Kirkconnell, K. Santos, T. Le-Nguyen, P. Hovey, S. Ng, D. Getman, B. Weinbaum; Hologic, San Diego,
CA
Abstract Body:
Background: Mycoplasma genitalium (Mgen) is a sexually transmitted bacterium linked to cervicitis,
pelvic inflammatory disease, urethritis, and increased transmission of HIV and other STIs. With no
culture method or FDA-approved NAAT assay currently available, there is no gold standard method to
use as a comparator for the validation of new Mgen assays. Here, we demonstrate the clinical and
analytical performance of three new Research Use Only Mgen NAAT assays for use as a composite
comparator for clinical validation of Mgen IVD assays. Methods: Three transcription-mediated
amplification (TMA) assays were developed and validated for detection of 16S or 23S rRNA from Mgen,
using Aptima-format General Purpose Reagents on the Panther instrument or manual DTS platform
(Hologic). Analytical sensitivity (95% LOD) was determined by Probit analysis of serial dilutions of Mgen
lysate and in-vitro RNA transcript in screened-negative clinical specimen matrix or Specimen Transport
Medium (STM), respectively. Analytical specificity was determined by testing 16 panels consisting of 54
non-target bacteria, protozoa, and viruses. Specificity panels were tested in the absence and presence of
low titer Mgen to evaluate potential assay cross-reactivity and interference. Clinical performance of the
3 assays was determined by testing residual vaginal swabs and female and male urine specimens
(N=1,400) obtained from adults (age 16+) from several US clinical sites. Agreement between the CE-
marked Aptima Mycoplasma genitalium (AMG) TMA assay and the 3 comparator TMA assays was
determined individually as well as using a composite determination based on the consensus result of the
three assays. Results: Analytical sensitivity of the assays using Mgen lysate in clinical specimen matrix
ranged from 0.0027 to 0.0057 CFU/mL. Sensitivity with IVT RNA in STM ranged from 23.5 to 61.9 copies
RNA/mL. No tested organisms cross-reacted with any assay or interfered with detection of Mgen at the
tested concentrations. Positive, negative, and overall agreements between AMG and the 3-assay
composite comparator was 98.8%, 100%, and 99.9%, respectively. Of the 1,400 specimens tested, only
one specimen yielded a discordant result between AMG and the composite comparator. Conclusions:
The comparator assays were sensitive and specific for Mycoplasma genitalium, with very good clinical
agreement. These assays may be useful for validation of future IVD NAATs for this organism.
Session Number: 220
Session Number: 220
Abstract Title:
Reproducible Semi-Quantification of Pneumonia Pathogens in Contrived Sputum and Bal Specimens
with No Sample Pre-Treatment in An Automated Multiplex Pcr System
A. Clark, D. Goldgar, E. Huynh, A. Hillman, J. Green, S. Thatcher; BioFire Diagnostics, Salt Lake City, UT
Abstract Body:
Traditional methods of testing bronchoalveolar lavage (BAL) and sputum for pneumonia yield varied
results due to difficulties related to sample manipulation. Repeated cultures of clinical specimens show
high variability between replicates of the same specimen. Many pneumonia causing bacteria require
quantification to differentiate pathogenic loads from commensal carriage but culture methods are slow,
subjective, and identifying co-infections is difficult. A simple sampling workflow requiring no specimen
pre-treatment paired with the FilmArray® Pneumonia Panel (FA Pneumo) provides rapid identification of
26 pneumonia causing pathogens (viral and bacterial) and 7 antibiotic resistance markers (ARM) with
accurate, reproducible semi-quantification of typical bacteria in log10 units from 104 - >107 copies/ml.
Clinical remnant specimens (78) were spiked individually with multiple dilutions of mixes containing 7-8
organisms each. Organisms were quantified by plate enumeration and/or qPCR prior to pooling. Each
mix was tested in several BAL and sputa using a prototype FA Pneumo Panel. Specimens were
introduced into the system with no pre-treatment using a flocked swab. Semi-quantification by the FA
Pneumo Panel of typical bacteria in contrived specimens was compared to other methods over 3
independent culture events. Accurate identification of all on-panel organisms and associated ARMs was
evaluated. Of 14 typical bacteria tested at titers of ~104 - 108 copies/ml by qPCR the FA Pneumo Panel
detected 774/932 (83%); log10 quantity reported was within 1 log for 736/774 (95%) replicates. Of
replicates tested at titers of ~104 - 107 cfu/ml by plate enumeration the FA Pneumo Panel detected
555/595 (93%); log10 quantity reported was within 1 log for 407/555 (73%) replicates. Replicates
reported are those tested at >103.5 U/ml as determined by each reference quantification. Correct ARMs
were identified in 337/338 (99.7%) replicates when the linked organism was detected. Of 11 atypical
bacteria and viruses tested at titers >103 copies/ml, the FA Pneumo Panel detected 714/720 (99.2%)
replicates. The FA Pneumo Panel provides reproducible semi-quantification and accurate identification
of multiple pathogens within a single specimen. A simple workflow requiring no sample pre-treatment
delivers reliable results for a variety of complex lower respiratory specimens. Compared to traditional
methods the FA Pneumo Panel is a rapid, objective alternative that eliminates much of the variability
associated with testing lower respiratory specimens.
Session Number: 220
Session Number: 220
Abstract Title:
Evaluating the Utility of Repeat Testing of Multiplex Respiratory Pathogen Panels from Nasopharyngeal
Swabs: What is An Appropriate Cutoff?
C. Salmon, C. Bender, C. Doern; VCU Hlth.System, Richmond, VA
Abstract Body:
Multiplex respiratory pathogen panels (RPP) offer rapid turnaround times and the capability to detect
numerous viral and bacterial pathogens. Despite their utility, these tests are expensive and can be
subject to over-utilization, such as with unnecessary repeat testing. As a molecular test that detects
nucleic acid, and not viable organism, the appropriate time frame has not been established for repeat
testing. In this study, we retrospectively reviewed patients’ results who had repeat testing performed
within 7 days of the initial test to determine the utility of this practice and establish an appropriate cut-
off for repeat testing. We reviewed all RPP results performed on the BioFire FilmArray Respiratory
Pathogen Panel between June 2014 and May 2017 and identified patients with repeat testing from
nasopharyngeal swabs specimens within 48 hr and 7 days of the prior test. Two hundred and fifty-one
episodes of repeat testing were identified, 24 and 107 were repeated within 48 hr and 7 days,
respectively. Of those repeated within 48 hr, there were two (8%) that converted from negative to
positive (RSV [43 hr], Coronavirus [7 hr]) and none that converted from positive to negative. Of 107 tests
repeated within 7 days, there were 22 (21%) that differed from the original result (12 pos to neg, 11 neg
to pos). Interestingly, the average time for a positive to negative conversion was 148 hr (6.2 days). Only
a single conversion occurred in less than 135 hr (5.6 days). Results were mixed for negative to positive
conversions with an average time to a conversion of 110 hr (4.6 days). Taken together, these data
suggest that a 7-day cutoff for repeat testing is too long and that 1 in 5 patients may have a different
result inside that time frame. In contrast, implementing a 48-hr cutoff for repeat testing would have
only missed 2 negative to positive conversions and zero positive to negative conversions. Importantly,
no influenza results changed with a repeat test inside 48 hours. This suggests that this test should not be
used as a test of cure inside that time-frame and that implementing a 48-hr cut-off would have minimal
impact on patient care.
Session Number: 220
Session Number: 220
Abstract Title:
Precision of the Filmarray® Pneumonia Panel - Considerations for Interpreting Relative Abundance of
Bacterial Nucleic Acids in Lower Respiratory Specimens
M. F. Hockin, M. Buccambuso, T. J. Edwards, J. Arce, R. Lems, L. O'Connor, J. Larsen, J. Manwaring, A.
Fratto, D. Abbott, J. P. Southwick, A. Judd, M. Brooks, E. Amiott; BioFire Diagnostics, Salt Lake City, UT
Abstract Body:
The FilmArray Pneumonia Panel rapidly identifies viruses, bacteria, and antimicrobial resistance genes in
lower respiratory specimens. To aid in the diagnosis of infections from complex specimens, the panel
provides molecular estimates of abundance for 15 bacteria. Values are reported as a bin result in 1-log
intervals over a clinically relevant range (Not Detected, 10^4, 10^5, 10^6, and ≥10^7 copies/mL). Bin
results are useful for assessing relative abundance of bacteria in polymicrobial specimens and can be
interpreted similarly to quantitative and semi-quantitative culture methods currently used in the
diagnosis of lower respiratory infections. The 1-log range of each bin result provides accuracy within ±
0.5-log, but precision will vary from 50-100% based on the concentration of the bacteria relative to the
bin limits. For example, when an analyte concentration is near the center of the 1-log range, precision
with repeated testing will be high (>90%). Precision will also be high at the extremes of the reported
range (Not Detected and ≥10^7 copies/mL). By contrast, when analyte concentration approaches a
boundary between two bins, both bin results are equally probable and accurate (within ± 0.5 log).
However the precision of the bin result will decrease and may be as low as 50%. Precision testing for the
FilmArray Pneumonia Panel was performed on samples containing multiple bacteria at different
concentrations from 10^2 - 10^7.5 copies/mL. Ninety replicates per sample were tested over several
days on multiple reagent lots and different FilmArray® Instruments. Precision was measured as the
distribution (%) of replicates per bin per concentration. Precision of relative reporting between analytes
in the same sample was also evaluated. Testing demonstrated that, as expected, precision was 98-100%
for the high and low bins (Not Detected, ≥10^7 copies/mL), was ~90-100% for inputs near the bin center
and was as low as 57% when at bin boundaries. In samples with two analytes at the same concentration,
bins were concordant in >90% of replicates when inputs were near a bin center and in 50-75% of
replicates for inputs at a bin boundary. Bin accuracy was reproducibly ± 0.5-log of the input value. These
data describe expectations for the accuracy and precision of FilmArray Pneumonia Panel estimates of
bacterial abundance and reliable assessment of relative abundance in polymicrobial specimens. Note:
Testing was performed with Investigational Use Only reagents. The FilmArray Pneumonia Panel has not
been evaluated by the U.S. FDA or other regulatory agencies.
Session Number: 220
Session Number: 220
Abstract Title:
Seasonality of Percent Positivity and Experience with Reflexive Culture with A Clinically-Performed
Legionella Pcr Assay
S. Rucinski, M. Murphy, J. Mainella, K. Kies, S. Cunningham, A. Schuetz, R. Patel; Mayo Clinic, Rochester,
MN
Abstract Body:
We have performed an in-house developed LightCycler-based real-time PCR assay targeting the
Legionella 5S ribosomal RNA (rRNA) gene using FRET hybridization probes for product detection, on
sputum, tracheal secretions, bronchial washes and bronchoalveolar lavage fluid from Mayo Clinic and
Mayo Medical Laboratories patients for 10 years. PCR-positive specimens of sufficient quantity have
been reflexively submitted to Legionella culture by plating to BCYE agar with incubation at 35°C for 7
days. Suspect Legionella colonies have been identified using partial 16S rRNA gene sequencing or
MALDI-TOF MS. The positivity rate of our Legionella PCR assay over a 96 month period has shown an
apparent seasonal pattern, which we present here. Results of reflexive culture and their association with
assay crossing point (CP) values, as well as specific species isolated are also described. From Jan. 2010 to
Dec. 2017, 44,773 Legionella PCR tests were performed, with an overall positivity rate of 1.3%. Positivity
was highest in the summer and lowest in the winter (Figure). 490 positive specimens were submitted to
reflexive culture, of which 195 were positive and 295 were negative. The following species were
isolated: L. pneumophila (n=164), L. longbeachae (n=11), L. bozemanii (n=9), L. micdadei (n=5), and
other or non-identified Legionella species (n=6). 52% and 34% of the specimens with no growth had CP
values >35.00 and 30.01-35.00 cycles, respectively; 16%, 38% and 32% of the culture-positive specimens
had CP values of 20.01-25.00, 25.01-30.00 and 30.01-35.00 cycles, respectively. In conclusion,
approximately 40% of Legionella PCR-positive specimens were culture positive for Legionella species,
with the most common species found being L. pneumophila. Legionella PCR positivity has an apparent
seasonality, with higher rates in summer than in winter.<p><a
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Session Number: 220
Session Number: 220
Abstract Title:
Retrospective Analysis of Bronchoalveolar Lavage (Bal) Bacterial Culture Results to Optimize Culture
Incubation Time and Clinically Significant Reporting of Results
J. Witmier, D. S. S. Wijetunge, D. Myers, D. Craft; Penn State Hlth., Hershey, PA
Abstract Body:
Introduction: BAL obtained during bronchoscopy is minimally invasive and yields a fluid with high
diagnostic value for detection of lower respiratory tract pathogens. Bacterial culture incubation
protocols to final report may be dependent upon the pathogens frequently isolated from specific
patient populations. Optimizing incubation times can lead to lean work flow efficiencies and decreased
time to final reporting which can influence the clinical management of the patient. Our incubation
protocol for BAL cultures is 5 days before final report. The objective of this study was to evaluate the
optimal incubation time for BAL cultures in our patient population. Methods: All positive BAL cultures
obtained at our medical center from Oct 2016 to Oct 2017 were retrospectively reviewed for first
observation of growth, date of final report, and pathogens reported. Reports were quantitative (>10,000
cfu/ml cut-off) and organisms were identified and susceptibility tested based on etiology (pathogen) and
if part of normal respiratory flora (NRF). Results: 151 cultures were positive for one (n=109) or multiple
(n=42) pathogens. 20 different pathogens were recovered to include commonly isolated GPCs, glucose
non-fermenting GNRs and many different GNRs of the Enterobacteriaceae family. The most frequently
isolated pathogen was S. aureus (44), followed by M. catarrhalis (23) and H. influenzae (22).The majority
of pathogens were recorded within 2 days. 3 of 4 pathogens detected beyond 3 days were associated
with quantities near the quantitative cut-off value for clinical significance and in the presence of greater
quantities of NRF and/or other pathogens. One culture with >100,000 cfu/ml of both M. catarrhalis and
NRF at day 5 had no work-up comment until day 5. Additionally, a culture of P. aeruginosa at day 4 was
accompanied by a separate culture of tissue obtained simultaneously in which the same phenotype of P.
aeruginosa grew at day 2. Conclusions: Our study showed that 89.73% of all BAL pathogens were
detected within 2 days and 97.7% within 3 days in our patient population. Although 4 clinically
significant pathogens were reported after 3 days, all were associated with greater amounts of NRF or
other pathogens. Our data suggests that a 3 day incubation for BAL cultures is the most cost effective
and workflow efficient protocol that does not compromise clinically significant reporting. Additionally,
by finalizing negative cultures at 3 days, our clinicians will be able to narrow or deescalate to
appropriate therapy up to 2 days earlier (personal communication).
Session Number: 220
Session Number: 220
Abstract Title:
Automated Multiplexed Molecular Sys. for Detections of Bacteria and Viruses in Patients with
Pneumonia: Performance Confirmation by Alternate Methods
D. Judd, B. Graham, D. Sadrija, O. Cham, C. Li, J. Stone, K. Broadbent, C. Graue, M. Jones, J. Cloud, M.
Rogatcheva; BioFire Diagnostics LLC, Salt Lake City, UT
Abstract Body:
The FilmArray® Pneumonia Panel plus (BioFire Diagnostics, LLC) is intended to identify pneumonia
causing agents in unprocessed sputum (including endotracheal aspirate) and bronchoalveolar lavage
(BAL) specimens. This test detects 18 bacteria (15 reported semi-quantitatively when present at ≥ 104
copies/mL), 9 viruses, and 7 antibiotic resistance markers. Clinical performance was evaluated using an
Investigational Use Only version in a multi-center study. Bacteria reported semi-quantitatively were
compared to quantitative bacterial culture and quantitative Next Generation Sequencing (NGS) while
viral detections were compared to the standard of care results and two molecular assays. Any
discordant detections were investigated in this study to determine the root cause of false positive (FP)
and false negative (FN) results using additional molecular methods. Nucleic acids from residual
specimens with discordant results were extracted and tested using molecular assays targeting genes or
gene regions different from the FilmArray targets. Bi-directional sequencing of the amplified region(s)
was used for confirmation. Additionally, FN specimens were retested multiple times on the FilmArray
Pneumonia Panel plus. In a preliminary analysis of 1683 specimens (847 BAL and 836 sputum), 1174
bacterial and 438 viral pathogens were detected. 133 of these detections were FP (69 bacterial and 64
viral). All but three bacterial FP results have been sequence confirmed as true positive (TP) detections.
These samples contained the organism of interest but at a concentration below the quantitative
threshold of the comparator assay. To date, 35 viral FP’s have been confirmed as TP. The discrepant
analytes were present in these samples at a concentration below the limit of detection (LOD). There
were 50 FN results (32 bacterial and 18 viral) identified by comparator methods but not by FilmArray.
Analysis of the FilmArray data indicated that the many of the bacterial FN detections were on the
borderline-level or below the quantitative threshold. The majority of the samples with FN detections
showed positive detections for the analyte of interest when re-tested. Discrepancy investigation
substantiated that the FilmArray Pneumonia Panel plus detects targeted bacteria and viruses with high
sensitivity and specificity. The vast majority of the discrepant samples resulted from organism
concentrations below the LOD of the comparator and FilmArray assays. The FilmArray Pneumonia Panel
plus has not been evaluated by the FDA or other regulatory agencies
Session Number: 220
Session Number: 220
Abstract Title:
Comparison of Two Multiplex Panels for the Detection of Respiratory Pathogens
G. Berry, F. Zhang, M. Olszewski, S. Juretschko; Northwell Hlth.Lab., Lake Success, NY
Abstract Body:
Background Multiplexed testing for respiratory pathogens are becoming more common in laboratory
testing. We evaluated one of these multiplexed testing platforms, the ePlex respiratory pathogen (RP)
panel (GenMark), and compared it to the Filmarray RP panel (BioFire) to determine performance
characteristics. Accuracy, precision, and correlation with previously characterized patient specimens
were assessed. Materials and Methods Testing was performed following the FDA-approved package
insert instructions. Accuracy and precision for the ePlex RP panel were established using the
ZeptoMetrix quality control (NATtrol™ QC) (ZeptoMetrix) and the Maine Molecular Quality Control
(ePlex RP Control M306) (Maine Molecular) panels. A patient correlation was also done using a
combination of frozen retrospective (n= 52) and fresh prospective (n=41) patient specimens in Universal
Transport Media (UTM) during October of 2017. Results Overall, the sensitivity of the ePlex RP panel
ranged from 85.71% to 100% and specificity ranged from 97.22% to 100.00% when using the
ZeptoMetrix panel. Specifically, there were three targets that showed reduced sensitivity (Coronavirus,
Mycoplasma pneumoniae, parainfluenza virus type 4), potentially due to target sample titers in the
mixed positive specimen pool being near the limit of detection for the assay. The Maine Molecular panel
was also tested and achieved 100% reproducibility for all targets. Ninety-three clinical specimens were
evaluated by both the ePlex RP panel and the Filmarray RP panel as a patient correlation. While
sensitivity and specificity of frozen specimens ranged from 75% to 100% and 98% to 100%, respectively,
prospective specimens showed 100% correlation between both methodologies (although not all targets
were detected during the testing timeframe). Further analysis of discordant results revealed that 2
coronavirus and 1 Metapneumovirus targets were not detected by the ePlex, but were detected by the
Filmarray, and 1 Parainfluenza virus type 4 was detected by the ePlex, but was not detected by the
Filmarray. This can be attributed to stated sensitivity differences between the two assays listed in the
ePlex FDA-approved package insert. In addition, one Influenza A H3N2 was also detected by the
GenMark, but was untypable and negative upon repeat on the Filmarray (consistent with a positive, but
low level viral titer). The overall concordance between the two assays was 95.7% (89/93). Conclusions
Overall, the GenMark ePlex RP panel performed comparably to the BioFire Filmarray RP panel.
Session Number: 220
Session Number: 220
Abstract Title:
Multicenter Validation of A Chromogenic Medium for Screening of Staphylococcus aureusIn Respiratory
Specimens from Cystic Fibrosis Patients
M. Gaskin1, A. Ferroni2; 1Hamilton Hlth.Sci., Hamilton, ON, Canada, 2Hosp. Necker Enfants Malades,
Paris, France
Abstract Body:
Background: Staphylococcus aureus (S. aureus) causes purulent bacterial infections, many of which can
lead to serious complications resulting in significant morbidity and healthcare costs. A quarter of the
population carry S. aureus asymptomatically and its early detection is vital in preventing transmission
and subsequent infection. In patients with cystic fibrosis, S. aureus is one of the most commonly isolated
pathogens and is associated with advanced pulmonary disease. The objective of this study was to
validate the use of CHROMagar™ Staph aureus agar to screen for S. aureus in nasal surveillance
specimens and respiratory specimens from cystic fibrosis patients. The study was carried in two
hospitals: Hamilton (ON - Canada) and NECKER (Paris - France). Material/Methods: In this study a total
of 200 clinical specimens were collected and seeded onto CHROMagar Staph aureus agar plates and
incubated for 20 hours in an aerobic environment at 35 degrees C at which point analysis was
performed. Maldi-ToF was performed on target and non-target colour colonies. Results were compared
to the same samples set up on Mannitol Salt agar incubated at 35 degrees C for 20 hours. Results: Of the
200 specimens tested, 81 were positive for S. aureus in both agars, an additional 9 specimens tested
positive for S. aureus only on CHROMagar ™ Staph aureus and 1 only on Mannitol Salt agar, for a total of
91 positive specimens. 18 specimens showed non-target colour growth, usually white or blue, on
CHROMagar™ Staph aureus. These colonies were identified as Staphylococcus haemolyticus, E.faecalis.
Two light pink colonies identified as Staphylococcus schleiferi and Staphylococcus epidermidis.
CHROMagar™ Staph aureus agar showed a sensitivity of 99% (95%CI 0.94-1) and a specificity of 100%
(95%CI 0.97-1) as compared to Mannitol Salt agar which showed a sensitivity of 89% (95%CI 0.81-0.94).
Conclusions: Results showed CHROMagar™ Staph aureus had a significantly greater sensitivity than
Mannitol Salt in isolating S. aureus from nasal surveillance specimens and respiratory specimens from
cystic fibrosis patients. S. aureus colonies were easily differentiated as mauve colour and most
breakthrough growth was inhibited.
Session Number: 220
Session Number: 220
Abstract Title:
Evaluation of the Filmarray Meningitis/Encephalitis (Me) Panel
S. Mineau1, M. Kissoon1, B. Willey1, J. Augustine1, S. Poutanen1, J. Ng1, D. Boyd2, J. Gubbay3, E.
McLachlan2, T. Mazzulli1; 1Univ. Hlth.Network/Sinai Hlth.System, Toronto, ON, Canada, 2Natl.
Microbiol. Lab. / Publ. Hlth.Agency of Canada, Winnipeg, MB, C
Abstract Body:
Background: Meningitis and encephalitis are serious medical conditions that can be caused by numerous
microorganisms and for which a rapid and accurate etiologic diagnosis is of utmost importance. The
FilmArray ME Panel is a multiplex PCR assay that can detect 14 bacterial, viral and fungal
microorganisms in cerebrospinal fluid (CSF) samples with short turnaround time. The objective of this
study was to evaluate the performance of this assay. Methods: CSF that tested negative for all the
microbiology tests ordered by the responsible physician at the time of collection were selected: 40
samples were spiked with a total of 4 to 9 microorganisms per sample and 10 samples were not spiked
with targets included in the assay. In addition, CSF known to be positive for HSV-2, HHV-6 and VZV were
selected. For bacteria and yeasts, isolates recovered from invasive clinical infections were used to spike
study samples with a targeted concentration of 10 to 100 times the lower limit of detection of the assay.
Serogroup B, C, Y and W135 N. meningitidis were included as well as 11 different serotypes of S.
pneumoniae. For viruses, enterovirus, HSV-1, HSV-2, VZV and human parechovirus positive cultures, as
well as a CMV positive plasma, were used to spike study samples. Reproducibility was assessed by
testing selected samples in triplicate. Discrepancies were resolved by testing with another method that
was considered to be validated or verified. Results: The sensitivity for each microorganism, with the
denominator corresponding to the number of different isolates tested, is shown in the table. No false
positive results were obtained. The reproducibility was 100%. Conclusions: The FilmArray ME Panel
showed adequate sensitivity, specificity and reproducibility in our study. Further study is needed to
better evaluate the detection of those targets with small number of samples used in this study. The
clinical role and impact of this assay also need to be determined in the patient care setting.<br /><p><a
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Session Number: 220
Session Number: 220
Abstract Title:
Application of Real Time Pcr in the Diagnosis of Acute Bacterial Neonatal Meningitis in A Tertiary Hlth.
Care Ctr. in India
M. S. Raza1, A. Kapil1, V. Goyal1, R. Lodha1, S. Sood1, H.Gautam1,, K. Saigal2, A. Ghos2, 2 CNBC, New
Delhi, S. Mohapatra1, R. Chaudhry1, B. K Das1; 1All India Inst. of Med. Sci., New Delhi, India
Abstract Body:
Background: Acute bacterial meningitis in neonates is known to have significant morbidity and mortality.
One in ten neonates die from meningitis and majority of the survivors develop significant lifelong
complications including seizures, impaired vision and hearing loss. Diagnosis of neonatal meningitis is
technically challenging, invasive and time-consuming as the majority of babies receive antibacterial
therapy before getting admitted at the centre. Real time PCR accelerates the diagnostic approach.
Methods: 25 CSF were collected from March, 2015 to December, 2017. All samples were processed in
the department of Microbiology, AIIMS, following standard Microbiological diagnostic procedure. Blood
agar and MacConkey agar were used for culture. Brain heart infusion broth with 0.5% yeast extract was
used for culture of L . monocytogenes. DNA was extracted from CSF by using QIAamp DNA mini Kit
(Qiagen). Conventional as well as Real time PCR were performed to test each sample for S. agalactiae, E.
coli and L. monocytogenes. Results: Overall 25 CSF samples were processed by conventional and
molecular methods, four CSF (16.1%) were positive for organisms by molecular methods. Out of the four
organisms detected, three were E. coli (two were detected by Real time PCR, and one detected by both
conventional and Real time PCR) , and one was S. agalactiae ( detected by real time PCR). All samples
were culture negative for S. agalactiae, E. coli and L. monocytogenes. Mean glucose and protein level
was (38 and 184/dl) respectively. PMN cell count was high. Conclusions: Real time PCR has becoming the
powerful tool for the rapid diagnosis of acute bacterial neonatal meningitis where there is the need for
urgent diagnosis and treatment. It will help in saving the life of the neonates.
Session Number: 220
Session Number: 220
Abstract Title:
Rapid Identification of Organisms from Sterile Sites in Pediatric Patients Using Rapid Molecular Blood
Culture Identification Panel
S. L. Hamilton1, K. Messacar2, A. M. Prinzi1, J. C. Mitchell1, E. D. Beil1, E. B. Dowell1, S. R. Dominguez2;
1Children's Hosp. Colorado, Aurora, CO, 2Univ. of Colorado, Aurora, CO
Abstract Body:
Background: The impact of rapid identification methods, such as the Biofire FilmArray Blood Culture
Identification panel (BCID), in decreasing time to organism identification and antibiotic utilization for
patients with bacteremia has been well established. The performance and impact of the BCID panel on
other sources has not been well studied. Methods: This was a two-part study that ran concurrently. For
part one, 91Gram stain positive broth cultures (BD Peds Plus blood culture bottles inoculated with
sterile body aspirates and tissues) were tested prospectively by BCID in parallel with bacterial culture
and organism identification by Bruker Maldi-TOF. Broth cultures were from the following sites: 46
tissues (including abscess, cellulitis, and lymph nodes), 21 bones, 15 cerebrospinal fluids from
ventriculoperitoneal shunts, 5 synovial fluids, and 4 pleural fluids. The second part involved a “spiking
experiment” utilizing organism pools that encompassed all targets on the BCID panel. Source pools were
created utilizing residual patient specimens to account for matrix interferences. A negative control was
prepared from each source pool inoculated in a blood culture bottle without organisms. Results: For
each source, all targets from organism pools were detected and the negative pool had no targets
detected. Of 91 samples tested by BCID, 90 (99%) agreed with bacterial culture. One discrepant result
was obtained from a bone sample which had polymicrobial growth of 3 organisms. Two samples were
reported from BCID as MRSA due to the detection of S. aureus and mecA gene, but the cultures were
positive for MSSA and oxacillin resistant coagulase negative staphylococcus (CONS). There were 12
(13%) samples that grew organisms on culture that were not targets included on the BCID panel and 4
(4%) samples that grew organisms that where only flagged with the Enterobacteriaceae target.
Conclusions: The Film Array BCID system displayed excellent sensitivity and specificity on positive broth
cultures from sterile site tissues and aspirates from pediatric patients and can easily be adapted to use
on these sources. Limitations of this assay included decreased sensitivity for detection of organisms in
polymicrobial cultures, inability to distinguish MRSA from MSSA with CONS carrying the mecA gene, and
organism detection limited to included targets Adding a rapid identification to sterile site sources has
the potential to improve time to identification for causative agents and facilitate timely antibiotic
intervention.
Session Number: 220
Session Number: 220
Abstract Title:
Reduction of Blood Culture Contamination Using Initial Specimen Diversion Device
J. Blakeney; Beebe Hlth.care, Lewes, DE
Abstract Body:
Background: Contaminated blood cultures remain a significant problem at our institution despite
repeated sterile technique education efforts. Our historical blood culture contamination (BCC) rate is
2.8% (about 250 contamination events/year), with higher rates (>4%) in some months. False positive
blood culture results can lead to unnecessary antibiotic treatment, longer stays, and increased costs
(estimated to be $3,500/event). The SteriPath (SP) device (Magnolia Medical Technologies) is designed
to reduce contamination by diverting and isolating the initial 1.5-2 mL of blood, which is most likely to
contain skin contaminants. We instituted a trial of this device in an effort to reduce the incidence of
contamination events. Methods: The study was conducted at Beebe Healthcare over 16 weeks. Blood
cultures drawn in the ED, ICU, and by the phlebotomy team were collected using either the SP device or
standard method (SM). The collection method was recorded for each sample, and the number of false
positive events was recorded for each method. A culture was considered contaminated if normal skin
bacteria grew in only one culture set within a three-day period. Results: A total of 2639 blood cultures
were collected between 5/30/207 and 9/16/2017, with a total of 38 contamination events (1.44%). SP
was used to collect 1837 cultures; SM was used for the remaining 802. The SP group had 14
contamination events (14/1837, 0.76%). The SM group had 24 contamination events (24/802, 2.99%).
There was a 75% reduction in the BCC contamination rate in the SP group compared to the SM group
(χ2=18.029, p<.0001) Conclusion: Use of the SP device led to a significant decrease in the BCC rate in all
groups: ER, ICU, and phlebotomy team. The overall contamination rate for the 16-week period was
1.44% (using both SP and SM), a 49% reduction compared to the historical rate of 2.8%. These results
led us to adopt the SP device as standard practice. With SP we have maintained an average BCC rate of
1.5% in the months following the study.
Session Number: 220
Session Number: 220
Abstract Title:
Evaluation of the T2bacteria Panel (T2bp) Compared to Standard Blood Culture At Ochsner Med. Center
M. Ogawa, C. J. Wargo, H. Marty-Vigo, R. Hooper, D. S. Ashcraft, S. Simar, G. A. Pankey; Ochsner Clinic
Fndn., New Orleans, LA
Abstract Body:
Background: The T2Dx fully-automated instrument performs whole blood DNA amplification and uses
novel magnetic resonance technology with the T2BP for direct detection of species-specific amplicons of
Acinetobacter baumannii, Entercoccus faecium, Escherichia coli, Klebsiella pneumoniae, Pseudomonas
aeruginosa and Staphylococcus aureus at a limit of detection as low as 2 CFU/mL in 3-5 h. The goal of
our study was to validate the T2BP compared to standard blood culture (BC) in patients at the Ochsner
Medical Center. Methods: From March - August 2017, 3.0 mL K2EDTA whole blood samples were
collected from 178 consented patients from the same site and needle (immediately after an ordered
blood culture). BACTECTM FX BC isolates were identified by MALDI-TOF. T2BP positive and negative
external controls were tested daily, and an internal control was included with each sample for
monitoring performance in each reaction. Results: T2BP detected 32 organisms (12 E. coli, 9 P.
aeruginosa, 6 S. aureus, 5 K. pneumoniae) from 31 of 178 patients. BC identified 10 (4 P. aeruginosa, 4 S.
aureus, and 2 E. coli). 14/22 T2BP-positive/BC-negative samples were considered positive since
temporally related BCs or other site cultures grew the same bacteria. The remaining 8 were considered
false positives. No A. baumannii or E. faecium were identified. The 147 T2BP negative tests had no
growth on BC (true negatives). T2BP demonstrated an overall average 99% specificity and 100%
sensitivity when compared to BC.<table class="AbstractTable" id="{89C45F83-6390-4603-ACC6-
ED7F43B291E8}"><caption class="AbstractTableCaption"></caption><tr><td rowspan="1"
colspan="1">Organism</td><td rowspan="1" colspan="1">T2BP<br />positive</td><td rowspan="1"
colspan="1"># BC positive/<br /># Positive by Chart Review</td><td rowspan="1" colspan="1">Total<br
/>Positive<br />by culture</td><td rowspan="1" colspan="1">% Specificity of T2BP: <br />BC/ BC +
Positive by Chart Review </td></tr><tr><td rowspan="1" colspan="1">A. baumannii</td><td
rowspan="1" colspan="1">0</td><td rowspan="1" colspan="1">0/0</td><td rowspan="1"
colspan="1">0</td><td rowspan="1" colspan="1">100/100</td></tr><tr><td rowspan="1"
colspan="1">E. coli</td><td rowspan="1" colspan="1">12</td><td rowspan="1"
colspan="1">2/4</td><td rowspan="1" colspan="1">6</td><td rowspan="1"
colspan="1">94/97</td></tr><tr><td rowspan="1" colspan="1">E. faecium</td><td rowspan="1"
colspan="1">0</td><td rowspan="1" colspan="1">0/0</td><td rowspan="1" colspan="1">0</td><td
rowspan="1" colspan="1">100/100</td></tr><tr><td rowspan="1" colspan="1">K.
pneumoniae</td><td rowspan="1" colspan="1">5</td><td rowspan="1" colspan="1">0/5</td><td
rowspan="1" colspan="1">5</td><td rowspan="1" colspan="1">97/100</td></tr><tr><td rowspan="1"
colspan="1">P. aeruginosa</td><td rowspan="1" colspan="1">9</td><td rowspan="1"
colspan="1">4/3</td><td rowspan="1" colspan="1">7</td><td rowspan="1"
colspan="1">97/99</td></tr><tr><td rowspan="1" colspan="1">S. aureus</td><td rowspan="1"
colspan="1">6</td><td rowspan="1" colspan="1">4/2</td><td rowspan="1" colspan="1">6</td><td
rowspan="1" colspan="1">99/100</td></tr><tr><td rowspan="1" colspan="1">Total</td><td
rowspan="1" colspan="1">32</td><td rowspan="1" colspan="1">10/14</td><td rowspan="1"
colspan="1">24</td><td rowspan="1" colspan="1"></td></tr></table> Conclusion: Based on limited
testing, the T2BP seems very promising. T2BP did not miss any growth from BC of a bacteria species on
panel (100% sensitivity) and detected 24/32 true positives (99% average specificity). The T2BP false
positives may represent transient bacteremia, dead intact bacteria, contamination, instrument error, or
true bacterial infections too low in count to be detected by standard BC. Our study suggests that the
T2BP can provide highly sensitive and specific results in hours vs days for a standard BC. Enabling faster
appropriate antibiotic choice for bloodstream infections and decreasing unnecessary antibiotic use
should make T2BP a valuable, complementary tool to use with traditional BC, which remains necessary
for bacterial susceptibility testing.
Session Number: 220
Session Number: 220
Abstract Title:
Direct Pcr Testing of Blood Cultures to Predict Resistance of Gram-Negative Enterobacteria to β-Lactam
Antibiotics
C. J. McIver, R. P. Stevens, P. C. Taylor; New South Wales Hlth.Pathology, Sydney, Australia
Abstract Body:
Background: Third generation cephalosporins, β-lactam-β-lactamase inhibitor combinations (e.g.
piperacillin + tazobactam) or carbapenems, alone or in combination, are used as first-line treatment for
sepsis due to Gram-negative enterobacteria. Early accurate prediction of resistance to β-lactam
antibiotics assures optimal treatment selection and may influence clinical outcome and antibiotic
stewardship decisions. We describe a low cost method for rapid detection of resistance genes direct
from positive blood cultures. Methods: Real time multiplex PCR assays targeting three classes of
plasmid-mediated β-lactamases (mPCR): carbapenemases (bla-IMP, -OXA-48, -NDM, -KPC, and -VIM),
ESBLs (bla-CTX-M-1 & -9, -SHV, and -VEB) and ampC (bla-ACT, -MIR, -LAT, -DHA, and CMY-2), and a
uniplex: bla-TEM were configured for the direct analysis of flagged BactALERT® blood cultures
(bioMerieux). mPCR assays detect the β-lactam class without specifying the putative gene, allowing 5
targets to be detected in the LightCycler 2.0 (Roche) saving both time and money. Bacteria were
harvested and tested directly from blood cultures without prior genomic extraction as previously
described1, a process taking less than one hour. Harvested isolates were also identified by MALDI-TOF
MS analysis (Bruker Daltonics). Assays were validated in 32 culture bottles (16 aerobic/anaerobic) spiked
with enterobacteria harbouring one or more of the putative genes before application to 53 clinical blood
cultures (26 aerobic / 27 anaerobic). Molecular results were compared to phenotypic susceptibility
determined by the CDS method2. Results: Putative genes were detected in all 32 spiked-blood cultures
and in 52/53 clinical isolates. There was no difference in detection between bottle types. Prediction of
resistance to β-lactam antibiotics concurred with phenotypic testing. Failure to detect bla-TEM in one
isolate of E. coli prevented prediction of ampicillin resistance. Conclusions: In 98.1% of 53 clinical blood
cultures tested, this low cost method rapidly detected resistance to β-lactam antibiotics used as a first-
line treatment of septicaemia caused by Gram-negative enterobacteria. Application can potentially
improve clinical outcome with implications for antibiotic stewardship.
Session Number: 220
Session Number: 220
Abstract Title:
A Prospective Clin. Study on Optimization of Blood Culture Sampling
D. Yu1, Å. Parke1, C. Unge1, C. Henning2, A. Somell1, J. Sundén-Cullberg1, K. Strålin1, V. Ozenci1;
1Karolinska Univ. Hosp., Stockholm, Sweden, 2Sahlgrenska Univ., Borås, Sweden
Abstract Body:
Background: Optimal blood culture (BC) sampling is probably the most important factor in the outcome
of blood culture based diagnosis of sepsis. The aim of the present study was to analyze the effect of
patient selection criteria and different sampling strategies in order to improve blood culture sampling.
For a final sepsis diagnosis, an established infection and an increase of ≥2 between baseline and acute
sequential organ failure assessment score was required (sepsis-3). Methods: A new triage model for
patients suspected with sepsis was established at the ER of Karolinska University Hospital. In total six
blood culture (BC) bottles (four from the first arm, BC1, BC2, BC3, BC4, and two from the other, BC5,
BC6) were collected from each patient that fulfilled the criteria for triage. BC1, BC3 and BC5 were
aerobic BacT/ALERT FA Plus whereas BC2, BC4 and BC6 were anaerobic BacT/ALERT FN Plus bottles. The
effect of numbers of bottles and single vs. two sampling on blood culture positivity was analyzed. In
addition, correlation between the triage model and consequent clinical diagnosis of sepsis and blood
culture positivity was studied. Results: A total of 121 patients were enrolled in the triage model. 14
patients had less than 6 BC bottles and were excluded. 642 bottles from 107 patients were analyzed.
There were 44 patients with 219/642 positive BC bottles. Nine patients had contaminant bacteria in
16/642 (2.49%) bottles. Of the remaining 35 patients with clinically relevant bacteria, 30 had
monomicrobial and 5 had polymicrobial growth. In 203/642 (31.62%) bottles growth of clinically
relevant bacteria was observed. Growth of clinically relevant bacteria could be observed in 24 BC1, 30
BC2, 31 BC3, 28 BC4, 33 BC5, and 29 BC6 bottles. All clinically relevant bacteria detected in BC5 and BC6
except one Pseudomonas aeruginosa that grew only in BC5 were also detected in at least one of the
four BC bottles from the first sampling. In contrast, two Enterococcus faecium, one E. faecalis and one
Enterococcus spp. were only detected in at least one of the four BC bottles from first sampling but not in
BC5 and BC6 from the other. Clinical sepsis was observed in 26/35 (74.29%) patients who had clinically
relevant growth and 34/72 (47.22%) who had no growth or contaminants in BC bottles. Conclusion: The
triage model worked successfully with a high proportion of sepsis patients with clinically relevant
bacteria in blood culture. Single sampling model seems to be non-inferior to double sampling strategy.
Session Number: 220
Session Number: 220
Abstract Title:
Fourteen Day Blood Culture Incubation Aids in the Diagnosis of Cutibacterium Acnes Endocarditis
B. Dylla, M. Sohail, B. Pritt, A. Schuetz, R. Patel; Mayo Clinic, Rochester, MN
Abstract Body:
Blood cultures may be negative in a significant percentage of cases of infective endocarditis (IE).
Clinicians frequently request that the clinical laboratory extend the duration of incubation of blood
cultures in cases of suspected IE. Moreover, Clinical and Laboratory Standards Institute (CLSI) guidelines
recommend a longer incubation or terminal subculture if blood cultures are negative at 5 days in some
patients with suspected IE. However, the clinical utility of these practices is unclear. We performed a
prospective study from December 15, 2015 to December 21, 2017 to evaluate the utility of prolonged
blood culture incubation and blind subculture on blood cultures for cases in which clinicians requested
extended blood culture incubation, typically for suspected endocarditis. Routine blood cultures were
performed using the Becton Dickinson BD BACTEC FX™ platform with a blood culture set consisting of
two BD BACTEC™ Plus Aerobic/F bottles and one BD BACTEC™ Lytic Anaerobic/F bottle. Over the study
period, clinicians requested extended blood culture incubation on 134 blood cultures from 65 patients.
The incubation duration was extended from the standard 5 days to 10 or 14 days for 17 and 117
cultures, respectively. Bottles were removed briefly on day 5 of incubation, subcultured to chocolate
blood agar, and returned to the blood culture instrument to fulfill the extended incubation period.
Subcultures were incubated for 5 days at 37°C in CO2. Terminal blind Gram and acridine orange stains
were performed on 105 negative cultures on the last day of incubation. Results of all blind subcultures
and terminal Gram and acridine orange stains were negative. Beyond 5 days of incubation, there were 5
positive blood cultures in three patients, all for Cutibacterium acnes. One patient had a single positive
culture after 138 hours of incubation, determined clinically to be a contaminant. The other two patients
had two positive cultures each, after 153 and 168 hours of incubation for one and 178 and 182 hours of
incubation for the other; both were diagnosed with prosthetic aortic valve endocarditis, confirmed by
histopathology, with findings compatible with C. acnes infection. The second case additionally had a
positive valve culture for C. acnes. In conclusion, a 5 day standard blood culture incubation time is
adequate for recovery of almost all cultivable causes of endocarditis except C. acnes, for which extended
incubation is useful. Blind subculture at day 5, and terminal Gram and acridine orange stains of negative
blood culture bottles are not recommended.
Session Number: 220
Session Number: 220
Abstract Title:
Comparative Evaluation of Bactec Fx, Bactalert Dual-T, and Usp<71> Sys. for Sterility Testing of Cellular
Therapy Products
M. England, F. Stock, J. Gebo, K. Frank, A. Lau; NIH, Bethesda, MD
Abstract Body:
Introduction: Biopharmaceutical programs encompassing cellular engineering and immunotherapies for
targeted cancer treatment in large academic centers has led to an increasing demand on clinical labs to
assist with product sterility testing. We performed a large-scale growth promotion evaluation of three
test systems (compendial USP<71>, Bactec, and BacTAlert Dual-T), to assess performance, measuring
time to positivity (TTP) for bacteria, yeast, and mold. Methods: 109 challenge isolates (47 Gram positive
bacteria, 21 Gram negative bacteria, 7 yeasts, and 34 molds), representing organisms from the NIH
cGMP environment or from previously contaminated products, were evaluated. Bottles were seeded
with <100 CFU. For USP<71>, each of two TSB and Thio broth culture sets were incubated at 25°C and
35°C (compendial method specifies only TSB at 25°C; Thio at 35°C). Similar conditions were applied for
two sets of aerobic and anaerobic plus bottles for the BacTAlert Dual-T. One set of aerobic and
anaerobic plus bottles was incubated at 35°C for Bactec. TSB and Thio bottles were inspected daily for
turbidity. All positive bottles were confirmed by Gram stain morphology. Per USP, growth promotion
was acceptable if growth of bacteria and fungi was detected within ≤3 days and ≤5 days, respectively.
Results: <table class="AbstractTable" id="{B0621517-244E-477B-99DC-4BB7C02FA099}"><caption
colspan="7">Growth detected (%)</td></tr><tr><td rowspan="2" colspan="1">TTP</td><td
rowspan="2" colspan="1">Organism Type (N)</td><td rowspan="1" colspan="3">USP<71></td><td
rowspan="1" colspan="3">BacTAlert</td><td rowspan="1" colspan="1">Bactec</td></tr><tr><td
rowspan="1" colspan="1">Both 35°C</td><td rowspan="1" colspan="1">Both 25°C</td><td
rowspan="1" colspan="1">TSB 25°C<br />Thio 35°C</td><td rowspan="1" colspan="1">Both
35°C</td><td rowspan="1" colspan="1">Both 25°C</td><td rowspan="1" colspan="1">Aer 25°C<br
/>Ana 35°C</td><td rowspan="1" colspan="1">Both 35°C</td></tr><tr><td rowspan="5"
colspan="1">≤3 Days Bact.<br />≤5 Days Fungi</td><td rowspan="1" colspan="1">GP (47)</td><td
rowspan="1" colspan="1">74.5</td><td rowspan="1" colspan="1">51.1</td><td rowspan="1"
colspan="1">72.3</td></tr><tr><td rowspan="1" colspan="1">GN (21)</td><td rowspan="1"
colspan="1">76.2</td></tr><tr><td rowspan="1" colspan="1">Yeast (7)</td><td rowspan="1"
colspan="1">71.4</td></tr><tr><td rowspan="1" colspan="1">Mold (34)</td><td rowspan="1"
colspan="1">32.4</td></tr><tr><td rowspan="1" colspan="1">Total (109)</td><td rowspan="1"
colspan="1">60.6</td></tr><tr><td rowspan="5" colspan="1">14 Days</td><td rowspan="1"
colspan="1">GP (47)</td><td rowspan="1" colspan="1">85.1</td><td rowspan="1"
colspan="1">91.5</td><td rowspan="1" colspan="1">83.0</td></tr><tr><td rowspan="1"
colspan="1">GN (21)</td><td rowspan="1" colspan="1">61.9</td><td rowspan="1"
colspan="1">Yeast (7)</td><td rowspan="1" colspan="1">42.9</td><td rowspan="1"
colspan="1">Mold (34)</td><td rowspan="1" colspan="1">11.8</td><td rowspan="1"
colspan="1">Total (109)</td><td rowspan="1" colspan="1">55.0</td><td rowspan="1"
colspan="1">90.8</td><td rowspan="1" colspan="1">68.8</td></tr></table> Conclusions: The USP<71>
compendial method yielded a 56% success rate. The BacTAlert system (bottles at 35°C) performed best
(72.5%) across all organism groups; detection improved to 86.2% if growth was extended to 14 days (as
indicated for product sterility testing). Our data shows that mold detection across all systems is
suboptimal; alternate methods must be applied for high-risk products. For 11 bottles (10.8%), mold was
visibly present but failed to be detected with the automated systems. 42 isolates were detected across
all culture combinations; of these, TTP was faster with the BacTAlert at 35°C by a range of 2-48 h. This
large dataset demonstrates the limitations of culture test systems for the detection of environmental
microbial contaminants that are different than routine clinical isolates. Thorough assessment of
performance is essential for ensuring patient safety.
Session Number: 220
Session Number: 220
Abstract Title:
Benefits of Adding Anaerobic Blood Culture Bottles in the Diagnosis of Bloodstream Infections in
Children
J. Dien Bard1, N. Lichtenfeld2, H. Choi3, K. Manshadi2, T. Chang1, A. Festekjian1; 1Children's Hosp. Los
Angeles; Univ. of Southern California, Los Angeles, CA, 2Children's Hosp. Los Angeles, Los Angeles, CA,
3Western Univ., Pomona, CA
Abstract Body:
Background: Anaerobes account for up to 20% of all bloodstream infections. In contrast to adults, the
addition of an anaerobic (ANA) blood culture (BC) to the standard-of-care aerobic (AER) BC for pediatric
patients is controversial. Recent studies have demonstrated a striking increase in ANA BSI in both adult
and pediatric patients. We sought to assess the potential benefits of including ANA BCs in diagnosing
pediatric BSI at a free-standing, tertiary care pediatric medical center. Methods: A full BC set consisting
of ANA and AER BC bottles was implemented in the Emergency Department (ED) on February 1, 2017.
Prevalence of aerobic and anaerobic bloodstream infections was determined, and the performance of
ANA and AER BCs in the recovery of facultative anaerobes was compared. Statistical analysis was
conducted using unpaired t test and Fisher’s exact test. Results: A total of 4829 blood culture bottles
were collected in the ED between February and December, 2017; 2490 AER and 2339 ANA. Compliance
rate for inclusion of ANA BC bottle was 93.9%. A total of 363 (7.5%) positive BCs were identified, 172
(47.4%) of which were from ANA bottles. 120 organisms were recovered from both AER and ANA
bottles. An additional 71 (19.6%) and 52 (14.3%) positives were recovered from only AER and ANA BCs,
respectively. Of the 52 positive by ANA BCs only, 23 (44.2%) were considered true pathogens, including
Enterobacteriaceae (7), Streptococcus pneumoniae (2), Staphylococcus aureus (4). Recovery of true
pathogens in AER BCs only were lower at 31.0% (22/71) and were predominantly Enterobacteriaceae (9)
and other gram-negative organisms including, Haemophilus influenzae (3), Acinetobacter spp. (2), and
Moraxella catarrhalis (2). The prevalence of anaerobes was low at 5.2% (9) with recovery of obligate
anaerobes (3) and Cutibacterium acnes (6). The mean time to positivity was 3 hours faster in ANA BCs
(17.7 vs 14.6 h; P = 0.0874). ANA BCs flagged positive first 56.9% of the time compared to 25.7% with
AER BCs (P = 0.0001). Recovery of probable contaminants was comparable between bottles at 43.6% for
ANA BCs and 50.8% for AER BCs (P = 0.2065). Conclusions: Addition of ANA BC bottles proved to be
beneficial in children presenting to the ED. Despite the low prevalence rate of anaerobes, inclusion of
ANA BC bottles allowed for the recovery of a number of true pathogens that were negative in AER BC
bottles. Moreover, ANA BC bottles were likely to detect bacterial growth faster than AER BC bottles.
Session Number: 220
Session Number: 220
Abstract Title:
Prognostic Value of Time Durations for Positive Blood Culture Signals in Bacteraemia
A. A. Shittu1, S. B. A. Oseni2, A. O. Aboderin2; 1Ekiti State Univ., Ado-Ekiti, Nigeria, 2Obafemi Awolowo
Univ. Teaching Hosp. Complex, Ile Ife, Nigeria
Abstract Body:
Background: The aim was to determine the association of bacteria growth in blood culture sample with
the outcome of infection in children, for improvement of patients’ care in clinical practice. Methods:
Ethical approval for the study was given by the Ethics and Research Committee of Obafemi Awolowo
University Teaching Hospitals Complex (OAUTHC), Ile-Ife. Consecutive children, 14 years of age and
below presenting with fever of ≤48 hours duration, were recruited through the emergency units of
OAUTHC. Patients’ blood sample was taken for culture using BACTEC 9050. Relevant demographic and
clinical information including treatment outcomes were obtained using a proforma. Time duration for
positive blood culture signal was also noted. Data was analyzed using appropriate descriptive and
inferential statistics using SPSS version 22 IBM. Results: Three hundred and forty three patients were
studied within 13 months period. Of the 117 positive blood cultures, 80 (68.4%) were positive within 24
hours. The time to positivity of cultures ranged from 24 to 120 hours with the median growth time of 24
hours. The mortality rate was 15% in those that were positive within 24 hours as against 8.1% for those
that were positive after 24 hours (OR, 2.3; 95% CI, 1.09 to 4.84). Positive blood culture signal within 24
hours is a risk for mortality (P = 0.028). Conclusion: The time to positivity in bacteraemia provides useful
prognostic information. Positive blood culture signal within 24 hours is a predictor of mortality among
cases of bacteraemia; efforts should therefore be made to intensify the treatments of patients in this
category so as to mitigate mortality. Riad Khatib, Kathleen Riederer, Sajjad Saeed, Leonard B. Johnson,
Mohamad G. Fakih, Mamta Sharma, M. Shamse Tabriz and Amir Khosrovaneh. Time to Positivity in
Staphylococcus aureus Bacteremia: Possible Correlation with the Source and Outcome of Infection.
Clinical Infectious Diseases 2005; 41: 594-598.
Session Number: 220
Session Number: 220
Abstract Title:
Analysis of the Characteristics of Blood Culture Contaminants in A Tertiary Care Hosp. Laboratory
N. Aygul1, K. Wallgren1, P. Dinnétz2, V. Ozenci1; 1Karolinska Univ. Hosp., Stockholm, Sweden,
2Södertörn Univ., Stockholm, Sweden
Abstract Body:
Background: Analysis of factors influencing contamination of blood culture (BC) bottles is important in
establishing strategies to avoid contamination and early interpretation of contamination in the clinical
routine. The aim of the study was to analyze several factors in relation to contamination of BC bottles.
Material/Methods: In this prospective study we analyzed contaminated BC bottles in relation to: (i) age
and gender of the patient; (ii) bottle type; (iii) the day of the week; (v) sampling time and (vi) volume of
blood in the bottle. The data was analyzed with linear mixed models. BacT/ALERT-3D BC system and -FA
Plus and -FN Plus bottles were used. Contaminated BC bottles were defined as the growth of one of the
following bacteria; Staphylococcus epidermidis and other coagulase-negative Staphylococcus spp.,
Bacillus spp., Propionibacterium acnes, Corynebacterium spp., Micrococcus spp in (i) 1/2 or 1/4 bottles
or (ii) 2/2 or 2/4 bottles from the same BC set and without any growth in any other clinically relevant
microbiological sample in a period of 5 days after sampling. Growth of above mentioned potentially
contaminant bacteria in several BC bottles and/or in combination with other relevant samples was
regarded as clinically relevant. Results: A total of 10,671 individual BC bottles from 2,723 adult patients
were included. There were 192/10671 (1.80%) contaminated BC bottles and 192/10671 (1.80%) BC
bottles with clinically relevant growth of potentially contaminant bacteria. There was no difference
between the 192 BC bottles that were contaminated and 192 BC bottles with clinically relevant growth
of potentially contaminant bacteria in age and gender of the patients, time of sampling and the day of
the week. Among the 192 contaminated BC bottles, the frequency of aerobic BacT/ALERT FA bottles was
four times higher than the anaerobic BacT/ALERT FN bottles (p<0.05). Mean (SD) time to detection was
longer, 33.9 (23.9) h in contaminated bottles compared to 22.9 (13.1) h in bottles with clinically relevant
growth. Mean (SD) blood volume that could be measured in 173 contaminated BC bottles was lower
than in 192 BC bottles with clinically relevant growth of potentially contaminant bacteria, 8.5 (4.5) ml vs.
9.6 (4.6) ml respectively. Conclusions: The bottle type, time to detection and volume of blood can be
relevant factors in estimating contamination in BC bottles with growth of potentially contaminant
bacteria.
Session Number: 220
Session Number: 220
Abstract Title:
Sustained Reduction in Blood Culture Contamination Rates in A Univ. Hosp. Emergency Department
I. Nachamkin1, D. Danoski2, J. Barnes3, J. Barger2; 1Univ. of Pennsylvania Perelman Sch. of Med.,
Philadelphia, PA, 2Hosp. of the Univ. of Pennsylvania, Philadelphia, PA, 3Penn Presbyterian Med. Ctr.,
Philadelphia, PA
Abstract Body:
Background: Proper and timely collection of blood cultures are critical for the evaluation of patients with
suspected bloodstream infections. Pre-analytical variables such as disinfection of the venipuncture site
are critical for minimizing blood culture contamination. False-positive blood cultures (i.e. contaminated)
have important downstream effects including misinterpretation of results, inappropriate antimicrobial
therapy, increased LOS and others. Emergency Departments are known for having some of the highest
rates of blood culture contamination. The purpose of this report is to demonstrate that with consistent
monitoring of contamination rates, feedback from the laboratory, and an engaged quality management
team in the ED, contamination rates can be reduced and sustained at a low level. Methods: The Hospital
of the University of Pennsylvania (HUP) is a tertiary academic center with 788 inpatient beds, 35,000
annual admissions and 62,000 ED visits. Blood cultures at HUP comprise a 2-bottle system (Bactec Plus
Aerobic/Anaerobic). Approximately 1000 blood cultures are performed monthly by the ED. We use CLSI
guidelines for defining contaminated blood cultures; any one of 6 microorganisms positive in a single
bottle within a 24 hour period is considered a contaminant. Rates are reported quarterly to the ED and
other departments. Results: From July-September 2014, the monthly BC contamination rates for the ED
were 2.21%, 3.24% and 3.92%, respectively. Following the initiation of a quality improvement project by
the quality team in the Emergency Department, the contamination rate dropped to 2.09% in October,
2014. Subsequently through September 2017, there were 20/35 months where the contamination rates
were <1.5% (0.78%-1.47%) and for 10 months near 1%. Conclusions: Lowering blood culture
contamination rates in an academic Emergency Department is achievable and maintained with continual
education and process improvement effort.<br /><p><a
href="http://files.abstractsonline.com/CTRL/08/a/db8/445/82e/43d/d8c/6c9/7d9/bb8/625/f3/g4739_1
src="http://files.abstractsonline.com/CTRL/08/a/db8/445/82e/43d/d8c/6c9/7d9/bb8/625/f3/g4739_1.j
Session Number: 220
Session Number: 220
Abstract Title:
Positive Blood Cultures after the Start of Iv Antibiotics
K. H. Rand1, S. G. Beal1, B. R. Allen1, T. F. Payton1, K. Rivera2, G. P. Lipori1; 1Univ. of Florida,
Gainesville, FL, 2San Juan Bautista Sch. of Med., Caguas, PR
Abstract Body:
Background: Current CMS sepsis core measure (SEP-1) compliance criteria include obtaining blood
cultures (BC) before starting antibiotics. While it is logical that prior antibiotics would reduce the
likelihood of a positive BC, we could not find data on how rapidly BCs are affected by prior antibiotic
treatment. Methods: We studied 30,743 patients who had a BC and were admitted through the adult
(>18y) Emergency Department to the UFHealth Shands Hospital, Gainesville FL between 8/2012-
12/2016. Blood cultures were done with BacTec aerobic, anaerobic and pediatric resin bottles,
incubated for 5 days. We calculated the hourly rate of positive blood cultures obtained before and after
the start of IV antibiotics by subtracting the time stamp in the electronic medical record (Epic) between
the 1st BC collection time and the start of the 1st IV antibiotic dose. We considered S. aureus, gram
negative rods, Beta-hemolytic Streptococci and Enterococci as significant pathogens and coagulase
negative Staphylococci, S. viridans, Propionibacterium sp, Micrococcus sp and Bacillus sp as
contaminants. Results: Of the 30,743 patients admitted with a BC, 6649 (21.6%) were coded as septic on
admission. Among septic patients 973/4284 (22.7%) were positive for significant pathogens in BC
obtained in the 3 hours before IV antibiotics, while 109/552 (19.7%) grew a significant pathogen in the
1st hour after IV antibiotics were started, Chi Sq p=0.129, NS. The positivity rate fell to 40/497 (8%) for
the subsequent 1-6 h after starting IV antibiotics (p< 0.001, vs pre-antibiotics). Mortality was not
statistically different for patients whose cultures were obtained 1-60 min before vs 0-59 min after
antibiotics were started (p=0.212). For the non-septic group, BC were positive in 3.0% for the 3 h prior to
antibiotics vs 1.3 % for the 1st h after antibiotics (p<0.001), and 1.95% for the 1 - 6 h after starting IV
antibiotics (p=0.011). The reduction in proportion of positive BC in the septic (13%) vs non-septic (57%)
groups 0 -3 h before vs the 1st hour after the start of IV antibiotics appears to differ between the
groups, possibly reflecting differences in severity of illness or effectiveness of antibiotics between septic
and not septic groups. Conclusion: Since the intent of the SEP-1 core measure is to optimize the quality
of care for septic patients, consideration should be given to revising the requirement to allow blood
culture collection within an evidence-based reasonable time after IV antibiotics are documented in the
electronic medical record.
Session Number: 221
Session Number: 221
Session Title: CPHM03 - Diagnostic Bacteriology: Pre-Analytical Methods and Automation
Abstract Title:
Comparative Evaluation of Puritan and Copan Sputum Solution for Recovery of Respiratory Pathogens
Abstract Body:
Liquefaction and thorough mixing of sputum specimens ensures a representative sample for culture.
Buffered phosphate solutions of dithiothreitol (DTT) are often used for this purpose. Tubes with DTT
that are adaptable to automated plating systems are now available from Puritan Medical Products (P)
and Copan Diagnostics (C). P (1.0 mL/tube) and C (0.5 mL/tube) sputum solutions (SS) were inoculated
with 100 uL of approx. 107 cfu/mL of ATCC and clinical strains of H. influenza (HI), M. catarrhalis (MC), S.
pneumoniae (SPN), S. pyogenes (SPY), P. aeruginosa (PA), S. aureus (SA), C. albicans (CA), K. pneumoniae
(KP), B. cepacia (BC), and S. maltophilia (SM). Tubes were plated using a BD Kiestra InoqulA delivering 10
uL of sample onto SBA or CHOC with zigzag streaking. Tubes were plated at 0, 2, 4, and 6 h. Changes in
colony counts of 2 log10 or more as compared to time 0 h were considered significant. Sputum samples
were also tested in SS. Using a transfer pipet, 100 uL of sputum was added to P and C SS and vortexed.
Tubes were monitored for liquefaction every 15 minutes. Once liquefied, samples were plated onto SBA,
CHOC and MAC using 30 uL with 4 quadrant streaking. Culture growth was graded 1+, 2+, 3+ and 4+ and
compared to non-liquefied, manually plated cultures. Differences in growth of 2 grades or more were
considered significant. Viability of SPY, PA, SA, CA, KP, BC and SM did not significantly change after
exposure to P and C SS for up to 6 h. However, for HI and SPN there were no viable organisms remaining
at 6 h when exposed to C with significant decreases starting at 2 h for some strains of HI and at 4 h for
some strains of SPN. There were also significant decreases in colony counts for MC exposed to C for 6 h.
For sputum samples, the time required for adequate liquefication ranged from 15 – 45 minutes. C
treated samples remained more viscous than with P after the same exposure time with fibrin clot
equipment errors. P and C treated samples yielded equivalent results as compared to nontreated
manually plated samples and compared to each other. C and P cultures often yielded higher colony
counts as compared to manually plated specimens. P and C SS tubes hold the potential of providing an
efficient mechanism for liquefaction and automated plating of sputum samples. Care must be exercised
with the length of time that samples are exposed to SS especially when using C. Sputum samples treated
with P and C were comparable to each other and to non-liquefied manually plated samples. The lower
volume of SS in C may cause mechanical issues with some automated plating systems due to higher
viscosity as compared to P.
Session Number: 221
Session Number: 221
Abstract Title:
Comparison of Streaking Patterns, Plating Consistency, and Isolation of the Wasp and Inoqula Benchtop
on Blood Agar, Macconkey Agar
M. L. Faron, H. Samra, B. Mesich, B. W. Buchan, N. A. Ledeboer; The Med. Coll. of Wisconsin,
Milwaukee, WI
Abstract Body:
Numerous publications have demonstrated that automatic plating creates more consistent streaking
patterns and isolates more bacteria than manual plating; however, it is unclear what streaking patterns,
systems, and plates are optimal for culture. In this study, we compared the WASP and benchtop InoqulA
for their ability to reproducibly inoculate plates and isolate colonies. To test precision of inoculum,
sterile saline was inoculated at concentrations ranging from 103 to 108 CFU/mL using 4 pathogens. Each
concentration was plated to blood and MacConkey agar, using the WASP 1uL and 10uL loop and a
calibrated pipette at 10uL on the InoqulA in triplicate. Plates were assessed for isolation of >5 colonies,
which is indicative enough organism to complete ID and AST. Urine cultures were then plated in singlet
following the above methodology. A technologist reviewed each plate recording preliminary results.
Significant growth was based on clean-catch urine culture guidelines. Furthermore, two technologists
were blinded to the streaking methods and ranked quality of streaking patterns from 1-3 to evaluate
technologist preference to streaking quality with 3 being the highest. Evaluation of plates for the need
to subculture demonstrated that >5 colonies were accessible for testing for all streaking methods when
plating between 104 -107CFU/mL. At 108 CFU/mL the inoqulA required subculture for 18% of specimens
tested. Out of 149 urine specimens, 8 differences were observed. Of these 8, 5 specimens had the same
pathogens, but workup differed because the organisms were near the significance threshold (10K/mL).
For 2 specimens 50% of the plates reported likely contamination, while the other 50% required workup
based on laboratory policy. The final specimen did not recover gram positive cocci using the WASP 10uL,
likely due to containing >100k of gram negative organisms. Overall head to head comparisons were
concordant in >97% of specimens tested. The need to subculture was similar for full plates ranging from
4-6% of specimens plated. Technologist preferred the WASP 1uL loop with an average score of 2.4
followed by the WASP 10uL loop (2.1), and InoqulA (1.9). These data demonstrated that in most cultures
the automated system will not affect results; however, use of 1uL for plating may improve sensitivity as
all pathogens were detected from this method. Use of 10uL for inoculation may decrease sensitivity and
increase turnaround time as these data demonstrated that over inoculation can reduce isolation and
inhibit detection of other pathogens.
Session Number: 221
Session Number: 221
Abstract Title:
Comparative Evaluation of Puritan and Copan Lim Broth for Recovery of Group B Streptococcus Using
Automated Plating
Abstract Body:
Reliable detection of maternal Group B Streptococcus (GBS) colonization is essential in preventing
neonatal disease. Screening for GBS using a vaginal/rectal swab cultured in Lim broth is commonly used
for this purpose. Subculture of Lim broth is typically performed manually but Puritan Medical Products
(P) and Copan Diagnostics (C) now offer Lim broth in tubes designed for use with automated plating
systems. S. agalactiae ATCC 12386 and clinical strains of GBS were tested alone or in combination with
mixtures of bacteria representing fecal and vaginal flora. P and C standard flocked swabs were
inoculated with 100 uL of approx. 107 cfu/mL and placed into Lim broth. After 24h incubation, tubes
were vortexed and loaded onto a BD Kiestra InoqulA programmed to dispense 10 uL of sample onto
sheep blood agar with magnetic bead zigzag streaking. After 24h incubation, plates were examined for
quality of GBS colony isolation. Quantitative counts were determined for all colony types growing on
plates. A one log10 or greater difference in colony count was considered significant when comparing P
and C. When tested alone, P and C yielded equal quantities of well isolated GBS colonies. P and C
recovery of GBS was comparable in mixed cultures but GBS colony counts and isolation varied
depending on the amount of growth of the other bacterial strains in the mixture. When present in mixed
cultures, Enterobacteriaceae growth was comparable for P and C with GBS colony counts and isolation
equal to cultures with GBS alone. Growth of Enterococcus species in mixed cultures was also
comparable for P and C, however, when this organism was present the quantity and isolation of GBS was
reduced compared to cultures with GBS alone. Colony counts of P. aeruginosa and some Staphylococcus
and Corynebacterium species were significantly higher in C than P in some cases making visualization of
isolated GBS colonies difficult. Growth of yeast was significantly higher in P than in C. There were no
mechanical problems associated with automated plating using P and C. P and C Lim broth allowed for
efficient and consistent plating on an automated platform. P colony counts of GBS were equivalent to C
when tested alone or when tested in combination with mixtures of bacteria representing fecal or vaginal
flora. With the exception of P. aeruginosa for C, the growth of Gram negative rods was equally
suppressed by P and C. For both P and C, overgrowth of Gram positive bacteria in some cases reduced
the number of well isolated GBS colonies but some strains of Staphylococcus and Corynebacterium were
better suppressed by P than C.
Session Number: 221
Session Number: 221
Abstract Title:
Comparison of Copan Wasp versus Bd Kiestra Inoqula in Isolating Colonies from Positive Urine Culture
Specimens
M. Noorbakhsh1, S. Novak_Weekley2; 1Sutter Hlth.Shared Lab., Livermore, CA, 2Copan Diagnostics,
Inc., Murrieta, CA
Abstract Body:
Background: Automated plating instruments become available in the last decade to reduce labor
associated with specimen inoculation and improve the quality of plating. We used the two most
common instruments on the market today, WASP® (Copan Diagnostics), and BD KiestraTM InoqulA.
Crucial to these instruments is the ability for the plater to produce enough isolated colonies so that
work up off the primary plates can be performed. In this study the ability of each instrument to produce
well isolated colonies (at least 3 or more colonies), so that culture work up could proceed without sub-
culturing and time delay. Methods: Total of 294 turbid urine specimens submitted for routine culture at
Sutter Shared Laboratory were plated on Blood and MacConkey agar plates by the WASP with 1µL loop
on whole plate (WW1), WASP with dual 1µL loop and on bi-plates (WB1) and by the InoqulA pipetting
(KW10). All plates were transferred to the WASPLab™ for incubation in CO2 and images were taken at
18 hours. Images from each set of plates were examined for the quality of the streaking through
different parameters presented in a questionnaire. Results: Out of the 294 urine samples, 211 were
found positive for WB1, 213 for WW1, and 207 for KW10. A concordance of 96% was calculated among
the clinical outcome of the 3 conditions analyzed. WB1 positive samples shown 272 isolates and 234 had
enough colonies for work-up (86%), for WW1 282 isolates were present and 250 had enough colonies
for work-up (89%), for KW10 270 isolates were present and 224 had enough colonies to work-up (83%).
To estimate the difference in colony isolation ability, a score was given (1colony= 1 score, >6 colony= 7
score): no difference was found between WW1 vs KW10 (p= 0.404) and in WB1 vs KW10 (p= 0.402) for
isolates with colony counts between ≥10,000 CFU/mL and <100.000 CFU/mL. A significant difference
was found in samples with a concentration ≥100,000 CFU/mL: the number of isolated colonies was
higher in WW1 in comparison with KW10 (p= 0.001). No statistical difference was found between KW10
and WB1 (p= 0.921). Conclusions: The ability of the instrument to isolate colonies for immediate work
up without subculture can impact when the preliminary or final results are given to the provider, while
poor isolation of colonies results in sub-optimal patient care and delay in turn-around-time. Based on
this study both the WASP and Kiestra InoqulA systems were comparable in terms of isolating colonies in
a positive urine specimen, however WASP performs slightly better (89% versus 83%) for providing more
colonies for further workup.
Session Number: 221
Session Number: 221
Abstract Title:
Evaluation of the Bd Kiestra Total Lab. Automation Sys. for Inoculation and Incubation of Throat Swab
Specimens
M. L. Yarbrough1, R. Jennemann2, C-A. D. Burnham1; 1Washington Univ. Sch. of Med., Saint Louis, MO,
2Barnes-Jewish Hosp., Saint Louis, MO
Abstract Body:
Background: The introduction of total laboratory automation (TLA) is changing the workflow of clinical
microbiology but must be validated prior to routine clinical implementation. The objective of this study
was to evaluate the performance of the BD Kiestra TLA system for inoculation and incubation of throat
swab specimens for culture. Methods: Prior to evaluation of accuracy and reproducibility, a number of
TLA-specific parameters were evaluated to optimize the TLA throat culture method, including different
inoculation volumes, streaking patterns, and incubation times. The accuracy of the Kiestra TLA system
was compared to conventional culture as the reference standard. One hundred (100) throat swab
specimens were evaluated; these specimens were collected using the ESwab collection and transport
system and submitted as part of routine clinical care. After inoculation of the specimen to a blood agar
plate (BAP), cultures were incubated at 35°C in CO2 and imaged at 18 hours and 48 hours. For
comparison, specimens were manually inoculated to BAP and examined after approximately 24 and 48
hours incubation at 35°C in CO2. To assess reproducibility, 20 contrived specimens (5 each of
Streptococcus pyogenes, S. agalactiae, S. dysgalactiae, and Arcanobacterium haemolyticum) were tested
in parallel with both TLA and manual methods, as described above. Results: The final optimized test
parameters used for TLA inoculation and incubation included plating a volume of 50 µL of specimen in a
quadrant streak pattern (spreading pattern 19). Out of 100 specimens tested, 4 cultures were positive
(all S. pyogenes) on the TLA, for 100% agreement with the reference method. No discrepancies in
pathogen identification occurred between the TLA and the reference method for the contrived
specimens. Review of TLA images taken at 18 hours incubation was sufficient to accurately identify β-
hemolytic colonies of Streptococcus species on the TLA. Notably, hemolysis of colonies of A.
haemolyticum was not readily visualized at 18 hours on the TLA, compared to slight hemolysis
discernable at 24 hours using manual methods. Hemolysis was clearly visible with either method at 48
hours. Conclusions: Automated processing of throat swab specimens with TLA improved standardization
and consistency of culture workup. High resolution imaging reduces the incubation time necessary prior
to analysis for the vast majority of positive specimens, which can facilitate decreased technologist
hands-on time and provide faster culture results.
Session Number: 221
Session Number: 221
Abstract Title:
Impact of Lab. Automation on Microbiology Specimen Processing and Culture Reporting in A Large
Integrated Healthcare Organization
N. Z. Khoury1, K. M. Ellyson2, A. R. Borowick2, R. L. Bergsbaken2, C. A. Janita2, D. A. Olson1;
1Hlth.Partners and Park Nicollet Med. Groups, Bloomington, MN, 2Regions Hosp., St. Paul, MN
Abstract Body:
The consolidated microbiology laboratory for the HealthPartners Medical Group, located at Regions
Hospital (RH) in St. Paul, MN, serves more than 55 primary care clinics, 22 urgent care locations, 55
medical and surgical specialties, nursing homes, and 6 hospitals spanning eastern Minnesota and
western Wisconsin. High culture processing volumes (>40,000 cultures per month) and a need for rapid
and efficient specimen processing, drove the microbiology laboratory decision to implement the BD
Kiestra™ Work Cell (WCA) system. This platform enables automated specimen processing, plate
incubation and digital imaging. Automated plating occurs 24/7 and culture interpretation with reporting
occurs from 5AM -11PM, daily. The WCA was validated for urine cultures, beta-cervical cultures, and
MRSA screens in September, 2016. Blood cultures were validated 12 months later (Sept 1, 2017). We
conducted a review of pre- and post-Kiestra go-live to evaluate volume/FTE and turnaround time
efficiencies earned, utilizing the WCA automation line. Volume data (2014-2017) demonstrate an
increase in specimens processed (Figure 1). However, the additional volume did not negatively impact
the critically important positive blood culture turnaround time. Despite off-site blood culture processing
and 4 hour courier delivery routes to RH, the turnaround time to final result for positive blood cultures
remained consistent (range 2-11 days), pre- and post-Kiestra go-live, without accruing significant
overtime and staying under budgeted FTE.<br /><p><a
href="http://files.abstractsonline.com/CTRL/30/5/fa9/84f/d89/4bf/69c/23e/a06/e77/394/b5/g7028_2.J
PG" target='_blank' address=no ><img
src="http://files.abstractsonline.com/CTRL/30/5/fa9/84f/d89/4bf/69c/23e/a06/e77/394/b5/g7028_2.JP
G" alt="" border="0" width="600" height="412" /></a></p>
Session Number: 221
Session Number: 221
Abstract Title:
Digital Detection of Group A Streptococcus Using Colorex Strep A Chromagar and Wasplab Chromogenic
Detection Module
J. Dien Bard1, J. Nelson2, K. Mata3, D. Thorpe2, S. Novak-Weekley2; 1Children's Hosp. Los Angeles;
Univ. of Southern California, Los Angeles, CA, 2Copan Diagnostics, Murrieta, CA, 3Children's Hosp. Los
Angeles, Los Angeles, CA
Abstract Body:
Background: Despite the availability of several diagnostics tools for the diagnosis of Group A
Streptococcus (GAS) pharyngitis, culture remains one of the primary methods in use today and is still
considered the gold standard for the detection of GAS from pharyngeal samples. This is likely because
culture has high sensitivity compared to rapid antigen tests and low cost compared to molecular
approaches. However, in large volume laboratories, screening for GAS by culture can be cumbersome
and streamlined approaches using automation is necessary. This study evaluates the capability of the
WASPLab Chromogenic Detection Module (CDM) to automatically detect and interpret orange GAS
colonies on a new chromogenic agar (Colorex Strep A agar, CHROMagar). Methods: A total of 100
remnant pharyngeal samples from pediatric patients presenting to the Emergency Department at
Children’s Hospital Los Angeles with presumed bacterial pharyngitis were enrolled in this study. Samples
were collected using ESwab Transport System (Copan Diagnostics) and initially tested for the presence
of GAS by PCR (Lyra Direct Strep Assay, Quidel). Remnant samples were inoculated onto blood agar and
Colorex Strep A, using the WASP specimen plater and incubated in the WASPLab Total Laboratory
Automation system (Copan Diagnostics) for 24 hours. Results from the WASPLab CDM were compared
to results obtained by manual read and PCR. Results: Of the 100 pharyngeal samples, 12 GAS positives
and 86 GAS negatives were detected by both PCR and Colorex Strep A. A total of 2 discrepant results
were identified; one culture positive that was PCR negative and one culture negative that was PCR
positive. Compared to PCR, Colorex Strep A had a sensitivity of 92.3% and a specificity of 98.9%. The
WASPLab CDM read was 100% comparable to manual reads but at a much faster speed. Conclusions:
This proof-of-concept study demonstrates that the Colorex Strep A by CHROMagar has comparable
sensitivity and specificity to PCR. More importantly, we demonstrate that the WASPLab CDM can
accurately and efficiently detect all positive GAS colonies, eliminating the need for manual reads. This
has the ability to dramatically improve workflow by reducing turn-around-time and redirecting
laboratory personnel to other more complex tasks.
Session Number: 221
Session Number: 221
Abstract Title:
Validation and Implementation of Colorex (Chromagar) Chromagaresbl Msupercarba Biplate on Wasp
Wasplab to Screen for Esbl Cpe
M. Gaskin; Hamilton Hlth.Sci., Hamilton, ON, Canada
Abstract Body:
Background: Identification of Extended Spectrum Beta-lactamase (ESBL) and Carbapenemase (CPE)
producing organisms in colonized patients from surveillance cultures reduces colonization, infection and
decreases the financial burden to the healthcare system. The introduction of WASP™/WASPLab™
automation in our laboratory supports time and cost-effective methodologies to prevent the spread of
ESBL and CPE in our hospitals. The objective of this study was to validate a new Colorex™
(CHROMagar™) CHROMagarESBL/mSuperCARBA (CCES) bi-plate for ESBL/CPE screening using the
WASP™ and WASPLab™. Methods: This study used 239 ESBL specimens collected with ESwab™ kits
processed on the WASP™ using a protocol (1ul dual loop and a twin loop 2/bi-plate streaking pattern
and CCES bi-plates). Reference strains (N=49) of ESBL, CPE, SPICE and AMP C were tested. The bi-plates
were incubated in the WASPLab™ for 20 hours then imaging analysis was performed. Results were
compared to current testing which uses a Colorex™ C3GR/KPC (CCK) bi-plate. Discordant results were
confirmed with ESBL disks and CPE. Results: Of the 239 samples tested, 90 were positive for ESBL and 7
for CPE by current method using CCK bi-plates, versus 91 positives for ESBL and 7 positives for CPE on
the new CCES bi-plates using the dual loop on the WASP™ and WASPLab™ imaging analysis. There was a
29% increase in no growth cultures and a reduction in breakthrough growth of AMP C-producing strains,
Citrobacter freundii and SPICE organisms on the Colorex™ ESBL side, 89%, 82% and 92% respectively.
Testing bi-plates on the WASP™ using a dual loop resulted in a 39% reduction in processing time
compared to a 2 plate protocol. 33 known CPE strains were tested all of which grew on the Supercarba
compared to 30 growing on the C3GR plates. Conclusions: The decrease in breakthrough growth on the
CHROMagarESBL side of the new CCES bi-plate reduced off-line testing, resulting in time and cost
savings. The 39% reduction in processing time on the WASP™ is of critical importance to the functional
capacity of the WASPLab™. The WASPLab™ segregation software has the capability to analyze images
and put no growth images into a separate screen for rapid resulting.
Session Number: 221
Session Number: 221
Abstract Title:
Comparative Evaluation of Puritan and Copan Cary Blair Medium for Recovery of Enteric Pathogens
Abstract Body:
Cary Blair medium (CB) is commonly used for preservation of fecal specimens. CB is typically manually
plated to selective and differential media for culture of enteric pathogens. Tubes with modified CB are
now available from Puritan Medical Products (P) and Copan Diagnostics (C) for use with automated
plating systems. ATCC and clinical strains of Salmonella (SA), Shigella (SH), Y. enterocolitica (YE), E.coli
O157 (ECO157), C. jejuni (CJ) and V. parahaemolyticus (VP) were tested alone and with a mixture of E.
coli (EC) and E. faecalis (EF) to simulate normal fecal flora (EC/EF). CBs were inoculated with 100 uL of
approx. 107 cfu/mL. At 0 h, CBs were loaded onto a BD Kiestra InoqulA programmed to dispense 30 uL
onto SBA, MAC, XLD, CIN, MACS, and CSM plates with magnetic bead 4 quadrant streaking. One set of
CBs was maintained at 22-25oC (RT) with plating at 24 and 48 h. A second set of CBs was maintained at
2-8oC (RFT) with plating at 24, 48 and 72 h. Growth was graded as 1+, 2+, 3+ and 4+ at each time point.
A change of 2 or more grades as compared to time 0 h was considered significant. When CBs were
maintained at RT the quantity of growth of SA, SH, YE and EC0157 alone did not change significantly at
48 h compared to 0 h for both P and C, however, when tested with EC/EF, there were significant
decreases in SA and SH most notably in C. For CJ, at 24 h there was no significant change in quantity of
growth for either P or C, however, for some strains, tested alone or with EC/EF, there were significant
decreases in growth at 48 h with both P and C. For VP tested alone or with EC/EF, growth from P was
significantly lower at 48 h. When CBs were maintained at RFT, growth of SA and SH at 48 h did not
change significantly for both P and C, however, at 72 h significant decreases were seen with some
strains of SA and SH when tested with EC/EF again most notably in C. For ECO157 alone or with EC/EF
significant changes were seen at 48 h in both P and C. For YE and CJ alone or with EC/EF there were no
significant changes in growth at 72 h for both P and C. For VP at 48 h the quantity of growth was
significantly decreased for P when tested alone or with EC/EF. P and C tubes containing modified Cary
Blair allowed for streamlined, consistent and quality streaking on an automated plating platform. In our
study, P performed better than C at maintaining viability of SA and SH for a longer time at RT and RFT in
contrived fecal samples. P and C were comparable at maintaining viability at RT and RFT for YE, ECO157
and CJ. For VP, C performed better than P at RT and RFT. For optimum recovery of enteric pathogens
using P or C, storage should be limited to 24 hours at RT and 48 at RFT.
Session Number: 221
Session Number: 221
Abstract Title:
Effects of Delay in Transport on Neutrophil Quantitation of Primary Gram Smears
J. Rueckert, C. LaValley, A. Theiss, C. Wojewoda; Univ. of Vermont Med. Ctr., Burlington, VT
Abstract Body:
Background: Accurate interpretation of the primary gram smear is pivotal for guiding the microbiology
laboratory in reporting final culture results. The quantitation of neutrophils present in the primary gram
smear allows the laboratory to make informed decisions as to whether the bacteria cultured from the
patient’s sample represents a true infection versus contamination with normal flora. Studies have been
performed evaluating the stability and viability of organisms in transport media, but the stability of
neutrophils has not been studied. We evaluated the effects of delay in transport on quantitation of
neutrophils in the primary gram smear of specimens submitted in ESwab™ Liquid Amies transport
media. Methods: Purulent aspirate patient samples received for routine bacterial cultures were
randomly selected to inoculate three ESwab™ Liquid Amies transport media to represent varying
neutrophil quantities (many, few and rare). Gram smears were prepared at the time of inoculation, 6,
12, 24, and 48 hours and stained per our laboratory procedure. For routine bacterial cultures, the
package insert allows for the acceptance of specimens up to 48 hours after their time of collection. The
smears were interpreted by three second year pathology residents, and an average of ten X100 oil
immersion fields was calculated for each smear. Results: The overall trend observed was a decrease in
neutrophil quantitation. A significant decrease was observed beginning at 12 hours in all three
specimens, with an average decrease of 91.8%. The average decrease at 24 hours for the three
specimens was 98.8%.<br /><p><a
href="http://files.abstractsonline.com/CTRL/56/3/ded/d03/7a7/4f3/e87/827/156/93c/2d2/26/g5591_1
src="http://files.abstractsonline.com/CTRL/56/3/ded/d03/7a7/4f3/e87/827/156/93c/2d2/26/g5591_1.j
pg" alt="" border="0" width="600" height="400" /></a></p><br />Conclusion: Our findings indicate
delay in transport decreases neutrophil quantitation on gram smear. If scoring systems are used in the
work up of swab cultures, this could affect how many organisms are reported. Caution should be used
when using neutrophil quantitation from specimens delayed in transport to make decisions with
potential clinical consequences.
Session Number: 221
Session Number: 221
Abstract Title:
Value of Gram Stain As A Predictor of Bacterial Pathogens in Endotracheal Aspirates and
Bronchoalveolar Lavage Specimens
S. Miquirray1, C. Michel1, R. Humphries1, P. Pancholi2, M. Jindra2, S. Karow2, B. Schmitt3, J.
Koehlinger4, R. Buckner4, I. Towler4, M. Parcina5, R. Jozic5, J. Gonzalez Lopez6, A. Mir6, T. Martin-
Gomez6; 1Accelerate Diagnostics, Tucson, AZ, 2The Ohio Sta
Abstract Body:
Introduction: The role of Gram stain to guide therapy for patients with suspected hospital onset and
ventilator associated pneumonia is unclear. This study evaluated correlation of Gram stain of
endotracheal aspirates (ETA) and bronchoalveolar lavage (BAL) specimens with culture of a potential
pathogen (PP) for 466 patient specimens across 4 laboratories. Methods: 232 ETAs and 234 BALs were
tested by Gram stain and culture according to local laboratory standard operating procedures. Results:
260 (55.8%) specimens were negative for a PP: 35.8% negative for any growth, and 19.9% with normal
respiratory flora. PP included Klebsiella species (KLE) (n=40/206; 19.4%), S. aureus (SAU) (n=37/206;
17.9%), P. aeruginosa (PAE, 28/206, 13.5%), Candida albicans (20/206; 9.7%), Enterobacter species
(12/206, 5.8%), and Streptococcus pneumoniae (8/206, 3.8%). The majority of specimens yielded a
single PP (153/206; 74%). 46/206 (22.3%) yielded 2 PPs, 5/206 (2.4%) yielded 3 PPs, 1/206 each yielded
4 and 5 PPs, respectively (0.4%). The most prevalent combination of PP was PAE and yeast (n=4).
Correlation of gram stain with PP is shown in table. Sensitivity and specificity of gram stain was poor, but
negative predictive value was >90%. Of 78 specimens with a negative gram stain, 25 yielded gram
negative (GN) PPs and 16 gram positive PP, 20 yeast, and 17 polymicrobial with at least 1 PP. For
specimens negative for gram-positive cocci on gram stain, 4 yielded scant (1+) growth, 4 few (2+), 3
moderate (3+) and 3 heavy (4+) growth of SAU. For specimens negative for gram negative bacilli, 2
yielded scant (1+), 2 few (2+), 4 moderate (3+) and 2 heavy (4+) growth of PAE.<br /><table
class="AbstractTable" id="{12A9F814-0A1A-452A-8927-C5F82A37BC47}"><caption
rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1">Culture Result</td><td
rowspan="1" colspan="4">Gram Stain Performance</td></tr><tr><td rowspan="1"
colspan="1"></td><td rowspan="1" colspan="1">Sensitivity</td><td rowspan="1"
colspan="1">Specificity</td><td rowspan="1" colspan="1">PPV</td><td rowspan="1"
colspan="1">NPV</td></tr><tr><td rowspan="1" colspan="1">SAU</td><td rowspan="1"
colspan="1">59.5(42.2-75.0)</td><td rowspan="1" colspan="1">83.2 (79.3-86.6)</td><td rowspan="1"
colspan="1">23.4 (15.5-33.5)</td><td rowspan="1" colspan="1">96.0 (93.3-97.6)</td></tr><tr><td
rowspan="1" colspan="1">PAE</td><td rowspan="1" colspan="1">50.0 (31.1-69.0)</td><td
rowspan="1" colspan="1">86.3 (83.0-89.3)</td><td rowspan="1" colspan="1">18.9 (11.1-30.0)</td><td
rowspan="1" colspan="1">96.4 (94.0-98.0)</td></tr><tr><td rowspan="1" colspan="1">Gram-negative
bacilli</td><td rowspan="1" colspan="1">57.9 (47.8-67.3)</td><td rowspan="1" colspan="1">94.9
(92.1-96.7)</td><td rowspan="1" colspan="1">74.3 (63.3-83.0)</td><td rowspan="1" colspan="1">89.8
(86.4-92.4)</td></tr></table><br />Conclusion: Gram stain is a poor predictor of the presence of S.
aureus and P. aeruginosa on ETA and BAL cultures. In this study, the majority of specimens were
monomicrobial for PP, with KLE, SAU and PAE as the most commonly isolated PPs.
Session Number: 221
Session Number: 221
Abstract Title:
Eswab Or Liquid Amies Medium: Identifying the Appropriate Specimen for Detection of Group A
Streptococcus by Rapid Antigen Test
M. Gripka, N. Kanwar, K. Shaw, R. Selvarangan; Children's Mercy, Kansas City, MO
Abstract Body:
Background and Aim: Group A Streptococcus (GAS) is the most common cause of bacterial pharyngitis.
Rapid and accurate detection is important to ensure appropriate antibiotic treatment. Rapid antigen and
sample-to-answer molecular tests allow rapid detection of GAS from throat swabs. Copan ESwab in
Liquid Amies medium have been used for collection of throat swabs by rubbing the ESwab on the throat
and inserting it in Liquid Amies medium for elution of clinical material. It is unclear if ESwab or Liquid
Amies medium is the appropriate specimen for GAS antigen detection; hence we tested both ESwab and
the paired Liquid Amies medium on Quidel Quickvue+ Strep A rapid antigen test (QD) and compared
results with Luminex ARIES Group A Strep Assay and culture. Methods: Patients with symptoms of
pharyngitis were enrolled in the clinical trial for evaluation of Luminex ARIES Group A Strep Assay.
Copan flocked ESwab was used to collect throat specimen and eluted in 1 ml Liquid Amies medium.
Aliquots of Liquid Amies medium were tested by the ARIES Group A Strep Assay and inoculated on
routine bacteriological media for culture. The QD antigen test was performed using ESwab (ESWAB-QD)
and 100µl Liquid Amies medium (Amies -QD) and results were compared with ARIES GAS assay and
culture. Results: The sensitivity of ESwab-QD test compared to culture was 88% (CI 67.7-96.8) and to
PCR was 92.3% (CI 73.4-98.6). Specificity compared to culture was 50% (CI 35.4-64.6) and to PCR was
54% (39.5-67.9). The sensitivity of Amies-QD test compared to culture was 66.7% (CI 44.7-83.6) and
compared to PCR was 64% (CI 42.6-81.3). Specificity compared to culture was 89.6% (CI 76.6-96.1) and
compared to PCR was 90% (CI 77.4-96.3). Conclusion: Rapid antigen testing using ESwab was more
sensitive than testing Liquid Amies medium, but specificity was higher testing Liquid Amies medium vs.
ESwab when compared to culture and ARIES Group A Strep Assay. Neither specimen type provided the
optimum test result for GAS antigen detection. ESwab in Liquid Amies medium may not be suitable for
collecting throat swabs for GAS detection by antigen assay, but it is acceptable for PCR and culture.
Session Number: 221
Session Number: 221
Abstract Title:
Accuracy of Bacterial Isolate Recovery from Eswabs Submitted to A Central Lab. for Identification in An
Acute Skin Infection Clin. Trial
J. Nary, S. E. Dale; ACM Global Lab., Rochester, NY
Abstract Body:
Transporting bacterial isolates to a central laboratory for identification and susceptibility testing in
pharmaceutical clinical trials may involve creation of frozen cryovials and dry ice shipments. Our
laboratory performed testing for an acute bacterial skin infection study with sites in the United States.
Local laboratories prepared pure isolates with ESwabs in place of traditional glycerol/nutritive cryovials
to help eliminate expensive supplies, shipments and -80° C freezer storage. We evaluated the accuracy
of organism recovery from Eswab samples by comparing the local and central laboratory’s
identifications to determine if this is an efficient method of isolate shipment and recovery for future
studies. Local laboratories inoculated one ESwab and prepared two backup Microbank cryovials for each
organism identified from wound samples. ESwabs were sent to the central laboratory in ambient
overnight mailers and plated promptly upon receipt. If the organism did not grow or did not match the
site identification, one backup cryovial was requested from the local laboratory. The second cryovial was
requested if there was no growth or the discrepancy did not resolve with the first backup cryovial. Of
the Eswab samples cultured for the study, 86.5% (859/993) recovered the expected organism. Aerobes
and facultative anaerobes showed 90.3% (821/909) agreement between the local and central
laboratory’s identification. For anaerobes, 54.8% (46/84) required the backup cryovial culture to resolve
discrepancies compared to 9.7% (88/909) of the aerobes and facultative anaerobes. Discrepancies
resolved from the first backup cryovial for 22.6% (7/31) of anaerobes and 27.1% (16/59) of aerobes and
facultative anaerobes. Few identification discrepancies resolved using the second cryovial requested;
anaerobes 25% (2/8) and aerobes and facultative anaerobes 5.3% (1/19). Sites did not send requested
backup cryovials for 32.6% (15/46) of anaerobes and 33% (29/88) of aerobes and facultative anaerobes
(29/88). This submission algorithm saved 43% in overall cost compared to frozen cryovial shipments.
Our data shows that isolate submission with Eswabs shipped under ambient conditions is efficient with
>90% accuracy in recovering aerobic and facultative anaerobic isolates. For trials with this isolate mix,
we recommend submission of one Eswab for isolate recovery and creation of a single cryovial for the
occasional discrepancy or enhanced recovery of anaerobic and facultative anaerobic organisms.
Session Number: 221
Session Number: 221
Abstract Title:
Evaluation of Uriswab™ For the Collection, Transport and Preservation of Urine Specimens According to
the Clsi M40-A2 Guideline
A. Giambra, E. Orio, S. Castriciano; Copan Italia Spa, Brescia, Italy
Abstract Body:
Background: Accurate bacteriuria in urine is essential for the treatment of urinary tract infections. Urine
should be refrigerated or preserved during transport to prevent organisms overgrowth and support
pathogens viability. Copan developed the UriSwab™ (US), a collection, transport and preservation device
for urine specimen’s compatible with manual or WASP automated culturing methods. US has a leak-
proof screw-cap-tube with 2 or 3 sponges on a plastic stick attached to the cap. The sponges contain
preservatives salts to stabilize the original organisms load and are highly hydrophilic to absorb and
retain urine during transport and. The objective of this study was to evaluate the performance of the
UriSwab™ as per CLSIM40-A2 guidelines. Methods: ATCC strains of C. albicans (ATCC®24433), E. coli
(ATCC®25922), E. faecalis (ATCC®29212), P. aeruginosa (ATCC®27853), P. mirabilis (ATCC®7002), S.
saprophyticus (ATCC®15305), C. freundii (ATCC®8090), C. glabrata (ATCC®MYA-2950), E. cloacae
(ATCC®13047), M. morganii (ATCC®25829), S. agalactiae (ATCC®13813) were used for this study. From
each fresh sub-culture strain, a 0.5 McF suspension and six 10-fold serial dilutions were prepared in
sterile filtered synthetic urine to obtain a final count at time-zero of 25 to 250 organisms per plate.
Three lots of US were inoculated by submerging the sponges in 1.5x104, 1.5x103 and 1.5x102 CFU/ml
suspensions for 5 seconds and then returned into the tube. For each strain 15 US (3 at time zero, 6 at
24hs and 6 at 48hs at both 20-24°C and 2-6°C) were inoculated per lot. At each time-point, the US tubes
were centrifuged to release the urine and 100µl each were plated in triplicate onto appropriate agar.
After plates incubation, CFUs were counted to verify that each strains concentration in US at different
conditions remained within 1 log compared to time-zero as per guidelines. Results: Data obtained at all
conditions/times were within the acceptance criteria. Log reductions at 48hs for 2-6°C and 20-24°C were
-0.27 and -0.37 for C. albicans, -0.28 and -0.71 for E. Coli, -0.09 and -0.31 for E. faecalis, -0.15 and-0.66
for P. aeruginosa, -0.13 and -0.47 for P. mirabilis, -0.28 and 0.28 for S. saprophyticus, -0.13 and -0.19 for
C. freundii., -0.39 and -0.67 for C. glabrata, -0.29 and 0.15 for E. cloacae, -0.22and -0.21 for M. morganii,
-0.28 and -0.31 for S. agalactiae Conclusion: UriSwab™ device is fully compliant to the CLSI M40-A2
standard for all the strains tested. UriSwab™ is operator’s safe since absorbs spontaneously the urine
sample eliminating the use of a needle.
Session Number: 221
Session Number: 221
Abstract Title:
Patient Collected Vaginal Samples, Clinic-Based Universal Screening Protocols, and Home Collected
Samples: Evaluation of Care Models Designed to Improve Detection of Sexually Transmitted Diseases
(Std) in Female Patient Populations
N. Z. Khoury1, P. J. O'Keefe2, A. Coughlin2, G. Fedio3, D. A. Olson1; 1Hlth.Partners and Park Nicollet Lab.
System, Bloomington, MN, 2Hlth.Partners Central Lab., Eden Prairie, MN, 3Park Nicollet Hlth.Services,
St. Louis Park, MN
Abstract Body:
Background: STD screening challenges disproportionally impact women and minorities, and despite
national primary care and targeted public health programs, rates of STDs have increased over the last
decade in Minnesota. The HealthPartners (HP) Laboratory in partnership with the HP & Park Nicollet
(PN) Medical Groups identified an opportunity to improve patient care through alternative care models,
in the setting of STD. Our care models include laboratory-based testing and licensed medical
professional follow-up, to guide appropriate patient and partner care. Methods: We designed an IRB-
approved crossover study comparing patient experience and test results using FDA-approved clinic
collected “gold standard” swabs, and patient home collected “study swabs” for the detection of
chlamydia and gonorrhea. We also evaluated the impact of clinic-collected vaginal swabs and
maintenance of certification incentives for recruiters, to improve screening. Data collected from May 5,
2016 to Dec 31, 2017 were included in the analysis. The goal of this study was to evaluate both universal
screening protocols and home collections for detection of STDs. Results: Eligible female patients >16
years of age assessed during a physician office visit for STD (screening protocols or per reason of the
visit) were asked to participate. Patients in agreement to enroll for home collection were handed a
home-collection kit. FDA approved clinic- and home-collected samples were tested using the Hologic
Aptima Combo2 Assay Reagents on the Panther Instrument platform. Conclusions: Test comparison data
and patient feedback on vaginal swabs indicated that this model of care was perceived as safe and easy.
The clinic- and home-collected vaginal swabs also provided a patient-and provider-accepted method for
CT/NG screening. Further analysis is underway to expand and implement this alternative model of care
and roll-out via annual screening protocols or online diagnosis and treatment with nurse practitioners
(virtuwell.com). <table class="AbstractTable" id="{93F02CED-512E-4FEE-B3EB-
229E7A810590}"><caption class="AbstractTableCaption"></caption><tr><td rowspan="1"
colspan="1">Patients Enrolled</td><td rowspan="1" colspan="1">537</td></tr><tr><td rowspan="1"
colspan="1">Kits Returned</td><td rowspan="1" colspan="1">372</td></tr><tr><td rowspan="1"
colspan="1">Kits Available to Analyze</td><td rowspan="1" colspan="1">354</td></tr><tr><td
rowspan="1" colspan="1">Clinic & Home Collect Negative</td><td rowspan="1"
colspan="1">341</td></tr><tr><td rowspan="1" colspan="1">CT Result Agreement</td><td
rowspan="1" colspan="1">10</td></tr><tr><td rowspan="1" colspan="1">CT Discordant Result</td><td
rowspan="1" colspan="1">1</td></tr><tr><td rowspan="1" colspan="1">GC Result Agreement (0
Discordant)</td><td rowspan="1" colspan="1">2</td></tr><tr><td rowspan="1" colspan="1">Home
Collect Agreement: Gold Standard:</td><td rowspan="1" colspan="1">0.997</td></tr></table>
Session Number: 221
Session Number: 221
Abstract Title:
Impact of Specimen Transport Time on Identification of Pathogens: Comparison of A Pcr-Based
Diagnostic Platform to Routine Microbiological Cultures
C. Pickens, C. Qi, H. Donnelly, M. Breganio, R. Wunderink; Northwestern Univ., Chicago, IL
Abstract Body:
Background: Prompt identification of pathogens is essential for optimal management of patients with
suspected lower respiratory tract (LRT) infections. Delays in time from collection to receipt of specimens
by the microbiology lab can significantly affect recovery of pathogens from culture, particularly more
fastidious microorganisms. Whether this is true for more virulent pathogens is unclear. New molecular
diagnostics may be less affected by transport time delays. We therefore compared the performance of a
polymerase chain reaction (PCR)-based diagnostic platform to standard of care (SoC) culture methods
for pathogen detection in relation to length of transport time. Methods: The Unyvero LRT Lower
Respiratory Tract Cartridge detects 20 pathogens and 16 resistance markers in approximately five hours
directly in clinical specimens via PCR technology. Specimen transport time from patient sampling to
arrival in the lab was recorded on 1,653 prospectively collected tracheal aspirate and bronchoalveolar
lavage samples from a validation study at nine US sites. We focused on five nosocomial pathogens (E.
coli, Acinetobacter spp., S. aureus, P. aeruginosa, and S. maltophilia). Samples were tested by an
independent PCR/sequencing for panel organism and compared to Unyvero and SoC results. Results:
Average LRT specimen transport time was 4.5 hrs. Transport time >2hrs significantly increased the false
negative rate for the SoC with substantially fewer false negatives with Unyvero PCR. The effect of
transport time is greatest for S. aureus for both technologies. 32-42% cultures are falsely negative for
critical nosocomial pathogens such as Acinetobacter and Pseudomonas when transport time is delayed
over 2 hrs. Conclusion: Based on multiple independent PCR assays followed by bi-directional sequencing,
delays in transport time of LRT specimens to the microbiology laboratory results in loss of viable
pathogens, including virulent nosocomial pathogens. Molecular technologies, such as Unyvero PCR, are
less affected and identified causative agents even after long transport times.
Session Number: 221
Session Number: 221
Abstract Title:
Assessment of Anaerobic Culture Incubation Time
Y. Maldonado, P. Hamilton, L. Baumgartner, J. Aslanzadeh; Hartford Hosp., Hartford, CT
Abstract Body:
Background: Currently, there is no consensus among microbiology laboratories on ideal incubation time
for anaerobic cultures. In a recent survey of CliniMicroNet, respondents reported anaerobic culture
incubation times from 2 to 7 days. The goal of this study was to determine the ideal anaerobic culture
incubation time. Methods: In a prospective study, 838 consecutive anaerobic cultures obtained at
Hartford Hospital, an 869-bed level 1 trauma center, were Gram stained and planted on anaerobic
blood, colistin and nalidixic acid (CNA), and kanamycin and vancomycin (KV) agar plates (BD Diagnostics
Sparks, MD). Wound cultures were assessed for quality before proceeding to culture. These plates were
placed in an anaerobic jar that utilized the Anoxomat system (Advanced Instruments Norwood, MA) to
create anaerobic conditions. The jars were incubated at 35oC and were checked for growth after 2, 3, 5,
and 7 days. Results: Overall, 74 cultures from bronchoalveolar lavage (1), bone (2), fluid (21), tissue (14)
and wound (36) grew one or more anaerobic organisms. Fifty two (52) cultures grew 1 isolate, 15
cultures grew 2 isolates, 5 cultures grew 3 isolates and 1 culture grew 4 isolates for a total of 101
isolates. Of these isolates, 43 % were detected on day 2, 21% on day 3, 20% on day 5 and 17% on day 7.
Fourteen (14) cultures were positive only at day 5 (4 fluid, 3 tissue and 7 wound) and 12 cultures were
positive only at day 7 (2 fluid, 3 tissue and 7 wound). Overall, 60 % of all isolates were Gram negative
and 40% Gram positive. Seventy three (73) % of the Gram negative isolates were detected on day 2 or 3.
In contrast, 50% of the Gram positive isolates were detected on day 2 or 3. Conclusions: We conclude
and corroborate with CLSI recommendations that anaerobic cultures should be incubated for at least 5
to 7 days.
Session Number: 221
Session Number: 221
Abstract Title:
Performance Evaluation of the Stomacher for Automated Grinding of Tissues for Bacterial and Fungal
Culture
C. Muhumuza, A. Hill, C. Doern, R. Reed-Warren, S. Kannady, B. Forbes; VCU Hlth.System, Richmond, VA
Abstract Body:
Culture of biopsied tissue specimens is critical to the diagnosis of invasive bacterial and fungal
infections. Traditionally, processing of these specimens has been dependent upon a manual tissue
grinding procedure intended to release organisms and facilitate the inoculation of multiple media types.
Many laboratories will also routinely leave a piece of tissue intact so as not to rupture and kill fragile
fungal organisms, such as the zygomycetes. Due to specimen variability and user-to-user variation in
tissue-grinding practices, it is likely that inconsistent tissue culture results are generated. The objective
of this study was to evaluate the performance of the Stomacher, an automated tissue processing
instrument. A series of optimization experiments were performed to determine the minimal grinding
intensity and length of time that would generate consistent bacterial and fungal culture results, while
preserving organism viability. Initial optimization experiments assessed the effect of Stomacher grinding
on known quantities of the following organisms: Fusarium, Rhizopus, Mucor, and Aspergillus fumigatus,
Bacteroides fragilis, Pseudomonas aeruginosa, Enterobacter cloacae, Staphylococcus aureus, and group
B Streptococcus. Three speed settings (low, medium, and fast) and four time settings (30 s, 60 s, 120 s,
and 5 min) were tested for each organism. Following the grinding process, organisms were inoculated to
the appropriate media and incubated to assess the effect of each condition on viability. Our results
showed that the Stomacher had no deleterious effect on organism viability. Of interest was the finding
that zygomycete viability was unaffected by Stomacher grinding. To further explore this finding, we
conducted follow-up experiments to determine at what point Mucor, Rhizopus, A. fumigatus, and
Fusarium would be killed by grinding with both the Stomacher (using the same conditions as previously
described) versus a vigorous manual grinding process. Surprisingly, the only organism that was killed by
grinding was Fusarium. This is an interesting result as it contradicts the dogma that the zygomycetes are
at risk of being inactivated by vigorous grinding. With optimization experiments complete, a series of 10
clinical tissue specimens were processed using the Stomacher and the manual tissue grinder. The
Stomacher generated identical culture results to that of the manual process and as a result will be
implemented for clinical use in our laboratory.
Session Number: 221
Session Number: 221
Abstract Title:
Significance of Patient and Sample Characteristics in Explaining Concordance between A Quantitative
Multiplex Pcr Test and Quantitative Bacterial Culture
C. Rindlisbacher, S. Kerr, C. Graue, J. Nawrocki, B. Galvin, C. Zapata-Allegro, J. Jones, K. Bourzac; BioFire
Diagnostics, LLC, Salt Lake City, UT
Abstract Body:
Background: The FilmArray® Pneumonia Panel (BioFire Diagnostics, LLC) is a molecular diagnostic test
for rapid identification of bacterial and viral causes of lower respiratory tract infections (LRTI) using
native (unprocessed) sputum and bronchoalveolar lavage (BAL) specimens. Typical bacteria associated
with LRTI are reported semi-quantitatively over four bins centered on 10^4, 10^5, 10^6, and ≥10^7
copies/mL. Here, we apply machine learning algorithms to evaluate data collected during clinical
evaluation of the Pneumonia Panel to find predictors of concordance/discordance between the
Pneumonia Panel and quantitative bacterial culture. Method: The prospective clinical evaluation was
performed at 8 U.S. sites. Enrolled specimens were tested for typical bacteria using the Pneumonia
Panel and quantitative bacterial culture. A total of 847 BAL and 836 sputum specimens were collected.
Additional information, such as patient demographics, preexisting health conditions and antibiotic use,
was collected as part of specimen enrollment. A predictive-model was built using random forests to
determine which features of this dataset were most influential in predicting concordance between the
Pneumonia Panel and bacterial culture for on-panel targets. Results: Qualitative concordance between
the Pneumonia Panel and quantitative bacterial culture is given in Table 1 below, with an overall
sensitivity of 91.0% and specificity of 97.3%. Out of 733 discordant results, 658 (89.7%) were detected
by Pneumonia Panel but not by bacterial culture. Patient characteristics were found to have some
importance in predicting agreement between the Pneumonia Panel and bacterial culture. The most
significant predictor of concordant/discordant results, however, was found to be the identified
organism.<br /><table class="AbstractTable" id="{1D1CAFD8-EECC-4A3E-BF38-
788A258E2E71}"><caption class="AbstractTableCaption"></caption><tr><td rowspan="1"
rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1"></td><td rowspan="1"
colspan="1"></td><td rowspan="1" colspan="2">Quantitative Bacterial Culture</td></tr><tr><td
colspan="1">D</td><td rowspan="1" colspan="1">ND</td></tr><tr><td rowspan="1"
colspan="1">Pneumonia Panel</td><td rowspan="1" colspan="1">D</td><td rowspan="1"
colspan="1"></td><td rowspan="1" colspan="1">ND</td><td rowspan="1" colspan="1">75</td><td
rowspan="1" colspan="1">23753</td></tr><tr><td rowspan="1" colspan="4">Table 1: The number of
Pneumonia Panel detections compared to bacterial culture. D indicates detected, and ND indicates not
detected.</td></tr></table><br />Conclusion: As a molecular based test, the Pneumonia Panel is more
likely to identify typical bacteria in LRTI patients when compared with bacterial culture. Discrepant
results are significantly influenced by the organism present in the sample, which highlights the ability of
molecular based tests to detect organisms regardless of their ability to be cultured. Data presented are
from assays that have not been cleared or approved for diagnostic use.
Session Number: 221
Session Number: 221
Abstract Title:
Urine Preservation & Stabilization and its Application in Microbial Urinalysis
M. Suwoto, E. Kircher, S. Tang, R. Kemp, X. Jia; Zymo Res., Irvine, CA
Abstract Body:
Background: Urine is an attractive sample input for many applications, such as urinary tract infection
detection and cancers, because of its easy and non-invasive collection procedure. However, nucleic acid
detection in urine can be insensitive and problematic due to nucleic acid instability and bacteria
contamination in the sample. In addition, biased microbial lysis in urine hinders accurate sample
diagnosis. To fix these problems, improved urine collection and purification procedures should be
developed. Here, we demonstrate a complete solution for urine based molecular and microbial testing,
from sample collection to sample analysis. Methods: First, stool sample was spiked into a urine sample
to mimic stool contamination in urine. Next, Urine Conditioning Buffer™ (UCB™), a uniquely formulated
nucleic acid preservation and stabilization reagent, was added to the urine. To analyze the stability of
the urine sample, urine was stored at room temperature at different time intervals: 0 day, 2 weeks, and
1 month. At each time point, DNA was extracted using the ZymoBIOMICS™ DNA Miniprep Kit, an
innovative lysis system that enables efficient and unbiased lysis of microbes. The microbial composition
of the DNA was then profiled via 16s rRNA gene targeted sequencing. Results: The phylum composition
of UCB™ preserved urine was similar throughout the assigned time intervals (0 day, 2 weeks, and 1
month). This shows that UCB™ can both stabilize nucleic acid and inhibit microbial growth in the urine at
room temperature over a month period. Furthermore, we successfully performed unbiased lysis of
microbes in the urine sample. Both DNA from both gram-positive and gram-negative bacteria were
successfully extracted using the ZymoBIOMICS™ DNA Mini Kit. Conclusions: In this study, we present an
improved urine collection procedure to stabilize urine samples coupled to an unbiased microbial
urinalysis.
Session Number: 221
Session Number: 221
Abstract Title:
Improving Blood Culture Collection Volumes Through Near-Real-Time Feedback At A Tertiary-Care
Cancer Hospital
E. A. Powell1, M. Kamalska-Cyganik1, C. Otto2, S. Morjaria1, M. Gendron1, N. Babady1; 1Mem. Sloan
Kettering Cancer Hosp., New York, NY, 2SUNY Downstate Med. Ctr., Brooklyn, NY
Abstract Body:
Background: Optimal blood volume collection is critical to ensure adequate pathogen detection. For
each milliliter below the recommended volume (8-10 mL/bottle), the sensitivity of blood culture
decreases by 3%. Monitoring of blood culture volume helps identify areas for improvement. The goals of
this project were to: 1) Establish baseline blood cultures volume for the 4 hospital units with highest
number of blood cultures submitted 2) Improve clinical staff awareness of the optimal blood culture
volume 3) Increase the percentage of blood cultures with the recommended volume. Methods: Bottles
were weighed and the volume of blood in each bottle calculated based on the average weight of an
empty bottle and the density of blood. Automated monitoring by the instrument software which is
based on measurement of hematocrit was not a valid option in our cancer patients’ population. Baseline
data of blood culture volume and recommendations for collection were presented to nursing staff.
Information on optimal blood culture collection volume was added to the online test directory and
distributed to nursing leadership for further dissemination. Active monitoring was performed over two
time periods (period 1: 02/ 2016; period 2: 09/2016) for 4 consecutive weeks followed by interventions
(education of nursing staff: periods 1 and 2; addition of a comment for suboptimal draws to each blood
culture results: period 2 only). Routine, near-real-time monitoring and addition of the suboptimal
comment were implemented after the second intervention (period 3: May-2017). Results: At baseline
(period 1), 7% (range: 1-22%) of blood cultures bottles contained adequate volume. Following the first
intervention (staff education), monitoring was repeated and optimal volume increased to 17% (range: 4-
42%) of blood culture bottles (period 2) (p<0.05). Implementation of near-real-time monitoring and
feedback to clinical staff (2nd intervention, period 3) resulted in 59% (range: 56-77%) of blood cultures
with adequate blood volume (p<0.001). Conclusions: Routine monitoring of blood culture and
implementation of near-real-time feedback on blood culture volumes resulted in dramatically improved
blood culture volumes. As blood culture volume is a key determinant of pathogens recovery, adequate
collection of blood volume should result in better sepsis diagnosis of our immunocompromised patient
population. Continued monitoring and feedback to nursing staff will remain important to sustain the
observed improvement in blood culture volume collection.
Session Number: 221
Session Number: 221
Abstract Title:
The Effect of Inclement Weather on Testing Methods for Group A Streptococcus
K. Julmis, E. Mitchell, V. M. Torres, S. Juretschko, R. Khare, G. J. Berry; Northwell Hlth.Lab., Lake Success,
NY
Abstract Body:
Introduction: Inclement weather can influence clinical testing in numerous ways which includes factors
such as turn-around-time and specimen integrity. The US-Northeast, more specifically, New York
experienced freezing weather conditions in early January 2018. On the night of January 6, 2018 frozen
samples were received at Northwell Health Laboratories. Among these samples were 135 specimens
with orders for throat culture that were intended to be processed for Group A Streptococcus (GAS). In
order to assess the impact of these extreme weather conditions on the reliability of the culture result,
these specimens were additionally tested using molecular methods of detection (PCR). Materials and
Methods: The 135 throat specimens (24 culturette swabs and 111 ESwabs™) were thawed and plated
for culture, which consisted of plating to Selective Strep Agar and incubation at 37°C for 24-48 hours.
These samples were also processed using the Quidel Strep A C/G PCR Assay, which utilizes a manual
extraction, followed by a real-time PCR on the Cepheid Smartcycler. The Quidel assay detects GAS, as
well as group C/G Streptococcus. Results: Fourteen GAS positive specimens were identified by culture,
with PCR identifying 12 of these same specimens. Qualitative colony counts ranging from few to
numerous were documented and only GAS was identified. The 2 samples that were missed by PCR were
ESwabs™, suggesting that collection variation was not responsible for discordant results. One specimen
was also detected by PCR that was not identified by culture. In addition, 3 cases of group C/G
Streptococcus were identified by PCR and would not have been detected in our current culture workup.
Conclusions: These results show that even in conditions in which throat specimens froze, culture still
performed quite well, even identifying 2 additional specimens that were not detected by PCR. While
culture performed well in the detection of GAS, the PCR results also revealed that cases of group C/G
Streptococcus are being missed by culture. Overall, this data shows that freezing conditions had minimal
impact on culture results for GAS testing.
Session Number: 221
Session Number: 221
Abstract Title:
The Implementation of Diagnostic Microbiology Lab. in A Low Resource Setting, Experience from
Cambodia
J. Letchford, R. Martin, E. J. Baron, J. C. McLaughlin; Diagnostic Microbiol. Dev. Program, Los Altos, CA
Abstract Body:
Despite International Health Regulations 2005 and TB, Malaria and HIV programs, few diagnostic
microbiology laboratories exist in low resource settings (LRS) with the capacity for accurate results. We
describe the implementation of diagnostic bacteriology in Cambodia, using a balance of strengthening
and supportive activities. From 2008 to 2017, the Diagnostic Microbiology Development Program
(DMDP), in collaboration with Ministry of Health (MoH) and laboratory strengthening partners (LSP),
implemented diagnostic microbiology in five provincial hospital laboratories and strengthened three
national laboratories where a microbiology service was in place. We placed eight short term (3-6
months) and eight long-term (6-12 months) experienced expat mentors in laboratories to train more
than 40 government staff and eight Cambodian mentors. Cambodian mentors were recruited by DMDP
after graduating from the Technical School for Medical Care or University of Health Sciences. These
DMDP Cambodian mentors became the backbone of long-term onsite diagnostic strengthening. From
2013, we assisted Bureau of Medical Laboratory Services to strengthen the National Medical
Microbiology Laboratory Network by conducting quarterly meetings, encouraging sharing of experiences
and promoting continuous education. Other activities included implementation of a central media
production laboratory, review and feedback of monthly activity reports (MAR) and mentoring of hospital
doctors by DMDP expat and Cambodian doctors to improve awareness and understanding of a
diagnostic microbiology service. Government microbiology laboratories have increased from six in 2008
to 13 in 2017. Proficiency testing scores remain consistently above 85% and in 2016, laboratory
assessment scores of five laboratories ranged from 64% to 84%. Review of MARs led to development of
monthly cumulative pathogen reports and annual cumulative antibiograms allowing reporting of
pathogens (e.g., Burkholderia pseudomallei, Streptococcus suis) and resistance patterns (e.g., CRE, ESBL
and MRSA) to MoH. Laboratory strengthening requires expert mentoring for at least two years, group
meetings every quarter, close collaboration with international partners and fulltime in-country
leadership. The results of our collective efforts contribute to improved patient care and to national,
regional and global awareness of pathogen prevalence, emerging infectious disease and antimicrobial
resistance.
Session Number: 221
Session Number: 221
Abstract Title:
Validation of Group B Streptococcus Screening Cultures Using Total Lab. Automation
A. Crozier1, W. Lainhart2, C-A. D. Burnham2; 1Barnes-Jewish Hosp., Saint Louis, MO, 2Washington Univ.
Sch. of Med. in St. Louis, Saint Louis, MO
Abstract Body:
Background: Total laboratory automation (TLA) has the potential to standardize culture processing,
incubation, and work up of Group B Streptococcus (GBS) screening cultures. The objective of this study
was to optimize and evaluate the performance characteristics of TLA for GBS screening cultures.
Methods: For initial evaluation of TLA for GBS cultures, various sample inoculation volumes and
streaking patterns were evaluated. Patient specimens (n=67), collected using the BD Eswab, were
cultured using bioMérieux CHROMID® StreptoB agar and LIM broth for both manual and TLA (BD
Kiestra) processing/plating and culture methods. For each paired culture, the following metrics were
evaluated during this process validation: 1) accuracy of specimen label reading, and media selection and
labeling, 2) routing of media to the correct incubators, 3) streaking of the media, 4) imaging of media at
the correct incubation times, and 5) correlation of the results between the methods. To assess
reproducibility, 12 contrived specimens (3 Streptococcus agalactiae, 3 S. pyogenes, 3 S. porcinus, and 3
negative for Streptococcus) were each tested over 3 days. Results: Optimal TLA processing conditions
for GBS screening cultures included the use of 50 µL of specimen inoculated onto the solid (streaked
using pattern 19) and liquid media. The cultures were incubated at 35°C in air. The CHROMID® plate was
imaged at 18 hours. LIM broths of cultures without GBS detected on solid medium were subcultured (50
µL) after 24 hours of incubation using TLA Second Inoculation to a 5% sheep's blood agar plate. This
subculture was incubated at 35°C in 5% CO2 and imaged at 18 and 35 hours. Cultures were read only on
day shift (0700-1530). The TLA system performed well (both patient specimens and reproducibility
cultures) with 100% accuracy of specimen label reading, media selection, media labeling, routing of
media to the correct incubators, streaking of media, and imaging of media at the correct times. Twenty-
four (35.8%) cultures were positive, with 100% positive and negative agreement between the methods.
In reproducibility studies, all 36 cultures had comparable results (positivity and organism abundance).
Conclusions: Optimization of TLA for GBS screening cultures includes batched sample processing. TLA for
GBS screening is accurate compared to conventional methods, improves standardization of specimen
inoculation and reading, and reduces hands-on time for sample processing.
Session Number: 222
Session Number: 222
Session Title: CPHM03 - Diagnostic Bacteriology: Stool is Cool
Abstract Title:
Improved Detection of Campylobacter in Human Stool by A New Rapid Eia (Campylobacter Quik Chek™)
M. Cresse, J. Buss, E. Thacker, L. Chen, J. Herbein; TechLab, Inc., Blacksburg, VA
Abstract Body:
Background: Detection of Campylobacter in stool has long used culture optimized only for C. jejuni and
C. coli. Culture accuracy is limited by the tendency of Campylobacter to die erratically during handling
and by the difficulty of detecting microscopic colonies among competing fecal flora. Currently marketed
immunoassays are easier, but are burdened by high false positive rates and poor detection of low
Campylobacter concentrations. Multiplex molecular assays can generate costly diagnostic uncertainty if
they report co-infections of Campylobacter and other enteric pathogens. To overcome these concerns, a
new 30-minute EIA, the CAMPYLOBACTER QUIK CHEK™ was developed and assessed against culture and
an FDA-cleared immunoassay. Methods: Three clinical sites cultured 694 prospective patient specimens,
then shipped the specimens frozen to TechLab for testing on the QUIK CHEK™ test and Meridian
ImmunoCard STAT!® Campy (ICS), along with Luminex GPP™, 16S rRNA qPCR, species-specific qPCR and
bidirectional sequencing. Results: Analytical LODs of the QUIK CHEK™ test were 150-fold lower than the
ImmunoCard STAT!® for C. jejuni and 40-fold lower for C. coli, at 8.4 x 104 cfu/mL and 7.7 x 105 cfu/mL
respectively. These LODs are as good or better than culture of fecal specimens. The QUIK CHEK™ test
also detected C. upsaliensis, a pathogenic species that grows poorly in culture, but which was present in
10% of all clinical positive specimens. Cultures at the clinical sites gave 24 positives among 694
specimens; QUIK CHEK™ found 32 positives; and the ImmunoCard STAT!® found 75. The 4 molecular
methods identified C. jejuni, C. coli or C. upsaliensis in all 32 QUIK CHEK™-positive and 1 discrepant
culture-negative specimen. After this resolution, the QUIK CHEK™ showed a sensitivity of 100%,
specificity of 100%, and PPV and NPV of 100%. For the ImmunoCard STAT!®, the molecular testing
showed that it had identified 28 positives correctly, but had missed 4 of the 32 true positives and
reported 43 additional false positives, giving a PPV of 37.3% Conclusions: The CAMPYLOBACTER QUIK
CHEK™ test shows greatly improved reliability and a minimal false negative and false positive rate
compared to traditional culture or current immunoassays. The agreement of the QUIK CHEK™ test with
multiple molecular tests is excellent, with the added assurance that Campylobacter is present in a
meaningful clinical range. These characteristics and the speed of testing with the CAMPYLOBACTER QUIK
CHEK™ test provide dependable, rapid diagnostic confidence.
Session Number: 222
Session Number: 222
Abstract Title:
Low False Positives and High Sensitivity of A New Elisa (Campylobacter Chek™) To Detect Campylobacter
in Human Stool
E. Thacker, J. Buss, M. Cresse, L. Chen, J. Herbein; TechLab, Inc., Blacksburg, VA
Abstract Body:
Background: Despite being slow, limited by the tendency of Campylobacter to die erratically during
handling, and requiring well-trained personnel, culture of Campylobacter has remained the gold
standard for identification, in part because currently marketed immunoassays are prone to high rates of
false positives. A new 1-step, 50-minute ELISA, the CAMPYLOBACTER CHEK™, was designed to improve
accuracy and detection of Campylobacter sp. Methods: Prospective fecal specimens were cultured at 3
clinical sites, frozen, then tested at TechLab using the CAMPYLOBACTER CHEK™, Meridian Premier™
Campy ELISA, Luminex GPP™, 16S rRNA qPCR, species-specific qPCR and bidirectional sequencing. In
addition, culture-positive specimens were purchased (Discovery Life Sciences), confirmed to be positive
by all 4 molecular methods, and tested. Results: The CHEK™ assay’s analytical LODs were 5-fold lower
than the Premier™ test, at 2.1 x105 cfu/mL for C. jejuni and 1.6 x106 cfu/mL for C. coli. Additionally, C.
upsaliensis, a pathogenic species that traditional culture detects poorly, was recognized at 3 x 106
cfu/mL. Among 694 clinical specimens, culture detected 24 positives, whereas the CHEK™ assay
detected 33 and Premier™ detected 65. The 4 molecular methods identified C. jejuni, C. coli or C.
upsaliensis in all 33 CHEK™-positive and 1 discrepant culture-positive specimen. After this resolution,
the CAMPYLOBACTER CHEK™ test showed a sensitivity of 100%, specificity of 100%, PPV of 100% and
NPV of 100%. After molecular testing, 32 of the 65 Premier™-positive specimens remained negative,
giving a PPV of 50.8%. None of these 32 false positives were C. upsaliensis. Notably, culture missed 10 of
the 33 positives (30.3%) identified by the CHEK™ and Premier™ tests, and 4 molecular methods, giving a
sensitivity of 69.7%. To further characterize the ability of the CHEK™ assay to detect true positives, 30
confirmed-positive commercial specimens were tested. All 30 were CHEK™-positive, for a correlation of
100%. Conclusions: CAMPYLOBACTER CHEK™ test accuracy exceeds that of culture and has the highest
PPV among currently available Campylobacter EIAs. The CAMPYLOBACTER CHEK™ also agrees fully with
multiple molecular tests while providing information that an infection is clinically relevant. These
characteristics combined with the objectivity, ease of use and speed of the CAMPYLOBACTER CHEK™
assay make it possible to test specimens with confidence and, if required, subsequently culture only
positive specimens.
Session Number: 222
Session Number: 222
Abstract Title:
Clinical Evaluation of Campylobacter Sp. in Stool Specimens Using Two New Antigen-Based Tests
S. Young1, C. Griego-Fullbright1, P. Jim1, A. Wagner1, P. Illescas1, A. Trevizo1, J. Nicasio1, M. Phillips2, B.
Doyle2, L. Chen2, K. Schwab2, J. Stevens2; 1TriCore Reference Lab., Albuquerque, NM, 2TechLab,
Blacksburg, VA
Abstract Body:
Background: Campylobacter species are the most common cause of bacterial gastroenteritis, although
they are more difficult to culture than other diarrhea-causing bacteria. Detection of Campylobacter sp.
requires plating to special media, incubation in a microaerophilic atmosphere for up to 3 days and
encounters unpredictable viability losses. Multiplex molecular methods, while sensitive, are costly,
especially if they generate treatment uncertainty by reporting co-infections of Campylobacter and other
enteric pathogens. We evaluated two new antigen-based assays, the microwell plate-format
CAMPYLOBACTER CHEK™ and lateral flow CAMPYLOBACTER QUIK CHEK™ (TechLab, Inc., Blacksburg,
VA), in prospectively-collected patient stool specimens. Methods: Specimens in transport media were
cultured using Campy-selective media for 2 days in a microaerophilic atmosphere; colonies were
identified as Campylobacter by Gram stain. Specimens were also assayed using both the CHEK and QUIK
CHEK tests. Specimens with results discordant between culture and CHEK or QUIK CHEK tests were
analyzed using a composite reference method (CRM) of either Luminex™ GPP, or PCR specific for 16S
RNA, CadF, HipO and cpn60 genes, and sequence analysis of any resulting amplicon. Statistical analysis
and resolution of results discordant to culture were performed independently for each test. Results:
Among 966 specimens, culture detected 24 positives. The QUIK CHEK test detected 24 and the CHEK test
23 of the culture postives. The QUIK CHEK test detected 8 additional positive specimens; all positive by
the CRM, 7 C. jejuni and 1 C. upsaliensis. The QUIK CHEK test including C. upsaliensis was 100% sensitive,
100% specific, with a 100% PPV and NPV. The one culture-positive/CHEK-negative specimen was
confirmed culture-positive by CRM. The CHEK test also detected 8 additional positive specimens; all
positive by CRM, 7 C. jejuni and 1 C. upsaliensis. <u>The CHEK test including </u><u>C.
upsaliensis</u><u> as positive, had 96.9% sensitivity, 100% specificity, 100% PPV and 99.9% NPV.</u>
Conclusions: The CHEK and QUIK CHEK assays detected 29% and 33%, more positive specimens,
respectively than routine culture. In addition they detected C. upsaliensis, a pathogenic species that
culture usually misses. These results suggest that current culture-based estimates of Campylobacter
incidence are lower than actual rates and that significant numbers of patients are undiagnosed. The
CHEK and QUIK CHEK tests provide rapid single or batch testing with high accuracy and low false positive
rates.
Session Number: 222
Session Number: 222
Abstract Title:
Clinical Evaluation of Two New Antigen-Based Tests for the Detection of Campylobacter Sp. in Preserved
Stool Specimens
M. Mashock, D. Gerstbrein, M. L. Faron, N. A. Ledeboer, B. Mesich, B. W. Buchan; The Med. Coll. of
Wisconsin, Milwaukee, WI
Abstract Body:
Background: Campylobacter sp. is a leading cause of bacterial acute gastroenteritis but is often
undiagnosed because of inadequate diagnostic methods. Stool culture is widely used to identify
Campylobacter sp.; however, this approach is laborious, requires specialized media and incubation
conditions, and takes up to 72 hours for a reportable result. Molecular approaches are more rapid and
sensitive but can be costly and may not be practical for all laboratories. We evaluated two new antigen-
based assays, the microplate-based CAMPYLOBACTER CHEK and the lateral flow CAMPYLOBACTER QUIK
CHEK (TechLab, Inc., Blacksburg, VA), for detection of C. jejuni and C. coli in preserved stool specimens.
Methods: A total of 367 prospective stool specimens submitted in Para-Pak C&S preservative were
tested using CHEK and QUIK CHEK. Results were compared to stool culture using Campylobacter
Selective Medium (Remel, Lenexa, KS) incubated up to 72 hours in a microaerophilic atmosphere.
Characteristic colonies were identified by gram stain, oxidase, and MALDI-ToF MS. Specimens with
results discordant between culture and CHEK or QUIK CHEK were analyzed using Luminex GPP, or PCR
and sequence analysis specific for 16S RNA, CadF, HipO and cpn60 genes. Results for each test were
compared independently to culture for statistical analysis and discordant resolution. Results: Five (1.4%)
specimens were positive for Campylobacter sp. by culture; all were identified as C. jejuni by MALDI-ToF
MS. All 5 were positive by QUIK CHEK and 4/5 were positive by CHEK. The lone false negative CHEK
result was confirmed to be C. jejuni by PCR and sequencing. An additional 2 culture-negative specimens
were detected as positive by both CHEK and QUIK CHEK, and were confirmed to contain C. jejuni by PCR
and sequencing. One culture-negative specimen was positive by only QUIK CHEK but was confirmed to
contain C. jejuni. A separate culture-negative specimen was positive only by CHEK and was found to
contain C. upsaliensis by sequence analysis. Conclusions: CAMPYLOBACTER CHEK and CAMPYLOBACTER
QUIK CHEK detect Campylobacter sp. in 40-60% more stool specimens than routine culture. Compared
to combined culture and PCR results, CHEK was 85.7% sensitive (6/7) and 99.7% specific (359/360) for C.
jejuni or C. coli. QUIK CHEK was 100% sensitive (8/8) and 100% specific (359/359) for C. jejuni or C. coli.
These tests enable on demand rapid results (QUIK CHEK) or high throughput batch testing options
(CHEK) and can detect C. jejuni, C. coli and C. upsaliensis in culture-negative specimens.
Session Number: 222
Session Number: 222
Abstract Title:
Performance Evaluation of Two Commercial Enzyme Immunoassays (Eia) for Detection of
Campylobacter Antigens Directly from Human Fecal Specimens
M. Michael, D. S. S. Wijetunge, J. Vitale, D. Myers, D. Craft; Penn State Hlth., Hershey, PA
Abstract Body:
Introduction: Campylobacter is one the most common organisms associated with bacterial
gastroenteritis in humans resulting 1.3 million cases of diarrhea each year in the United States. Current
protocols for the laboratory diagnosis of Campylobacteriosis include stool culture and antigen detection
of Campylobacter spp. from fecal specimens. Although culture is considered the gold standard, antigen
detection offers high sensitivity and decreased turnaround time which may impact the therapeutic
management of patients. Sensitivity of stool culture may be impacted by unique culture conditions that
include slow growth and microaerophilic incubation at 42oC. The objective of our study was to evaluate
performance characteristics of CAMPYLOBACTER QUIK CHEK™ (CQCHEK) and CAMPYLOBACTER CHEK™
(CCHEK), (TechLab, Blacksburg, VA), for qualitative detection of C. jejuni and C. coli antigens in fecal
specimens. CQCHEK is a membrane associated rapid antigen detection method whereas CCHEK is a
micro-well enzyme-linked immunosorbent assay. Methods: Over a 4 month period, 219 fecal specimens
(fresh & Cary-Blair) were concurrently tested by conventional campylobacter stool culture, CQCHEK AND
CCHEK. In Phase I, aliquots of fecal specimens were sent to TechLab (n=154) for testing and in Phase II
(n=65), both EIAs were performed in-house according to manufacturer’s instructions. An aliquot was
frozen and stored for test discrepancy resolution as needed. Discrepancy analysis was performed at
TechLab and included in-house PCR, xTAG® Gastrointestinal Pathogen Panel (Luminex, Austin, TX) and
16sRNA sequencing (reference methods). Results: Out of 219 samples, 6 were culture positive for
Campylobacter spp. One culture positive specimen was negative by both EIAs and multiplex PCR/x-
TAG/16s rRNA sequencing. Sensitivity, PPV and PNV for both EIAs and culture were 100 %. Specificity
was 99.5% and 100% for culture and both EIAs, respectively. Conclusions: CQCHEK and CCHEK EIAs were
in agreement for 218/219 or 99.5% of the time. The specimen with discrepant results was a culture
positive for C. jejuni with a family member also culture positive. Both EIAs showed similar sensitivity and
specificity for detection of Campylobacter spp. in fecal specimens. The major advantage of both EIAs
was quicker turnaround time for detection (CQCHEK:30 min, CCHEK:1 hour) compared to culture (2-3
days). Our study indicates that CQHEK™ and CCHEK are rapid and reliable methods to detect
Campylobacter spp. from fecal specimens.
Session Number: 222
Session Number: 222
Abstract Title:
Outstanding Abstract Award: Detecting Cross-Contamination and False-Negative Results Using Fda-
Cleared Molecular Enteric Pathogen Tests: Automated Positivity Monitoring and Back-Up Culture
D. E. Bosch, J. Vong, R. J. Cybulski, L. Bourassa, A. B. Bryan; Univ. of Washington Med. Ctr., Seattle, WA
Abstract Body:
Background: Clinical enteric pathogens testing is rapidly evolving from culture-based techniques to more
highly sensitive and specific nucleic acid amplification tests. Distinguishing between a positive result
from a more sensitive assay and a false positive result can be challenging and is important, given
decreased use of culture-based methods, continued requirements for public health reporting, and
limited options to investigate closed FDA-cleared systems. Methods: Enteric pathogens testing was
performed on clinical stool specimens using the BioFire FilmArray® multiplex PCR platform coupled with
limited culture. Potential false positive STEC results were detected by expert technologist review.
Bayesian statistical models were used to detect non-random increases in positivity rates over time for
each organism. False negative results were identified by comparison to concurrent stool culture and
chart review. Culture isolates were identified with MALDI-TOF mass spectrometry and 16S rRNA gene
sequencing, and spiked into stool specimens for confirmatory testing. Results: Six enteric pathogens
panel PCR tests positive for Shiga-like toxin-producing E. coli (STEC) within 3 days raised suspicion for
false positive results. Confirmatory testing on an identical platform was negative in 5 of 6 cases. Analysis
of PCR amplification curves revealed a low Ct value for the initial true positive result and high Ct values
for subsequent samples, consistent with contamination by low-level nucleic acids. A Bayesian statistical
model was applied to positive enteric pathogens panel results over time, and identified significantly
elevated STEC positivity rates in association with cross-contamination. The statistical model is adapted
for prospective clinical lab surveillance. We identified a BioFire-negative sample with concurrent culture
growing 4+ Camplyobacter jejuni/coli by mass spectrometry and 16S rRNA gene sequencing. The culture
isolate was spiked into a stool specimen, and again tested negative by the enteric pathogens panel. The
findings are consistent with incomplete coverage of C. coli/jejuni strains, which may result from genetic
variations that interfere with PCR amplification. Conclusions: PCR-based enteric pathogens testing offers
major advantages over traditional stool culture. However, users should be aware of potential errors of
testing. Issues of contamination and incomplete coverage of pathogenic bacterial species can be
mitigated with novel quality control measures and concurrent limited stool culture.
Session Number: 222
Session Number: 222
Abstract Title:
Evaluation of Clin. Performance of Pannat® Nucleic Acid Stec Test for Detection and Differentiation of
Shiga-Toxin Expressing E. coli in Diarrheal Stool Specimens
N. Kanwar1, A. Nguyen1, E. J. Klein2, N. A. Ledeboer3, M. L. Faron3, M. Mashock3, P. A. Granato4, M.
Unz4, S. Young5, J. Dunn6, C. L. Johnson7, A. Lofquist8, T. Barbera8, R. Selvarangan1; 1Children's Mercy
Hosp. and Clinics, Kansas City, MO, 2Seattle Chi
Abstract Body:
Background: Shiga toxin producing E. coli (STEC) may cause life threatening illness among children and
older adults. The Centers for Disease Control estimates that 265,000 illnesses and 3600 hospitalizations
are attributed to STEC in the United States, annually. Prompt and accurate STEC detection helps with
appropriate patient management. The PanNAT® nucleic acid STEC test (PanNAT STEC) is a molecular
based sample-to-answer assay that detects and differentiates STEC by targeting shiga toxin genes 1 and
2 (stx1, stx2) and the O-antigen gene cluster specific to E.coli O157 (fcl) for identification. The aim of this
multicenter study was to evaluate the performance of PanNAT STEC test with reference to
ImmunoCardSTAT!® EHEC assay and sorbitol-MacConkey agar (SMAC) culture. Methods: Diarrheal stool
samples were obtained from both children and adults. One site actively enrolled subjects (n=84) upon
admission in the emergency department, and the other five sites examined left over samples (n=1248)
from subjects with gastroenteritis for which standard of care tests were performed. Samples were
divided into aliquots for PanNAT testing, SMAC culture and ImmunoCardSTAT!® EHEC assay following
enrichment in MacConkey broth. Clear or beige colonies on SMAC plates were shipped to a reference
site for O157 confirmation by latex agglutination and VITEK® 2 identification. Discordants were tested by
alternative PCR and Bidirectional Sequencing. Result: Data from 1280 samples were included for final
analysis; 52 exclusions included 31 protocol deviations and 21 invalid test results. The reference method
identified stx1, stx2, and O157 in 4, 7, and 5 samples, respectively. All positive samples were accurately
identified by PanNAT STEC test. Two samples were false positive for O157 target by PanNAT STEC; one
of which was confirmed true positive by discrepant testing. The overall positive percent agreement
(PPA) and 95% CI for stx1, stx2, and O157 was 100% (39.6-100), 100% (56.1-100), and 100% (46.3-100),
respectively. The negative percent agreement (NPA) and 95% CI for stx1 and stx2 was 99.4% (98.7-99.7)
and for O157 was 99.8% (99.4-100). Conclusion: PanNAT STEC test demonstrated a high PPA and NPA in
comparison with the reference methods for detection of STEC. The PanNAT assay provided accurate
results, with easy set up and rapid result availability within an hour. PanNAT assay is effective for near
patient/point of care settings to promptly detect all STEC producing stx1 and 2 toxins and specifically
identifying STEC E. coli O157 from stool specimens.
Session Number: 222
Session Number: 222
Abstract Title:
The Chicken Or the Egg: High Prevalence of Ulcerative Colitis among Patients with Stool Cultures Positive
for Aeromonas Species
I. Larsen, S. M. Butler-Wu; LAC+USC Med. Ctr., Los Angeles, CA
Abstract Body:
Background: The clinical significance of Aeromonas as a causal agent of diarrhea is unclear, although
Aeromonas have been reported to cause outbreaks of diarrheal illness. While clinical laboratories have
traditionally screened for the presence of Aeromonas in stool cultures, commercial multiplex
gastrointestinal PCR panels do not target this organism. To assess the clinical significance of Aeromonas
isolation from stool culture in a large, urban population, we investigated the association of these
organisms with infectious diarrhea at our institution. Methods: We performed a retrospective chart
review on patients with stool cultures positive for Aeromonas during a two-year period (2015-2016).
Immune status, antibiotic use prior to onset of diarrheal illness, duration and type of diarrhea,
associated symptoms, testing for stool leukocytes and other common stool pathogens were assessed to
determine the clinical significance of Aeromonas isolation. Results: A total of 2149 stool specimens were
cultured during the study period, of which 19 (0.9%) were positive for Aeromonas species. Two patients
were concomitantly positive for Campylobacter jejuni and Dientamoeba fragilis, respectively, and two
patients had toxigenic C. difficile detected. These patients were excluded from further analysis. Of the
remaining 15 patients, 13 (86.7%) had clinically significant diarrhea at the time of testing. A. sobria was
most frequently isolated (9/15, 60%), followed by A. hydrophila (5/15, 33.3%), and A. caviae (1/15,
6.7%). Importantly, seven patients (46.7%) had been previously diagnosed with Inflammatory Bowel
Disease (IBD). Of these, six (6) had a diagnosis of Ulcerative Colitis (UC), and one was diagnosed with
Crohn’s disease. The majority of patients with UC had bloody diarrhea (5/6, 83.3%) at the time of
testing. Conclusions: The majority of patients who tested positive for Aeromonas had clinically
significant diarrhea. Despite the relatively small sample size, we noted a high prevalence of UC among
patients with Aeromonas isolated from stool culture. Such a relationship was noted in a previously
published European study but this association has not been extensively described. Whether Aeromonas
acts as precipitating factor in the initial onset of IBD is unknown, and further studies with a larger
sample size are required to better characterize the potential association of Aeromonas with IBD.
Session Number: 222
Session Number: 222
Abstract Title:
Prospective Evaluation of Hardychrom Ss Nopro Agar for Salmonella and Shigella Isolation from Routine
Stool Culture
L. Tea, M. Umali-Wilcox, S. Butler-Wu; LAC+USC Med. Ctr., Los Angeles, CA
Abstract Body:
Background: Traditional stool culture media (e.g. Hektoen enteric agar) have high rates of isolation of
non-pathogenic microbiota that can mimic the colony morphologies of Salmonella and Shigella species.
Consequently, traditional stool cultures are associated with significant culture work-up and thus higher
demands on technologists’ time. HardyCHROM™ SS NoPRO agar is a selective chromogenic agar that is
approved for Salmonella and Shigella species isolation and differentiation from stool. HardyCHROM™ SS
NoPRO is inhibitory to Proteus species and would be anticipated to reduce the number of unnecessary
work-ups from stool cultures. Methods: From June-August 2017, all stools received for clinical stool
culture testing were inoculated in parallel on HardyCHROM™ SS NoPRO agar. Colonies requiring workup
from HardyCHROM™ SS NoPRO agar were directly identified by MALDI-TOF Mass Spectrometry (VITEK
MS, bioMerieux). Results of standard of care and HardyCHROM™ SS NoPRO agar testing were directly
compared. The number of cultures negative for Salmonella and Shigella but requiring further work-up
was assessed and a cost analysis was performed. Results: A total of 175 stool cultures were performed
during the study period. Nine specimens were positive for Salmonella and Shigella species by standard
of care culture (5.1% positivity rate): Shigella flexneri =6, Shigella sonnei =1 and Salmonella species =2.
HardyCHROM™ SS NoPRO was falsely negative for two specimens positive for S. flexneri (sensitivity
77%); SS agar was also falsely negative both of these specimens with the S. flexneri being isolated only
from MacConkey Lactose agar. 76 specimens required additional work-up with the standard of care
culture, compared with 28 specimens for HardyCHROM™ SS NoPRO (specificity of 54% and 83%,
respectively). In total, 90 colonies from SS agar required further work up (cost = $848) compared with 30
colonies from HardyCHROM™ SS NoPRO (cost = $157) during the study period. Conclusions: As with
other selective culture media, Shigella species may occasionally fail to be isolated with HardyCHROM™
SS NoPRO. Laboratories using HardyCHROM™ SS NoPRO should therefore consider inclusion of a non-
selective agar in their stool culture media. Compared with SS Agar, HardyCHROM™ SS NoPRO would
have reduced the number of cultures requiring work-up by 50% and reduced the overall costs associated
with stool culture by 80% when colonies were directly identified by MALDI-TOF.
Session Number: 222
Session Number: 222
Abstract Title:
Profiling of Shigella and Etec Diarrheal Cases in the Mal-Ed Study
J. Liu1, J. Platts-Mills1, E. Rogawski1, A. Lima2, G. Kang3, A. Samie4, R. Haque5, E. Mduma6, M. Kosek7,
J. P. Leite8, L. Bodhidatta9, N. Iqbal10, N. Page11, I. Kiwelu12, E. Houpt1, MAL-ED Network Investigators;
1Univ. of Virginia, Charlottesville, VA, 2
Abstract Body:
Background: Shigella and enterotoxigenic E. coli (ETEC) are leading causes of childhood diarrhea in
resource-limiting settings. Further analysis of Shigella speciation and ETEC colonization factors (CFs) may
provide guidance for vaccine development and intervention. Methods: 6877 diarrhea samples from
1715 children in the birth cohort study, MAL-ED, were tested for a broad panel of enteropathogens,
including Shigella/EIEC (targeting ipaH gene) and ETEC targeting heat-liable toxin gene (LT) and heat-
stable toxin genes (STh and STp), using real time PCR. Etiologies of diarrhea were determined by
calculating pathogen specific episode attributable fractions (as per Lancet 2016, (AFe) was defined as 1-
1/OR, where OR is odds ratio derived from logistic regression). A subset of diarrheal cases caused by
Shigella/EIEC and ETEC were further assayed for Shigella species (S. flexneri and S. sonnei) and ETEC CFs
including vaccine candidates CFA/I, CS1, CS2, CS3, CS5, and CS6, respectively. Results: Among 1241
diarrheal cases (from 671 children) with Shigella/EIEC attribution, a random subset of 562 cases (73%,
from 378 children) were further speciated, 178 (32%) S. flexneri and 129 (23%) S. sonnei. Among 1779
diarrheal cases (from 765 children) with ETEC attribution, 352 (from 256 children) were tested for CFs.
59% of cases (207/352) produced both LT and ST, with CF types: CS6 alone (17%), CFA/I (15%), CS1 and
CS3 (14%), CS2 and CS3 (14%), CS5 and CS6 (8%), 1-2% CS1 or CS2 or CS3, and 24% untyped. 21%
(73/352) produced only LT, with CS6 26%, CS1 8%, CFA/I 3%, CS3 3%, CS1+CS3 and CS5+CS6 1%, and 58%
untyped. 20% produced either STh or STp, with CFA/I 35%, CS6 31%, CS5+CS6 13%, and 22% untyped.
The distribution of these types across different study sites were also analyzed. Conclusions: S. flexneri
and S. sonnei accounted for 55% of Shigella/EIEC diarrhea cases. CFA/I, CS3, CS6 were the major ETEC
CFs, vaccine targeting these factors plus LT would cover ~95% of ETEC infections.
Session Number: 222
Session Number: 222
Abstract Title:
Phylogenetic Classification, Antibiogram and Plasmid Profiling of Enterotoxigenic Klebsiella Variicola And
Enterobacter Species Isolated From Iko River- Nigeria
A. O. Mmuoegbulam1, S. P. Antai1, V. M. Oliveira2, C. U. Iroegbu3, B. E. Asikong1, D. Belgini2; 1Univ. of
Calabar, Calabar, Nigeria, 2Univ. of Campinas, Campinas, Brazil, 3Cross River Univ. of Technology,
Calabar, Nigeria
Abstract Body:
Exploitation of natural resources has led to introduction of various pollutants into the environment.
Among the biota presumably affected by environmental pollution are the microorganisms. It is
speculated that oil pollution of the marine environment, for example, would create a preponderance of
antibiotic resistant survivors, which, on their part, would constitute an enormous public health problem.
One group of pathogens that could be affected in the event is enterotoxigenic enterobacteria, which
abound in Nigerian coastal waters and estuaries. Enterotoxigenic enterobacteria isolated from Iko River
in Akwa Ibom State, Nigeria, were evaluated for phylogenetic classification using the 16S rRNA
sequencing protocol. The sequence data generated from the PCR amplification and cycle sequencing
reaction were matched with available sequences in the ribosomal data project (RDP). Isolates 12A, 13
and EC6 (60%) were identified as Klebsiella variicola F2R9T (AJ783916), isolate 11A (20%) as
Enterobacter ludwigii EN-119T (AJ853891), while isolate 10 (20%) was identified to be either
Enterobacter asburiae JCM6051 (AB004744)/cancerogenus LMG 2693 T (Z96078). Enterotoxigenicity of
the isolates was established using the ligated ileal loop assay. All the isolates were found to be entero-
pathogenic with some histopathological effects. The plasmid profile of the isolates was also determined
using the Promega protocols and applications guide; and, with the exception of isolate 13, all the
isolates had plasmids. Antibiogram showed that the isolates were susceptible to Gentamycin,
Ciprofloxacin and Clavulin; however, minimum bactericidal concentration (MBC) determination showed
that Gentamycin and Ciprofloxacin were more effective than Clavulin. An evaluation of the relationship
between the presence of plasmid, antibiotic resistance/susceptibility and enterotoxigenicity, comparing
isolates from both oil-polluted and non-polluted water, showed no association between these factors.
This indicates that exposure to crude oil does not ameliorate or exacerbate antibiotic resistance,
enterotoxigenicity and plasmid acquisition.
Session Number: 222
Session Number: 222
Abstract Title:
Performance Evaluation of the Luminex Aries® C. Difficile Assay in Comparison to Two Other Molecular
Assays Within A Multi-Hospital Hlth. Care Center
S. Juretschko, R. Manji, R. Khare; Northwell Hlth.Lab., Lake Success, NY
Abstract Body:
Background: With about 500,000 infections annually, Clostridium difficile infections (CDI) remain a
serious issue in the U.S. (CDC). Both fast and accurate diagnosis of CDI is paramount to achieve
immediate Infection Control initiation, triaging and isolation, as well as appropriate antibiotic treatment.
However, both, over- and under-diagnosis can lead to adverse patient outcomes, such as unnecessary
administration of antibiotics or unwanted spread of spores in any hospital setting, respectively.
Methods: Between July 1st and October 15th 2017, Northwell Health Laboratories conducted a
prospective comparison study between three molecular assays: Luminex ARIES® C. difficile (tcdA &
tcdB), Cepheid® Xpert® C. difficile (tcdB) and BD -MAX™ (tcdB). 302 stool samples (Cary Blair) were
clinically tested with the Cepheid® assay. The de-identified remnant specimens were further tested in
parallel with the ARIES® and BD MAX™ assays. True positivity was determined as 2 out of 3 positives by
any assay. Discrepant results were further investigated with toxin specific sequencing. A time and
motion study, comparing hands-on time (HoT) and TAT for processing of one, six and 12 specimens on
each instrument was conducted. Results: Out of 302 specimens, 62 were resulted as positive for CDI
with the Cepheid® assay. Comparison results showed a positive and negative percent agreement
between ARIES® and Cepheid® of 95.2% (PPA, 59/62) and 99.2% (NPA, 238/240), respectively. PPA and
NPA between ARIES® and BD MAX™ were 91.8% (56/61) and 96.6% (230/238), respectively. Invalid rates
were determined to be 5% for BD -MAX™, 0.5% for ARIES®, and 0% for Cepheid®. TAT and HoT varied
significantly, depending on throughput and availability of instrumentation. Results shifted preferably
towards either assay, but Cepheid® shows a slight advantage for TAT, if sufficient bays are installed.
Taking repeat testing in account, the BD MAX™ assay with its high invalid rate showed a significant delay
in overall TAT. Conclusions: In terms of accuracy, the Luminex ARIES® C. difficile Assay performed
comparably with the Cepheid® assay, but was superior to the BD MAX assay. For low throughput and
immediate need of testing of single specimens the Cepheid® assay holds an advantage. However, with
its ease of use, speed, and accuracy, the ARIES® C. difficile Assay provides wider flexibility with a unique
mixture of batched and sample to answer testing.
Session Number: 222
Session Number: 222
Abstract Title:
Performing Reflexive Toxin A/B Enzyme Immunoassay in A Two-Step Algorithm for the Diagnosis of
Clostridium Difficile Infection Has Ltd. Utility
M. M. Mounajjed, B. Jung-Hynes, N. Safdar, D. J. Chen; Univ. of Wisconsin Sch. of Med. and Publ. Hlth.,
Madison, WI
Abstract Body:
Diagnosis of Clostridium difficile infection (CDI) via nucleic acid amplification testing (NAAT) for toxin
genes may result in overdiagnosis; however, the added value of other diagnostic modalities is currently
unclear. The purpose of this study was to determine whether addition of toxin enzyme immunoassay
(EIA) to specimens already tested by NAAT would differentiate patients with CDI from those who were
colonized by C. difficile. As part of routine diagnostic workup, unformed stool specimens are tested by
the hospital laboratory for the presence of C. difficile toxin B gene using the Cepheid Xpert C.
difficile/Epi Assay (PCR). For this study, stool samples, randomly selected based on investigator
availability from January to May 2017, were subsequently tested with the Alere C. DIFF QUIK CHEK
COMPLETE (EIA) to evaluate for the presence of glutamate dehydrogenase (GDH) and toxins A and B;
the EIA results were not provided to the treating clinicians. All corresponding medical charts were
reviewed while blinded to the EIA results for the following factors: severity of symptoms, 3 or more
unformed stools a day, antibiotic use within 90 days, prior history of CDI, antibiotic treatment for CDI,
and improvement of symptoms with treatment. Since there is no gold standard to diagnose CDI, these
factors were used to categorize patients into three groups: (1) CDI likely, (2) CDI unlikely, and (3) CDI
indeterminate. A total of 60 stool samples were tested from 60 patients. Clinical categorization of the
patients and the associated test results are shown in the Table. Clinical sensitivities for PCR, GDH EIA,
and toxin EIA were 100%, 81%, and 38%, and clinical specificities were 77%, 64-77%, and 86%,
respectively. The addition of GDH EIA and/or toxin EIA to PCR results did not help differentiate CDI from
colonization in this study. Several patients who were PCR-positive but GDH and toxin EIA-negative
benefited from treatment for CDI. Some categorized as CDI unlikely had positive PCR and EIA results,
probably representing detection of colonization. Two-step algorithms for CDI diagnosis may result in
undertreatement.<table class="AbstractTable" id="{F74CDAA1-7C83-4FC0-8142-
50B041486278}"><caption class="AbstractTableCaption"></caption><tr><td rowspan="1"
rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1"><u>Clinical
Category</u></td><td rowspan="1" colspan="2"><u>PCR</u></td><td rowspan="1"
colspan="3"><u>GDH EIA</u></td><td rowspan="1" colspan="2"><u>Toxin EIA</u></td><td
rowspan="1" colspan="1"><u>Total</u></td></tr><tr><td rowspan="1" colspan="1"></td><td
rowspan="1" colspan="1"><u>POS</u></td><td rowspan="1" colspan="1"><u>NEG</u></td><td
rowspan="1" colspan="1"><u>POS</u></td><td rowspan="1" colspan="1"><u>NEG</u></td><td
rowspan="1" colspan="1"><u>NOT DONE</u></td><td rowspan="1" colspan="1"><u>POS</u></td><td
rowspan="1" colspan="1"><u>NEG</u></td><td rowspan="1" colspan="1"></td></tr><tr><td
rowspan="1" colspan="1"><u>CDI Likely</u></td><td rowspan="1" colspan="1">26</td><td
colspan="1">5</td><td rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">10</td><td
colspan="1"><u>CDI Unlikely</u></td><td rowspan="1" colspan="1">5</td><td rowspan="1"
colspan="1"><u>CDI Indeterminate</u></td><td rowspan="1" colspan="1">12</td><td rowspan="1"
rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">4</td><td rowspan="1"
colspan="1"><u>Total</u></td><td rowspan="1" colspan="1">43</td><td rowspan="1"
colspan="1">43</td><td rowspan="1" colspan="1">60</td></tr></table>
Session Number: 222
Session Number: 222
Abstract Title:
Evaluation of An Ultrasensitive Immunoassay, Currently in Dev. for the Singulex Clarity System, for the
Detection of Clostridium Difficile Toxins A and B
A. Almazan, S. Abusali, S. Tam, E. Lee, A. Changavi, W. Trinh, K. Chau, J. Estis, B. Noland, J. Bishop, A.
Bartolome; Singulex, Inc., Alameda, CA
Abstract Body:
Background: Clostridium difficile infection (CDI) is the most common nosocomial infection. Currently,
immunoassays and PCR show deficits of either sensitivity or specificity in the diagnosis of CDI. The
Singulex Clarity® C. diff toxins A/B assay, in development for the Singulex Clarity® system and based
upon Single Molecule Counting technology, was designed to provide a highly sensitive, rapid, and
automated assay for the detection of C. difficile toxins A and B in stool. The analytical and preliminary
clinical performance of the assay is herein reported. Materials/Methods: C. difficile toxins were spiked
into buffer and stool and limits of detection (LoD) for toxins A and B were determined in both matrices.
Eight C. difficile strains, including A+/B+, A+/B-, A-/B+, and A-/B-, were tested to determine analytical
reactivity. Stability, potentially interfering substances, and cross-reactivity with common gastrointestinal
pathogens were evaluated. Repeatability was tested with triplicate samples. Assay reproducibility was
evaluated using samples subjected to three freeze-thaw cycles and for diluted samples stored at room
temperature for eight hours and refrigerated for two weeks. Samples from patients with suspected CDI
(n=103; TriCore Reference Laboratories) were tested and a clinical cut-off was obtained using cell
cytotoxicity neutralization assay (CCNA) as reference standard. Results: The LoDs for toxins A and B were
0.8 and 0.3 pg/mL in buffer, and 2.0 and 0.6 pg/mL in stool, respectively. The assay accurately detected
C. difficile toxins A and B from eight common strains. The assay was robust against 11 potentially
interfering substances, 51 bacteria (18 anaerobic, 30 aerobic, and 3 microaerophilic), 9 viruses, and one
fungus. For samples subjected to freeze thaw and storage, the qualitative results were reproducible and
the repeatability of triplicate qualitative results was 99.0%. Compared to CCNA, the assay had 96.3%
sensitivity and 97.4% specificity. Conclusions: The Singulex Clarity C. diff toxins A/B assay detects C.
difficile toxins A and B at very low concentrations and the assay has sensitivity and specificity
comparable to CCNA. The assay demonstrates reactivity to common C. difficile strains, is robust against
common interferents, and does not show cross-reactivity to common gastrointestinal pathogens. The
assay allows for the automated and reproducible ultrasensitive detection of C. difficile toxins A and B in
stool. Additional studies are required to confirm the performance against CCNA and clinical diagnosis.
Session Number: 222
Session Number: 222
Abstract Title:
Molecular Epidemiology of Hosp. Acquired Clostridium Difficile Infection in Two Regions of Zhejiang and
Evaluation of its Clin. Predicting Model
X. Wang1, L. Wang2, X. Xu3, Y. Luo4, X. Song4, J. Ye4, L. Zhao5, J. Huang6, Y. Tang7, D. Jin4; 1Hangzhou
First People,s Hosp., Zhejiang, China, 2Zhejiang Chinese Med. Univ. affiliated Hangzhou First People,s
Hosp.,, Zhejiang, China, 3Zhejiang Chinese Med
Abstract Body:
Clostridium difficile infection (CDI) as a major cause of antibiotic-associated diarrhea has been reported
in China recently. Detailed CDI symptoms are, however, not disclosed in Chinese people, and no
nomogram models are available to predict whether diarrhea samples need C. difficile test or not. We
performed a one-time cross-sectional study in Hangzhou First People’s Hospital Healthcare Facility
Group (HFPH) and Lishui Second People’s Hospital (LSPH) in Zhejiang from June 2015 to September
2017. Stool specimens collected from consecutive hospitalized patients with diarrhea were cultured for
C. difficile and the isolates were analyzed for the presence of toxin genes, multilocus sequence analysis
(ST) and antimicrobial susceptibility testing. CDI severities were defined by IDSA/SHEA guidelines
according to blinded medical record review. A total of 190 C. difficile isolates were recovered from 1,250
patients (15.2%). Of them, 110 (57.9%) had both tcdA and tcdB (A+B+), 71(37.4%) were negative for
tcdA and positive for tcdB (A-B+), and 9 (4.7%) had no toxin genes (A-B-). ST37 (19.5%) and ST54 (16.8%)
were the dominant genotypes. CDI severity is generally mild to moderate, and tcd A-B+ was the
dominant genotype with severity CDI (P < 0.001).All the isolates were completely susceptible to
vancomycin and metronidazole, and the antibiotic resistance of Clindamycin in LSPH (100.00%) was
higher than HFPH (84.40%)(P<0.001). Clinical data of 1250 patients with diarrhea were retrospectively
analyzed. Multivariate Logistic regression analysis revealed that, Neutrophils with less than 70%,
Lymphocyte with more than 40%, RBC with less than 5.5×1012/L, Hb with less than 160 g/L, and PLT
more than 300×109/L in blood were the independent clinical indicators for CDI(P<0.05). All the patients
were randomly divided into the model establishment group (n=1000, 80%) and validation group (n=250,
20%). The nomogram mode was established with a C-index of 0.727, and the C-index of the validation
group was 0.719, indicating that the nomogram model was qualified to predicting CDI in patients with
diarrhea. In conclusion, CDI has been undoubtedly a problem in hospitals in China, and the tcd A-B+
genotype should be emphasized in CDI control and prevention. The monogram model could play a role
on guiding clinician to predict CDI through common clinical indicators and driving clinical CDI diagnosis in
China. .
Session Number: 222
Session Number: 222
Abstract Title:
Ultrasensitive Detection of Clostridium Difficile Toxins A and B Using Single Molecule Counting
Technology
J. Sandlund1, A. Bartolome1, J. Bishop1, N. Nolan1, J. Todd1, F. Senchyna2, N. Banaei2; 1Singulex, Inc.,
Alameda, CA, 2Stanford Univ. Sch. of Med., Palo Alto, CA
Abstract Body:
Background: Current tests for the detection of free toxins in feces used for the diagnosis of Clostridium
difficile infection (CDI) lack sensitivity, while nucleic acid amplification tests lack specificity. We have
evaluated a prototype automated and rapid ultrasensitive immunoassay (UIA) for the detection of C.
difficile toxins A and B in stool based on Single Molecule Counting technology (limits of detection for
toxins A and B are 2.0 and 0.6 pg/mL, respectively). Materials/methods: Frozen stool samples from 311
patients with suspected CDI were tested with the UIA (Singulex Clarity® C. diff toxins A/B). Samples had
been previously tested with PCR (GeneXpert®), reporting also on 027 status of the strain. PCR+ samples
(n=211) were tested for the presence of free toxins with a rapid membrane enzyme immunoassay (EIA;
C. Diff Quik Chek Complete®), and EIA- samples (n=110) were further tested with cell cytotoxicity
neutralization assay (CCNA; 31 CCNA+ and 79 CCNA-). EIA+ or CCNA+ samples were considered true
toxin-positive and PCR- samples were considered C. difficile-negative. A cutoff that was previously
established on an independent sample set was used in this study. Results: The UIA yielded 97.7%
sensitivity (129 of the 132 true toxin-positive samples were positive by the UIA) and 100% specificity.
Eighteen of the 79 (22.8%) PCR+/EIA-/CCNA- samples were positive by the UIA. Furthermore, the UIA
reported that the median toxin concentrations in toxin+ samples in the 99 PCR+/EIA+, 30 PCR+/EIA-
/CCNA+, and 18 PCR+/EIA-/CCNA- samples were 4680.5 (IQR 1067.3–15839.2), 352.2 (IQR 124.6–540.2),
and 31.3 pg/mL (IQR 22.3–209.7; p <0.001), respectively. The median toxin concentration was higher in
samples with 027 (n=28) compared with non-027 (n=183) strains (2846 [IQR 96.4–7729] vs. 369.8 [IQR
5.0–3124] pg/mL; p=0.006). Conclusions: Compared with multistep toxin testing and PCR, respectively,
the UIA is ultrasensitive and highly specific and may offer a standalone solution for the rapid detection
and quantitation of free toxins in stool.
Session Number: 222
Session Number: 222
Abstract Title:
A Single Center’S Experience with A Novel Nucleic Acid Amplification Test for the Detection of the
Clostridium Difficile Toxin B Gene from Clin. Samples
C. Dang1, S. Lewis2, M. Olson1, A. Romesburg1, J. Liffers1, B. C. Ellis1, K. C. Carroll2; 1Johns Hopkins
Hosp., Baltimore, MD, 2Johns Hopkins Univ. Sch. of Med., Baltimore, MD
Abstract Body:
Background: C. difficile remains a formidable pathogen. While controversial, many laboratories use a
molecular test for the diagnosis of C. difficile infection (CDI). We report a single center’s experience from
a multi-center study for FDA 510K approval of a new qualitative real-time PCR assay, i.e. the GenePOC™
CDiff assay (GenePOC Inc., Québec, Canada). Methods: The Johns Hopkins Hospital (JHH) Microbiology
Laboratory tested 200 unformed stool specimens. To run the GenePOC CDiff test, approximately 5 μl of
the stool sample was tested according to the manufacturer’s package insert. 500 µl of the stool sample
was transferred to an Anaerobe Transport Medium and shipped to an off-site reference lab for direct
culture on CCFA agar and enriched culture using CCMB-TAL broth in case direct culture was negative.
Recovered isolates were determined to be cytotoxin positive by a cell culture cytotoxin neutralization
assay. In addition, since the standard-of-care in our clinical laboratory is the BD MAX™ Cdiff test (BD
Diagnostics, Inc., USA), the GenePOC CDiff assay was also compared with the BD MAX results. Results:
Two hundred samples were tested at JHH. Eleven samples were eliminated due to an exceeded transit
time to the reference lab for culture. Therefore, a total of 189 stool specimens had valid test results.
Fourteen samples were positive for toxigenic C. difficile by direct culture. All direct culture positive
samples were positive by GenePOC CDiff showing a sensitivity of 100%. An additional 11 specimens
were direct culture negative, but positive with GenePOC CDiff giving a specificity of 93.8%. Four of these
were positive by enriched culture. Compared to the BD MAX, the sensitivity and specificity of the
GenePOC test were 95.5% and 100%. Conclusions: The GenePOC CDiff test compares favorably to direct
culture on CCFA and to another FDA-cleared NAAT that detects the toxin B gene in unformed fecal
samples.
Session Number: 222
Session Number: 222
Abstract Title:
Outcome of Electronic Order Set Implementation Relative to Toxigenic Clostridium Difficile Pcr Analysis
and Hospital-Onset Clostridium Difficile Infection in A Multi-Hospital Healthcare System
T. K. Block1, K. L. Munson2, D. Block2, G. Land2, N. Riederer2, S. Rodriguez3, R. Stone2, A. Villalobos2, E.
Munson4; 1Wisconsin Clinical Lab. Network Technical Advisory Group, West Bend, WI, 2Ascension
Wisconsin, Milwaukee, WI, 3Metropolitan Methodist H
Abstract Body:
Background: The importance of accurate and clinically-significant detection of Clostridium difficile
infection (CDI) cannot be understated from both a clinical and infection control perspective. While
debate continues over the optimal laboratory assay/algorithm, laboratorians are also responsible for
assuring that appropriate specimens are tested. We report the impact of an electronic pre-analytic
intervention on CDI detection and prevention in a multi-hospital system. Methods: In a retrospective
analysis, data were obtained for C. difficile testing volume and detection rate, frequency of hospital-
onset CDI, and C. difficile standardized infection ratio (SIR) for five hospitals within a multi-hospital
healthcare system. Data were surveyed in six-month intervals from January 2015 through June 2017.
During the second half of 2016, a system-wide electronic order set was implemented limiting C. difficile
testing to patients with clinical diagnosis of pseudomembranous colitis or patients with clinically-
significant, sustained diarrhea without other identified causes (including laxatives, recent use of oral
contrast, enteral feedings). Laboratory detection of C. difficile occurred via toxigenic PCR throughout the
entire study interval. Results: During the initial six-month interval, the system-wide PCR testing volume
was 1578, the detection rate was 20.9%, and 88 cases of hospital-onset CDI were documented. During
the first six-month interval of 2017, 18.9% and 56.8% reductions in PCR testing volume and occurrences
of hospital-onset CDI were realized, respectively, versus data from early 2015. Overall PCR detection
rate decreased to 16.8%. System-wide inpatient census data did not change over the study interval (P =
0.99). Effects were also noted at the level of individual hospital. Linear regression analysis revealed
decreases in C. difficile PCR testing volume and detection rate, as well as hospital-onset CDI frequency
and SIR, in larger facilities. Changes observed within small-to-medium-sized facilities were similar to
those of larger facilities, but to a lesser degree. Reductions in hospital-onset CDI frequency and SIR
associated with larger facilities were significant (P ≤ 0.025). Conclusions: Pre-analytic factors may impact
not only the clinical microbiology laboratory but also the hospital infection prevention service in the
context of CDI. Ultimately, the prevention of healthcare-associated CDI may not strictly be incumbent
upon detection assay/algorithm offered by the clinical microbiology laboratory.
Session Number: 222
Session Number: 222
Abstract Title:
Quantifying the Limit of Detection of Clostridium Difficile Spores from Stool Using Anaerobic Culture
A. R. Thomas, V. B. Young, K. Rao; Univ. of Michigan, Ann Arbor, MI
Abstract Body:
Background: Anaerobic culture is currently the gold standard for detection of toxigenic C. difficile.
However, there is little published on the analytic sensitivity of this method against known
concentrations of spores in stool. We hypothesized that differences between strains and protocol
methods affect the limit of detection for C. difficile from stool. Methods: Spore stocks were prepared for
one lab strain (630) and one clinical ribotype (R027) of toxigenic C. difficile. Spore concentrations in
these stocks were quantified by plating serial dilutions in triplicate on taurocholate-cycloserine-
cefoxitin-fructose agar (TCCFA) and visually counting CFUs. To create our stool standards, we used stool
specimens that 1) tested negative in the clinical microbiology laboratory for C. difficile by polymerase
chain reaction (PCR) for tcdB (the toxin B gene) and 2) did not grow on TCCFA or in broth (TCCFB) after
an ethanol shock. Various concentrations of spore stock and PBS were mixed with stool to create the
final standards. These were plated in triplicate on TCCFA, inoculated into TCCFB, and incubated
overnight. For direct plating, the limit of detection was assigned when growth was no longer seen on
TCCFA. For broth enrichment, after overnight incubation a second set of plates were streaked from
TCCFB in triplicate, and re-incubated overnight. Results: The limit of detection for strain 630 when
plating directly onto TCCFA was 0.1 CFU/µL. For broth enrichment, the limit of detection improved to
0.01 CFU/µL. Limits of detection for R027 were identical to those of strain 630 both by direct plating and
broth enrichment. Conclusion: The limit of detection of anaerobic culture did not differ between strain
630 and R027, either by direct plating or by broth enrichment. However, broth enrichment improved the
limit of detection by an order of magnitude. Future work should quantify the effects of additional
variables, such as ethanol shock, antibiotic presence in stool, other strains, and time from collection to
culture. This sensitivity analysis can also be extended to PCR for tcdB, another common method for
identifying C. difficile infection.
Session Number: 222
Session Number: 222
Abstract Title:
Performance Comparison of Two Pcr Tests for Detection of Clostridium Difficile in Under A Hour
P. A. Granato1, G. Hansen2, E. Herding2, S. Chaudhuri3, S. Tang3, C. R. Rowell1, S. K. Garg3, J. J. Sickler3;
1Lab. Alliance of Central New York, Syracuse, NY, 2Hennepin County Med. Ctr., Minneapolis, MN,
3Roche Molecular Diagnostics, Pleasanton, CA
Abstract Body:
Background: Clostridium difficile infection (CDI) is a common cause of antibiotic-related cases of
diarrhea and antibiotic-associated pseudomembranous colitis. The timely and accurate diagnosis of CDI
allows for the rapid initiation of antibiotic therapy and the timely institution of infection control policies
to minimize disease transmission. Polymerase chain reaction (PCR) assays have become a preferred
modality for diagnosing CDI in the US. The cobas Liat Cdiff PCR test is a novel assay that can be
performed on-demand for institutional testing with a rapid 20-minute turnaround time from specimen
collection to result reporting. Methods: The current study compared the performance of 2 FDA-cleared
PCR assays, the cobas Liat Cdiff test and the Xpert C. difficile/Epi test, for their ability to detect the tcdB
gene in freshly collected, remnant stool specimens from 310 patients with signs and symptoms of C.
difficile-associated diarrheal disease. Overall, 55 specimens were C. difficile positive (17.7%). Results:
The cobas Liat Cdiff and Xpert C. difficile/Epi tests showed an overall percent agreement of 97.4%
(302/310; 95% CI, 95.0-98.9). Sensitivity and specificity of the cobas Liat Cdiff test were 94.5% (52/55;
95% CI, 84.9-98.9) and 98.0% (250/255; 95% CI, 95.5-99.4) respectively. Low specimen concentrations of
toxigenic C. difficile, indicated by significantly delayed PCR cycle threshold (Ct) values, explained most of
the discordance. Conclusions: The simplicity of use, rapid result reporting, and high sensitivity of the
cobas Liat Cdiff test makes it an attractive option for use in small to medium-sized laboratories.
Session Number: 222
Session Number: 222
Abstract Title:
C. Difficile Toxin B Pcr Cycle Threshold As A Predictor of Free Toxin
S. Lee, N. Nanda, R. She; Keck Sch. of Med. of the Univ. of Southern California, Los Angeles, CA
Abstract Body:
Background: Rapid, accurate diagnosis of toxigenic Clostridiodes difficile infection (CDI) is not yet fully
optimized. Several studies have suggested the use of toxin B PCR amplification cycle threshold (Ct) to
predict free toxin status in the stool. In this study, we investigate the validity of this approach in a
tertiary care, immunosuppressed patient population. Methods: We included patients who tested
positive by C. difficile/Epi PCR (Cepheid GeneXpert) from Jan. 2016-Jan. 2018 (n=225) at a tertiary care
facility specializing in surgical and cancer care (Keck Medical Center, Los Angeles, CA). Only samples
meeting Bristol Stool Scale type 7 were accepted for clinical testing. All stool samples were further
tested by glutamate dehydrogenase (GDH) and toxin B EIA (Alere Cdiff Quick Check) and a subset (Dec.
2016-Jan.2018) by cell cytotoxin neutralization assay (CCNA) (n=112). Median tcdB Ct values were
compared using the Mann-Whitney U test. Receiver operating characteristics (ROC) curve analysis was
performed. Results: Median tcdB PCR Ct values were significantly higher for GDH-negative vs.GDH-
positive samples (33.2 vs.26.7, P<0.01), EIA-negative vs.toxin EIA-positive samples (29.7 vs. 24.3,
P<0.01), CCNA-negative vs. CCNA-positive samples (29.6 vs. 26.65, P<0.05), and NAP1-negative vs.
NAP1-positive samples (28.1 vs. 26.1, P<0.05). The ROC curve comparing tcdB Ct values to toxin EIA
yielded an area under the curve (AUC) of 0.766. A Ct cutoff of 26.5 cycles had a sensitivity of 73.2% and
specificity of 79.2% for toxin EIA status. The ROC curve comparing tcdB Ct values to CCNA yielded an
AUC of 0.658 (no significant difference vs. AUC for toxin EIA). A Ct cutoff of 26.5 cycles detected CCNA-
positive samples with a sensitivity of 79.1% and specificity of 50%. Conclusions: We found that Ct values
had limited utility in predicting toxin status in stool, with an AUC of only 0.766 and 0.658. Although
potentially convenient to use, Ct values are not validated for quantitative measurement of bacterial load
in this FDA-cleared kit and could be subject to variations in swab sampling of the stool, specimen
consistency, and DNA recovery. Our findings argue against reporting of Ct value for clinical use and its
use to accurately predict results from toxin EIA or CCNA in a tertiary care setting.
Session Number: 222
Session Number: 222
Abstract Title:
Ability of C. Difficile Epidemic Nap1 Strain to Cause Outbreaks is Closely Related to Loss of Exosporium
Protein Function
G. Broukhanski, A. Chan; Publ. Hlth.Ontario Lab., Toronto, ON, Canada
Abstract Body:
Background: Over the last 20 years there was a significant increase in a number of cases and outbreaks
caused by Clostridium difficile (CD). It was attributed to an emergence of the NAP1/027 strain with an
increased production of toxins, higher sporulation rate, presence of a binary toxin and drug resistance
genes etc. One of its features is a deficiency in BclA, bacillus collagen-like protein, forming a hair-like nap
on spores which in other spore-forming bacteria (e.g. B. antracis) plays a critical role in adhesion and
aerosolation. As spores are considered the main route of CD dissemination these features might affect
spread of CD infection. We investigated structure of the BclA gene in CD isolates to identify if it plays a
role in outbreaks. Methods: 12 reference strains with a defined pulsotype and 140 clinical isolates from
the PHOL collection, classified as sporadic or outbreak by MMLVA, were used for DNA extraction and
measurement of spores’ adhesion properties. For BclA gene PCR published sequences of primers were
used. To determine variability in this region amplicons were sequenced. For generation of spores, NAP1,
NAP4 and NAP7 strains were grown on the Brucella Supplemented Agar for 9 days, spores washed 3
times with water and their adhesion was measured by vortexing of suspensions in micro tubes and
measuring concentration of residual spores using terbium-DPA time-resolved fluorescence. Results: PCR
for BclA gene demonstrated that the length of this region varies around 1800 bp but is not amplified in
pulsotypes 7 to 10. The shortest sequence (420 bp) was detected in NAP1 strains. Sequencing of
amplicons shows variability in the central region in non-NAP1 strains while in NAP1 there was a 1000 bp
deletion in this region and a point mutation created a stop codon at 142 bp position. Among 140 clinical
isolates this deletion was seen in all NAP1 isolates, identified as outbreak strains, but not in any other
strain. Adhesion assay show that NAP1 spores are least adhesive, NAP4 are intermediate and NAP7 has
the most adhesive spores. Conclusions: Our study demonstrates that NAP1 strains of CD associated with
outbreaks have the same truncated BclA gene structure not seen in any other strains we tested. As
spores of the NAP1 strains are least adhesive it could be easily spread, causing an outbreak. Use of the
stricter infection control measures such as thorough cleaning in hospitals preventing accumulation of
spores on fomites might be responsible for a significant decrease in detection of NAP1/027 strains and
frequency of CD outbreaks noted in the last 3 years.
Session Number: 222
Session Number: 222
Abstract Title:
Evaluation of Solana C. Difficult Assay for the Rapid Detection of Clostridium difficult in A Pediatric
Population
A. Hopper1, K. Pierce2, A. Phillips1, M. Dickey1, R. GrandPre1, K. Korgenski1, A. Pavia2, J. A. Daly1;
1Primary Children's Hosp., Salt Lake City, UT, 2Univ. of Utah, Salt Lake City, UT
Abstract Body:
Introduction: Clostridium difficile (CDI) continues to be a significant infectious pathogen in pediatric
populations, with infection rates increasing among children in both community and hospital settings.
Accurate diagnosis and treatment of CDI are associated with reduced treatment costs and duration of
convalescence. CDI infection in children is complicated by carriage, nonstandardized symptom
algorithms and a multitude of diagnostic test methods. The inauguration of nucleic acid amplification
tests (NAAT) with their improved sensitivity has enhanced diagnosis of CDI compared to enzyme
immunoassay and toxigenic culture. The Solana C. difficile Assay (Solana CDI) (Quidel Corporation, San
Diego, California), FDA approved for clinical use in October of 2017, is a molecular test designed to
detect the presence or absence of CDI toxin DNA from liquid stool within one hour of sample receipt.
This study was undertaken to compare Solana CDI to traditional toxigenic culture in terms of sensitivity,
specificity, accuracy, positive predictive value and negative predictive value. Methods: Unformed stool
samples obtained for CDI testing at PCH per standard of care and at physician discretion were tested
over a 7-week period in early 2017. A total of 115 samples were tested using the Solana CDI protocol.
Sample aliquots were then transported to Quidel Corporation for additional testing with the toxigenic
culture, to which the Solana CDI results were compared, as well as Lyra Direct C. difficile Assay (Lyra), an
alternate NAAT. Results: 17 samples were culture positive for CDI (14.8%). 27 samples were Solana CDI
positive. Of 98 culture-negative samples, 84 were also negative by Solana. Of the 18 discrepant samples,
Lyra showed the presence of CDI toxin DNA in 3 culture-negative samples; no CDI toxin DNA was
detected in 9 Solana-positive cases. Post discrepant resolution, Solana CDI sensitivity and specificity
were 94.1% and 90.8%, respectively. Accuracy was 91.3%; positive predictive value 64%, negative
predictive value 98.9%. Conclusion: The Solana CDI performed well in comparison to toxigenic culture.
Turnaround time, accuracy and flexibility of the Solana instrument make this assay an attractive assay
for CDI testing.
Session Number: 222
Session Number: 222
Abstract Title:
Copan FecalswabTm Validation for the Transport and Storage of Stool Specimens for the Detection of C.
Difficile Toxin with Meridian Illumigene Assay
A. Giambra1, S. Castriciano1, L. Hendrickx2, A. Lemmens2; 1Copan Italia Spa, Brescia, Italy, 2AZ Sint
Maarten Hosp., Mechelen, Belgium
Abstract Body:
Background: Diagnosis of gastro enteric infections can be complex due to the large variety of pathogens
involved, and the different testing methods, like culture, rapid antigens and toxins assays and nucleic
acids amplification assays. Stool samples are transported in large and bulky containers, Copan produces
the FecalSwabTM (FS), consisting of a flocked swab and a tube with 2ml Cary-Blair medium, that can be
used for rectal swabs collection or to transport stool. FS has been validated for culture, using manual or
WASPTM automated plating methods, for rapid antigen and toxins assays and for nucleic acid
amplification assays for the detection of enteric pathogens. The objective of this study was to evaluate
the performance of stool samples stored in FecalSwabTM for the detection of C. difficile toxins with the
Meridian Illumigene molecular amplification assay. Methods: In this study 75 stool samples were used,
28 C. difficile toxins positive stools stored frozen and 47 consecutive fresh stools submitted in dry
containers for C. difficile toxins testing with Meridian Illumigene assay. Samples were tested in
duplicate, as per method recommended by the manufacturer, and after transferring the stools in FS. The
FS stool were tested using 200µL and 500µL sample on the same day that were transferred in the
FecalSwabTM and after 5 days storage at 4°C±2 for stability. Discordant results were resolved by
retesting the stool sample. Results: In the frozen C. difficile toxin positive stools, 26/28 were positive
using 200µL and 500µL of FS while 2 were negative with the Illumigene assay. Comparing to the
recommended method, FS had a recovery of 92.85% C. difficile toxins. To date 47 fresh samples were
analyzed, 24 positive and 23 negative C. difficile toxins were detected by all sampling methods. The
recommended method detected 23 negative, 23 (95.8%) positive and one invalid result. The 200µL FS
detected 22 (91.7%) positive and 25 negative, while the 500µL FS detected 24 (100%) positive and 23
negative at the initial testing. After 5 days storage at 4°C±2 the 200µL of FS detected 21 (87.5%) positive
and 26 negative while the 500µL FS detected 23 (95.8%) positive, 23 negative and one invalid result.
Conclusions: Data obtained in this validation demonstrated that 500µL Copan FecalSwabTM is the
optimal sample volume for the detection of Clostridium difficile toxins with the Meridian Illumigene
assay, with minimal invalid results. Stool in FecalSwabTM had a good stability after 5 days at 4°C±2, are
easy to use, compatible with WASPTM automation and need little storage space.
Session Number: 222
Session Number: 222
Abstract Title:
Adoption of Commercially Available C. Difficile Primers/Probes on the Fully Automated Cobas®
6800/8800 Omni Utility Channel
R. Hein, S. McCune, S. Cagas, J. Osiecki, J. Engstrom-Melnyk; Roche Diagnostic Corp., Indianapolis, IN
Abstract Body:
Background: The cobas® 6800/8800 Systems are fully automated, sample-to-result systems for routine
or high-volume molecular testing that utilizes real-time PCR detection. The cobas omni Utility Channel
(UC), the open channel functionality of the system software, allows for the development and routine
performance of user-defined real-time PCR tests using TaqMan® technology. The UC supports a
complete, automated Lab Developed Test (LDT) workflow (sample in to result out) including sample
pipetting out of primary/secondary tubes, sample extraction, PCR reaction setup, amplification,
detection, calculation and result reporting. With commercially available primers and probes, there exists
the opportunity to incorporate LDTs onto the automated platform and into clinical laboratory workflows
without the need to design unique sequences. Here, we demonstrate the adoption of Clostridium
difficile primer/probe cocktail on the cobas® 6800 System. Methods: A C. diff primer/probe set was
obtained directly from a commercial vendor and added to the cobas omni Utility Channel Master Mix
Reagent 2 (UC MMx-R2) at four different probe concentrations. Using C. diff control material at five
target levels, the impact of varying the probe concentration was evaluated based on PCR amplification
results and curve morphology. Detection of C. diff by the pre-designed primer/probe set was further
assessed using simulated stool samples spiked into cobas® PCR Media Uni Swab Sample Kit. Results:
Reducing input quantities of the TaqMan probe resulted in corresponding and marked reductions in
amplification efficiency. We also observed a linear relationship between input probe concentration and
amplification curve morphology, with the lowest probe dilutions resulting in a complete lack of target
detection. Using one of the tested primer/probe conditions, excellent C. difficile detection rates were
observed using simulated stool specimens. Conclusions: Adoption of commercially available LDT
primer/probe sets is readily accomplished on the cobas® 6800 System using the UC, which allows for the
full automation of LDT testing while utilizing the same platform, consumables, and reagents as FDA-
approved IVD assays.
Session Number: 222
Session Number: 222
Abstract Title:
Performance Evaluation of the Singulex Clarity C. Diff Toxins A/B Assay (Currently in Development) and
Comparison to Cell Cytotoxicity Neutralization Assay, A Rapid Gdh and Toxin Immunoassay, and Pcr
A. Almazan, S. Abusali, E. Lee, J. Estis, M. Pagan, J. Sandlund, A. Changavi, W. Trinh, K. Chau, P. Sarma, J.
Bishop, A. Bartolome, S. Tam; Singulex, Inc., Alameda, CA
Abstract Body:
Background: Diagnostic algorithms and more recently stand-alone nucleic acid amplification tests
(NAAT) have replaced culture-based techniques previously used to diagnose Clostridium difficile
infection. However, immunoassays, NAATs, and cell cytotoxicity neutralization assay (CCNA) have known
deficits due to poor sensitivity, specificity, and/or long turnaround times. The Singulex Clarity® C. diff
toxins A/B assay, currently in development for the Singulex Clarity® system, is an automated and rapid
ultrasensitive immunoassay (UIA) for the detection of toxins in stool. We evaluated the clinical
performance of this novel test and a two-step algorithm against CCNA. Methods: Each toxin was diluted
in buffer or stool to determine the limit of detection (LoD) of the UIA. A derivation cohort consisting of
103 frozen stool samples (27 CCNA positive and 76 CCNA negative) was used to establish the assay’s cut-
off by calculating AuROC while optimizing overall diagnostic accuracy relative to CCNA. Clinical
performance of the UIA was independently evaluated using a cohort of 95 fresh stool samples (33 CCNA
positive and 62 CCNA negative). In this independent cohort, a two-step diagnostic algorithm was also
performed using a rapid immunoassay for detection of GDH and toxins A/B (C. Diff Quik Chek
Complete®), and any samples with GDH/toxin discordant results were further evaluated by NAAT (BD
MAX™ Cdiff). Results: UIA LoD for toxins A and B was 0.8 and 0.3 pg/mL in buffer, and 2.0 and 0.6 pg/mL
in stool, respectively. In the derivation cohort, the UIA had 96.3% sensitivity and 97.4% specificity
compared to CCNA. In the independently evaluated cohort, the rapid immunoassay resulted in 41%
(39/95) discordance and 67% (26/39) of discordant samples were positive by NAAT, yet 50% (13/26)
were false positive compared to CCNA. The two-step algorithm, had 97% sensitivity and 79% specificity
compared to CCNA. Clinical performance of the UIA, as evaluated using an independent cohort of
samples, had 97% sensitivity and 100% specificity compared to CCNA. Conclusion: Preliminary results
from the Singulex Clarity C. diff toxins A/B assay demonstrated ultrasensitive and accurate detection of
C. difficile toxins. This novel automated assay shows promise for providing a stand-alone solution to aid
in the diagnosis of CDI.
Session Number: 222
Session Number: 222
Abstract Title:
Effect of Storage Temperature and Time on Viability of Clostridium Difficile Vegetative and Spore Cells
from Stored Specimens
S. Nho, S-J. Kim, O. Kweon, C. Cerniglia; Natl. Ctr. for Toxicological Res., Jefferson, AR
Abstract Body:
Background: Incidence and severity of Clostridium difficile infection (CDI) have increased dramatically in
North America over the last decade. Currently several detection methods are available for C. difficile,
and their detection sensitivity depends mainly on the quality of the fecal sample, including cell viability.
Little information on the effects of storage conditions on the survival of C. difficile has been
documented. In this study, we evaluate the effects of storage conditions on viability of C. difficile
vegetative cells and spores in stool specimens for four weeks at four different temperatures. Methods: A
spiked stool sample was prepared by mixing 500 µL of bacterial suspensions (2× 108 CFU/mL) with 500
µl of an autoclaved stool sample. Aliquots of the spiked stool sample were stored at -70°C, -20°C, 4°C
and room temperature (RT). Aliquots were assayed for viable cells and spores before storage and at
days 1, 3, 5, 7, 14, and 28 by both plate counting and qPCR. Results: In plate counting for the vegetative
cells, the colony numbers after storage at -70 °C and -20 °C show a dramatic decrease after 24 h (on day
1, they were 56.5% and 53.8%, respectively) and then were stable in colony number (day 28, 46.8% and
50%, respectively). For the stool samples at 4°C and RT, the plate counts gradually decreased: 100% (day
0) to 80.3% and 80.3% (day 1) to 51.7% and 45.1% (day 2) to 45.6% and 37.5% (day 28). After day 7,
refrigeration at 4 °C and freezing at −20 °C and −70 °C were clearly superior to RT and the RT stool
samples showed the lowest count numbers. In plate counting of spore cells, no significant changes were
observed in the plate counts for all different storage conditions and times. On the other hand, qPCR-
based cell counts for all fecal samples showed similar numbers of cells (107-108 CFU/mL). Conclusion:
Viability of C. difficile vegetative cells and spores in stool samples is dependent upon the storage time
and temperature: in short-term storage, storage at 4 °C and RT was superior to storage under the
freezing conditions but after day 7, storages at 4 °C, -20 °C, and -70 °C were better. Storage temperature
and time should be considered when conducting initial or retrospective C. difficile detection tests.
Session Number: 222
Session Number: 222
Abstract Title:
Analytic Sensitivity and Accuracy of the Check-Points Check Direct Cpe, Cepheid Xpert Carba-R and
Chromid Carba Smart Chromogenic Agar for the Detection of Carbapenemase-Producing
Enterobacteriaceae
S. Vasoo1, P. Y. Hon1, J. P. Hsu1, T. H. Koh2, P. P. De3; 1Inst. of Infectious Disease and Epidemiology,
Tan Tock Seng Hosp., Singapore, Singapore, 2Singapore Gen. Hosp., Singapore, Singapore, 3Tan Tock
Seng Hosp., Singapore, Singapore
Abstract Body:
Background: Few head-to-head comparative data are available for the performance of commercial
assays used for carbapenemase producing Enterobacteriaceae (CPE) surveillance. We undertook a study
comparing the performance of the ChromID®CARBA SMART chromogenic agar (ChromID), Check-Points
Check Direct CPE (CDCPE) and the Cepheid Xpert Carba-R (Carba-R) RT-PCR assays for the detection of
CPEs. Methods: Eight reference isolates (Escherichia coli and Klebsiella pneumoniae) harboring the
following carbapenemases: KPC, NDM, VIM-1, IMP-1, IMP-4, OXA-48, OXA-181, OXA-232 were studied.
Simulated rectal surveillance swabs were prepared using swabs in Stuart’s media (Copan 139C) dipped
into a stool matrix diluted in Stool Transport and Recovery [S.T.A.R.] Buffer Reagent [Roche]) and spiked
with serial dilutions of the reference isolates from about 4 log to 1 log CFU/ml. These were subjected to
CDCPE and Carba-R, and culture on ChromID. For CDCPE, extraction was performed on the Roche
MagnaPure Compact instrument pre-PCR. Molecular assays were performed in triplicate for limit of
detection (LOD) determination (at least 1 of 3 replicates positive, with corroborating amplification
curve). Accuracy studies were done by spiking a panel of three sets of 30 simulated rectal swabs (~20
positives and 10 negatives for each target) with the reference bacteria near the LOD.<br />Results:<br
/><table class="AbstractTable" id="{25E0C8E8-F376-4A44-BA4C-5B842CDA5FB2}"><caption
rowspan="1" colspan="1">Species</td><td rowspan="1" colspan="1">Reference no.</td><td
rowspan="1" colspan="1">Resistance<br />mechanism</td><td rowspan="1" colspan="6">Limit of
detection (Colony forming units, CFU/ml), and corresponding Ct values for molecular
tests</td></tr><tr><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td
rowspan="1" colspan="1"></td><td rowspan="1" colspan="1">ChromID®CARBA SMART<br />OXA48-
side<br />CFU/ml</td><td rowspan="1" colspan="1">ChromID®CARBA SMART<br />CARB-side<br
/>CFU/ml</td><td rowspan="1" colspan="1">Check Direct CPE<br />(CDCPE) CFU/ml</td><td
rowspan="1" colspan="1">CDCPE Ct values</td><td rowspan="1" colspan="1">Xpert Carba-R<br
/>(Carba-R)<br />CFU/ml</td><td rowspan="1" colspan="1">Carba-R Ct values</td></tr><tr><td
rowspan="1" colspan="1">K. pneumoniae</td><td rowspan="1" colspan="1">6033-440078</td><td
rowspan="1" colspan="1">KPC</td><td rowspan="1" colspan="1">N.D.</td><td rowspan="1"
colspan="1">8</td><td rowspan="1" colspan="1">80</td><td rowspan="1" colspan="1">35.9-
36.3</td><td rowspan="1" colspan="1">800</td><td rowspan="1" colspan="1">35.3-
37.9</td></tr><tr><td rowspan="1" colspan="1">K. pneumoniae</td><td rowspan="1"
colspan="1">2073-318014</td><td rowspan="1" colspan="1">NDM</td><td rowspan="1"
colspan="1">N.D.</td><td rowspan="1" colspan="1">96</td><td rowspan="1" colspan="1">96</td><td
rowspan="1" colspan="1">42.9-43.7</td><td rowspan="1" colspan="1">960</td><td rowspan="1"
colspan="1">35.9-36</td></tr><tr><td rowspan="1" colspan="1">K. pneumoniae</td><td rowspan="1"
colspan="1">Kleb 377/14</td><td rowspan="1" colspan="1">VIM-1</td><td rowspan="1"
colspan="1">N.D.</td><td rowspan="1" colspan="1">59.6</td><td rowspan="1"
colspan="1">5.96</td><td rowspan="1" colspan="1">35.7-36.8</td><td rowspan="1"
colspan="1">596</td><td rowspan="1" colspan="1">35.7-37.2</td></tr><tr><td rowspan="1"
colspan="1">K. pneumoniae</td><td rowspan="1" colspan="1">EK52/96</td><td rowspan="1"
colspan="1">IMP-1</td><td rowspan="1" colspan="1">N.D.</td><td rowspan="1"
colspan="1">59.3</td><td rowspan="1" colspan="1">N.A.</td><td rowspan="1"
colspan="1">N.A.</td><td rowspan="1" colspan="1">593</td><td rowspan="1" colspan="1">37.8-
38</td></tr><tr><td rowspan="1" colspan="1">K. pneumoniae</td><td rowspan="1" colspan="1">Kleb
59/10</td><td rowspan="1" colspan="1">IMP-4</td><td rowspan="1" colspan="1">N.D.</td><td
rowspan="1" colspan="1">4</td><td rowspan="1" colspan="1">N.A.</td><td rowspan="1"
colspan="1">N.A.</td><td rowspan="1" colspan="1">N.D.</td><td rowspan="1"
colspan="1">N.D.</td></tr><tr><td rowspan="1" colspan="1">E. coli</td><td rowspan="1"
colspan="1">ATCC BAA-2523</td><td rowspan="1" colspan="1">OXA-48</td><td rowspan="1"
colspan="1">5.73</td><td rowspan="1" colspan="1">N.D.</td><td rowspan="1"
colspan="1">57.3</td><td rowspan="1" colspan="1">38.2-40</td><td rowspan="1"
colspan="1">57.3</td><td rowspan="1" colspan="1">35.4-36.1</td></tr><tr><td rowspan="1"
colspan="1">E. coli</td><td rowspan="1" colspan="1">Kleb 44/11</td><td rowspan="1"
colspan="1">OXA-181</td><td rowspan="1" colspan="1">680</td><td rowspan="1"
colspan="1">68</td><td rowspan="1" colspan="1">6.8</td><td rowspan="1" colspan="1">37.2-
37.7</td><td rowspan="1" colspan="1">6800</td><td rowspan="1" colspan="1">36.4-
37.9</td></tr><tr><td rowspan="1" colspan="1">K. pneumoniae</td><td rowspan="1"
colspan="1">5063-645495</td><td rowspan="1" colspan="1">OXA-232</td><td rowspan="1"
colspan="1">N.D.</td><td rowspan="1" colspan="1">46.3</td><td rowspan="1"
colspan="1">4.63</td><td rowspan="1" colspan="1">36.4-37.1</td><td rowspan="1"
colspan="1">463</td><td rowspan="1" colspan="1">32.7-35.7</td></tr></table><br />N.D. = not
detected; N.A. = not applicable For most panel-included targets, CDCPE had a LOD of 1-2 log CFU/ml
lower than Carba-R and was comparable to ChromID. For the spiked panel near the LOD, CDCPE had
100% sensitivity and specificity for all targets. IMP-4 was not detected by the Carba-R assay. The Carba-R
showed excellent sensitivity for KPC (95%), NDM (100%) and OXA-232 (100%), but lower sensitivity for
VIM-1 (70%), IMP-1 (20%), OXA-48 (10%), and OXA-181 (81%). Conclusions: The CDCPE showed
comparable analytic sensitivity compared to ChromID for the detection of CPE in our study. The Carba-R
assay’s LODs were generally 1-2 log higher. This could be due to inherent assay properties and
instrument threshold settings, resulting in diminished sensitivity for analytes near the LOD. Prospective
clinical and comparative studies are needed to determine the significance of these findings.
Session Number: 222
Session Number: 222
Abstract Title:
Fecal Carriage of Extended-Spectrum-Beta-Lactamase (Esbl)-Producing Escherichia coli and Klebsiella
Species among Med. and Non-Medical Students
S. K. Mishra1, B. Sapkota2, G. R. Dhungana2; 1Maharajgunj Med. Campus, Kathmandu, Nepal,
2Janamaitri Fndn. Inst. of Hlth.Sci., Kathmandu, Nepal
Abstract Body:
The emergence and rapid spread of extended-spectrum-beta-lactamase (ESBL) in gram-negative
bacteria has led to increased mortality, morbidity and economic burden worldwide. ESBL-producing
bacteria can colonize healthy intestine of human beings and hence can be disseminated in both
community and hospital. The aim of this study was to investigate the prevalence of faecal carriage of
ESBL-producing Escherichia coli and Klebsiella spp. among medical and non medical students. This study
was conducted on 104 medical students and 104 non-medical students of Janamaitri Foundation/Little
Angels' College of Higher Studies, Nepal. One stool sample from each of those students was collected
and processed for bacterial culture and sensitivity according to standard methodology. Each
morphotype was identified and characterized. Antimicrobial susceptibility profile of E. coli and Klebsiella
spp. isolates was determined by Kirby Bauer Disc Diffusion Technique. ESBL-production in those isolates
was tested by first culturing the stool sample in MacConkey agar and then placing 30 mcg ceftazidime
disc on it. Enterobacteria resistant to ceftazidime were further subjected to combination disc method,
for phenotypic confirmation of ESBL production, as recommended by Clinical and Laboratory Standards
Institute guideline. Ethical approval for this study was taken from Nepal Health Research Council (Ref.
756/2017). E. coli (n= 203, 86.7%) and Klebsiella spp. (n=31, 13.3%) were recovered from 208 stool
samples. Among those 234 isolates, 69 were positive for ESBL which included E. coli (n=66, 95.7%) and
Klebsiella spp. (n=3, 4.3%). Fifty (42.4%) out of 118 isolates from medical students, and 19 (16.4%) out of
116 from non-medical students showed ESBL production. When compared with non-ESBL-producers,
the producers showed increased resistance to ciprofloxacin (14.5% vs. 1.8%), cotrimoxazole (59.4% vs.
16.4%) and amikacin (10.1% vs 4.2%). Though all 234 E. coli and Klebsiella spp. isolates were susceptible
to meropenem, one-fourth of the non-ESBL-producers were resistant to ceftazidime. Prevalence of fecal
carriage of ESBL-producing bacteria was higher in medical students; however, there was considerable
number of such bugs colonizing non-medical students as well. This kind of iceberg phenomenon of
asymptomatic carriage of ESBL-producing pathogens might be acting as a source of infection in both
community and hospital. Therefore, surveillance of carriage of drug resistant bacteria should be done
regularly in different settings.
Session Number: 222
Session Number: 222
Abstract Title:
Development and Validation of A Multiplex Real-Time Pcr Assay to Detect Clostridium Perfringens And
Bacillus Cereus Group in Stool
K. Cummings, D. Wroblewski, J. Cole, H. Schwab, J. Carey, E. Snavely, N. Dumas, K. Musser, L. Mingle;
NYSDOH- Wadsworth Ctr., Albany, NY
Abstract Body:
Background: Clostridium perfringens and Bacillus cereus are two prominent causative agents of
foodborne illnesses. Both organisms are Gram positive, spore forming, enterotoxin producing bacilli that
are found in the environment. C. perfringens and B. cereus cause similar foodborne illness symptoms
and exhibit equivalent incubation periods. At the Wadsworth Center, classical culture methods are used
to test for the bacteria simultaneously, which is labor intensive and time consuming. A multiplex real-
time PCR screening assay was developed to screen for the detection of DNA of these bacteria in stool
specimens during foodborne outbreak investigations. Methods: Primers and probes targeting C.
perfringens (plc gene), B. cereus Group (nheC gene), and an inhibition control (bicoid) were designed
and validated for the real-time PCR screening assay. Stool specimens from previous C. perfringens and B.
cereus outbreaks were used in the validation. Healthy stools donated by volunteers were used as
controls in the study. Results: The real-time PCR assay was challenged with a panel of 54 organisms
including 17 Clostridium species, 20 Bacillus species, and 17 other organisms that are commonly found
in stool. The specificity of the assay was 100% specific for C. perfringens and 83.3% specific (5 of 6
strains) for B. cereus. The B. cereus primer-probe set detected four of seven members of the B. cereus
Group. The sensitivity of the assay in stool was 9.38 CFU/rxn for C. perfringens and 6.25 CFU/rxn for B.
cereus. Intra- and inter- accuracy verification studies for both organisms exhibited a coefficient of
variation of less than 5%. The B. cereus strain that was not detected was the result of 4 mismatches in
the forward primer, 4 in the probe, and 1 in the reverse primer. Accuracy verification was performed on
organism-positive clinical specimens and stool specimens spiked with an organism. Conclusions: The C.
perfringens and B. cereus real-time PCR screening assay we developed is both sensitive and specific. A
new screening algorithm was implemented which includes initial testing with this real-time PCR assay
followed by culture of PCR-positive specimens. This testing strategy provides a preliminary identification
in one day. This will greatly reduce the turnaround time for outbreak investigation reporting and will
also reduce the number of specimens that must be cultured.
Session Number: 222
Session Number: 222
Abstract Title:
Real-Time Pcr for Detection of Helicobacter Pylori in Fecal Samples and Characterization of Macrolide
Resistance
S. A. Cunningham1, E. Beckman2, J. N. Mandrekar1, V. Slepnev2, R. Patel1; 1Mayo Clinic, Rochester,
MN, 2Meridian BioSci., Inc., Cincinnati, OH
Abstract Body:
Background: Antimicrobial resistance in H. pylori is an emerging challenge. Typically, H. pylori infection is
diagnosed using stool antigen or urea breath testing, which do not provide information on antimicrobial
susceptibility. Susceptibility testing is currently only available by culture and susceptibility testing of a
gastric biopsy specimen, which requires an invasive procedure. A rapid, non-invasive method for
detecting H. pylori in fecal samples with prediction of first-line drug resistance is needed; this is
theoretically achievable using molecular methods. Methods: We are developing a H. pylori-specific real-
time PCR assay (MC-PCR) targeting the 23S rRNA gene region, allowing for specific organism detection
alongside detection of single nucleotide polymorphisms (SNPs) associated with macrolide resistance.
Our assay includes a species-specific hydrolysis probe for H. pylori detection, alongside variant sensitive
melting curve analysis using a SimpleProbe® (TIB MOLBIO) for macrolide resistance-associated SNP
detection. Nucleic acids (NA) are extracted with a modified Maxwell RSC PureFood GMO and
Authentication kit (Promega) and assayed on a LightCycler 480 II (Roche). Here, we compared our assay
to the MB Premier Platinum HpSA® PLUS assay using 117 fecal samples. In addition, the samples were
tested using analyte-specific reagents (ASR) from Meridian Bioscience (MB) intended for detection of H.
pylori and macrolide resistance using PCR (MB-PCR) for the purpose of assessing resistance detection.
For MB-PCR, NA were extracted using the QIAamp Fast DNA Stool Mini Kit (Qiagen) and tested on a
Rotor-Gene (Corbett). Resistance prediction agreement was calculated for those specimens that tested
positive for H. pylori by both assays. Results: Compared to the MB Premier Platinum HpSA® PLUS results,
MC-PCR had a sensitivity and specificity of 93.9 and 95.5%, respectively in 111 samples, with 7 inhibited
samples. Resistance prediction was compared in the 52 samples positive by both PCR assays; macrolide-
resistant H. pylori was detected by both assays in 19 samples and MC-PCR alone in one sample; no
samples were positive by MB-PCR alone (resistance prediction agreement, 98.1%). Conclusions: H. pylori
can be detected and assessed for macrolide resistance directly from stool.
Session Number: 222
Session Number: 222
Abstract Title:
Association of Oral Helicobacter Pylori Strains with Gastric Complications
S. A. Ansari1, T. A. Khan2, S. U. Kazmi3; 1Dow Dental Coll., Dow Univ. of Hlth.Sci., Karachi, Pakistan,
2Univ. of Karachi, Karachi, Pakistan, 3Dadabhoy Inst. of Higher Ed., Karachi, Pakistan
Abstract Body:
Background: Helicobacter pylori are micro-aerophilic gram negative spiral bacteria consider as a
responsible pathogen for chronic gastritis, duodenal and peptic ulcer and other gastric complications. H.
pylori colonizes in the lining of the gastro intestinal mucosa and in the oral cavity that may also act as
the first site for initial colonization in patients with weak oral hygiene and gastric re-infection.However,
previous studies established a correlation between H. pylori infection, gastritis and periodontitis but a
definite association has not been recognized as yet. The purpose of this study was to investigate the
incidence of H. pylori gene in oral mucosa and find out the relationship between oral H. pylori infection
and gastric complications. Material and Method: This study included 574 subjects in the age group
between 20-80 years with gastro duodenal and oral complications who reported at Civil Hospital -
Karachi . Oral health status of each patient was recorded by Oral hygiene index (OHI), probing depths
(PD) and clinical attachment loss (CAL). Gastric biopsy and plaque samples were taken from every
patient and subjected for H. pylori detection using rapid urease test,culture and PCR with different
primers specifically β globulin, 16SrRNA, bab A, CagA, UreA, UreC and VacA gene. The PCR products
were analyzed by gel electrophoresis. All statistical analysis was done on IBM SPSS 22 at a significance
level <0.05. The association between Oral HP gene and Gastric complications among cases and controls
was calculated using Pearson’s χ2. The risk of gastric complications was calculated using binary logistic
regression analysis. Results: Analysis of results revealed that 53% plaque samples and 24% biopsy
samples were positive for H. pylori infection by Helicourease Assay . and 46% biopsy and 36% plaque
samples tested positive by culture on selective media. Lower detection of H. pylori by culture in biopsy
samples might be due to exposure of plaque samples to oxygen which may have converted the
organisms to living non cultureable coccoid form. By PCR, approximately 59.75% and 59% of biopsies
and plaque specimen, were found to be positive for H. pylori. Oral H. pylori strains increase the risk of
developing Gastro esophageal reflux grade II, gastro esophageal reflux with normal upper GIT and
duodenal ulcer. UreA, UReC, babA, CagA and VacA genes are the important genes may enhance the
severity of the gastric infection. Conclusion: This study provides an evidence that oral pathogenic H.
pylori strain might be associated to the odds of gastric infections.
Session Number: 222
Session Number: 222
Abstract Title:
Effect of Probiotics in Modulating Inflammation in Invitro Ibd Model
D. Ashwarya Kuttappan, M. Amalaradjou; Univ. of Connecticut, Storrs, CT
Abstract Body:
Inflammatory bowel disease (IBD) is a chronic disease condition of the gastrointestinal tract manifested
as severe diarrhea, fatigue and weight loss. Various factors including genetic susceptibility, mucosal
immune dysregulation, and gut dysbiosis play a significant role in the pathogenesis of IBD. Current
treatment regime is predominantly symptomatic and provides limited relief. However, due to inherent
anti-inflammatory and immune modulatory effect, the use of probiotics could be a potential strategy
against IBD in humans. This study investigated the anti-IBD effect of three commercial probiotic strains
using a cell culture model. Three probiotic cultures, Bifidobacterium animalis ssp. lactis (BB-12®),
Lactobacillus acidophilus (LA-5®) and Lactococcus lactis ssp. lactis NRRL 663 (LL) were used in this study.
These cultures were grown at 37 °C in deMan, Rogosa Sharpe broth for 24 h. Caco-2 cells were cultured
in DMEM with 20% fetal bovine serum at 37°C for 16 - 20 days. Following differentiation, the
monolayers were exposed to probiotics (~ 7 log CFU/mL) for 24 h. The cells were then treated with a
cytokine cocktail (IL-1β- 25 ng/ml, TNF-α- 50ng/ml, IFNγ – 50 ng/ml and LPS-10µg/ml) for 24 h, to
activate maximal inflammatory response. The supernatant from the monolayer was collected and
analyzed for IL-8, and extracted nuclear protein were used to quantitate pNF-κB using ELISA.
Additionally, intestinal Caco-2 monolayers grown on a transwell system were used to measure the effect
of probiotics and inflammation on transepithelial electrical resistance (TEER) and intestinal permeability.
The study was replicated three times with duplicate samples, and the data were analyzed using Proc
mixed procedure of SAS 9.4. Pre-exposure to probiotics prior to cytokine treatment significantly reduced
the activation and nuclear translocation of NF-κB, compared to cytokine control (P <0.05). Further, the
reduction in pNF-κB was found to associated with a significant reduction in pro-inflammatory cytokine
production namely, IL 8 (P<0.05). Moreover, probiotics also protected the Caco-2 monolayer from
inflammation induced increase in permeability. While the inflammatory stimuli resulted in a TEER
reduction of 47% compared to the negative control (no inflammation and no probiotic treatment),
treatment with probiotics was associated with 11-18% loss in TEER. The results of our study suggest that
these commercial probiotic strains could be employed in the control of IBD; however, follow up in vivo
studies are warranted to validate the results.
Session Number: 222
Session Number: 222
Abstract Title:
Performance of A Fully Automated Sys. for Processing Stool Specimens Collected in Two Fecal Swab
Transport Systems
J. Snyder*1, G. Thomson2, M. Bruce2; 1Univ. of Louisville, Louisvillle, KY, 2Univ. of Louisville Hosp.,
Louisville, KY
Abstract Body:
Background: Total Laboratory Automated Systems (TLA) have revolutionized the processing of multiple
specimen types for bacteriological culture. Their suitability for processing fecal specimens directly from
a transport container or fecal swab collection and transport system is unclear. The primary objective of
this study was to determine if a fully automated mode of inoculation is a reliable method in processing
liquid stool specimens. A secondary objective was to evaluate the potential of using an automated
inoculation process for fecal specimens received in two commercial fecal swab collection devices.
Materials and Methods: Frozen stool specimens (N=100), previously found to be negative by manual
culture for select gastrointestinal pathogens (Escherichia. coli O:157, Camplobacter jejunii, Shigella spp.,
and Salmonella spp) were “spiked” with a standardized inoculum (0.5 McFarland Standard) of E. coli
O:157 (ATCC 35150), Campylobacter jejunni (ATCC 33291), Salmonella enterica serovar typhimurim
(ATCC 14028), and Shigella flexneri (ATCC 12022). Swabs from each commercial collection and transport
system were “dipped” into the “spiked” stool specimens and returned to their respective system.
Samples were then processed and inoculated on the BD Kiestra TLATM . For each challenge organism
and fecal swab (25 were individually “spiked” with each challenge pathogen; 25 “unspiked” raw stool
specimen served as negative controls; an additional 25 uninoculated devices served as controls for
sterility and interference by the transport medium. A total of 400 fecal swabs (200 Copan and 200
Puritan consisting of 100 “spiked” and 100 “unspiked” with raw stool specimen per challenge organism)
were run through the Kiestra TLATM . Results: 14 initial processing failures occurred (9 Copan and 5
Puritan swabs) that resulted in an error alert by the Kiestra TLATM due to foam or clotting of stool. Of
these, 11 were successfully processed on the second attempt and 3 required semi-automated
processing. Overall, the percentage of recovery of the four challenge organisms was 100%, of which
96.5% (N = 386/400) were recovered during the first attempt with the remainder detected on the
second attempt or following semi-automated processing. Summary: 1) Inoculation of liquid stools using
the fully automated mode of the BD Kiestra TLATM is reliable for processing stools collected and
transported in the two commercial fecal transport systems evaluated in this study.
Session Number: 222
Session Number: 222
Abstract Title:
Comparison of Two Swab Transport Sys. to Maintain Viability of Clinically Relevant Enteric Pathogenic
Bacteria
H. Fernandez, R. Widen, S. Silbert; Tampa Gen. Hosp., Tampa, FL
Abstract Body:
Background: Studies have indicated that rectal swab specimens can provide accurate test results when
used with stool culture and molecular testing of enteric pathogens. The aim of this study was to
evaluate two fecal swab systems to transport and maintain clinically relevant enteric pathogenic
bacteria: FecalSwab™ Transport System (Copan, USA) and Opti-Swab® System (Puritan, USA). Methods:
The following ATCC enteric pathogenic bacteria strains were evaluated for survival after incubation at
room temperature (25°C) using the two transport swab systems described above: Escherichia coli ATCC
25922, Shigella flexneri ATCC 12022, Salmonella typhimurium ATCC 14028, Shigella sonnei ATCC 9290
and Yersinia enterocolitica ATCC 9610. A vortex elution protocol proposed by the CLSI standard M40-A2
was performed. This CLSI standard document provides a method of quality control testing, together with
acceptance criteria not only for viability but also for overgrowth of bacteria. Briefly, an initial 0.5
MacFarland suspension of each strain was prepared, followed by six 10 fold dilutions (1.5 x 107 to 102
CFU/mL). The last three suspensions with final concentrations of 1.5 x 104 to 102 CFU/mL of each strain
were used as working suspensions. Both, FecalSwab and Opti-Swab systems were inoculated in triplicate
with 100µL of each organism working suspensions. Swabs were held at 25°C for 0h, 24h and 48h. Each
inoculated swab was cultured and bacterial survival was evaluated after 24h-48h incubation at 35°C.
Results: Cultures from all swabs were averaged and results were compared with the M40-A2 CLSI
document acceptance criteria. Bacterial recovery from swabs held at 0h were within the limits accepted
by the M40-A2 CLSI document for both, Copan and Puritan fecal transport devices. Additionally, all five
strains tested were recovered from all swab systems after 24h and 48h of incubation. Copan FecalSwab
was able not only to maintain the organisms viable, but to keep the growth stable for up to 48h of
incubation. On the other hand, organisms’ overgrowth were observed after 24h and 48h of incubation in
all five species inoculated on the Puritan Opti-Swab System. These overgrowth cultures didn’t remain
within the 2 log10 of the initial microorganism concentration, as required by the CLSI M40-A2
document. Conclusion: Only Copan FecalSwab transport system was able to comply with all criteria for
each enteric pathogenic organism’s survival at all time points as described by the CLSI standard M40-A2.
Session Number: 223
Session Number: 223
Session Title: CPHM08 - Diagnostic Public Health Microbiology: Circulating, Emerging, and Reemerging
Pathogens
Abstract Title:
Use of Whole Genome Sequencing During A Multistate Outbreak of Multidrug Resistance
Campylobacter jejuni Linked to Pet Store Puppies
L. A. Joseph1, L. Francois Watkins2, J. Chen3, K. Tagg3, C. Bennett3, H. Caidi1, J. Folster1, M. Laughlin1,
L. Koski4, R. Silver4, L. Stevenson5, S. Robertson1, J. Pruckler1, M. Nichols1, H. Pouseele6, H. A.
Carleton1, C. Basler1, C. Friedman1, A. Geissle
Abstract Body:
Background: Campylobacter jejuni is a leading cause of bacterial foodborne and zoonotic illness in the
United States. Pulsed-field gel electrophoresis (PFGE) has historically been used for Campylobacter
surveillance and outbreak investigation; however, PulseNet is implementing whole genome sequencing
(WGS) for surveillance/outbreak investigation. In 2017, CDC and several state health departments
investigated a multistate outbreak of campylobacteriosis linked to pet store puppies. In this
investigation, we used whole genome multi-locus sequence typing (wgMLST) to link outbreak-associated
human and canine C. jejuni isolates. We also used the WGS data to predict the antimicrobial
susceptibility of these isolates. Methods: We sequenced human and canine C. jejuni isolates using the
Illumina MiSeq. Sequences were analyzed using the Campylobacter wgMLST v.5 allele database in
BioNumerics 7.6, developed in collaboration with domestic and international partners. WGS analysis,
using an in-house antimicrobial resistance workflow (based on ResFinder 3.0), was used to determine
predictive resistance. Antimicrobial susceptibility testing (AST) was performed by the National
Antimicrobial Resistance Monitoring Team using the broth microdilution method. We performed PFGE
on a subset of C. jejuni isolates using the PulseNet Campylobacter protocol. PFGE data were analyzed in
BioNumerics 6.6.10 and named using PulseNet nomenclature guidelines. Results: The wgMLST analysis
differentiated epidemiologically-linked C. jejuni isolates from PFGE-indistinguishable sporadic isolates
and separated the outbreak isolates into three clades (59-282 alleles different). Clade 1 contained four
clinical isolates from four states (4–30 alleles different). Clade 2 contained 11 clinical isolates from six
states (1–21 alleles different). Clade 3 contained 18 clinical isolates from 11 states and nine canine
isolates from two states (0–32 alleles different). Five PFGE patterns were produced by isolates in clades
two and three. All isolates were multidrug resistant using WGS data which was confirmed by AST.
Conclusions: Our study showed that wgMLST analysis provided greater resolution and epidemiological
concordance compared with PFGE during this outbreak investigation. Predicted resistance results were
comparable to AST and were available in real-time. This study demonstrated the power of WGS data for
linking outbreak-associated C. jejuni isolates and determining the antimicrobial resistance profiles of
these isolates in a single workflow.
Session Number: 223
Session Number: 223
Pathogens
Abstract Title:
The Worlds Largest Waterborne Campylobacteriosis Outbreak
B. Gilpin; Insitute of Environmental Sci. & Res., Christchurch, New Zealand
Abstract Body:
Background: It is estimated that over 5500 cases of campylobacteriosis occurred in Havelock North, New
Zealand in August 2016 as a consequence of the consumption of untreated drinking water contaminated
with sheep faeces. Methods: Multiplex Binary Typing (MBiT) and whole genome sequencing (WGS) was
undertaken on Campylobacter jejuni isolates from 284 notified cases of campylobacteriosis from the
outbreak region. Genotypes were compared with isolates from reticulated water, the supply bore and
sheep faeces. Results: 208 cases with at least 10 different genotypes of C. jejuni were linked to the
outbreak (Figure 1). MBiT analysis confirmed a link between cases and the water 1 day after receipt of
primary isolation plates, could be performed on non-viable isolates, and allowed screening of almost
500 isolates, of which WGS was performed on a subset. WGS was completed on the first set of isolates
within 4 days of isolation. Comparisons between the two methods suggested that MBiT genotypes
differing by 1 or 2 alleles should be considered as potentially indistinguishable. WGS using multilocus
sequence typing (MLST) or SNP analysis produced equivalent results. Six of the genotypes had less than
2 wgMLST differences from isolated recovered from the water and/or sheep faeces confirming the
source of contamination. Conclusions: Combined use of epidemiology and genotyping was better able to
define the exposure period, with genotyping helping exclude cases with other causes of
campylobacteriosis. There was no evidence of significant secondary transmission beyond initial outbreak
period. Genotyping of campylobacter isolates was essential for refining case definition, confirming
exposure period and connecting cases with reticulated water and with the contamination sources.
Investigating and genotyping geographically separated cases with limited exposure periods contributes
strongly to determination of exposure period and sources.<br /><p><a
href="http://files.abstractsonline.com/CTRL/6b/f/447/fbb/d22/4c4/5a3/678/e2f/a36/d9d/bc/g7010_1.j
src="http://files.abstractsonline.com/CTRL/6b/f/447/fbb/d22/4c4/5a3/678/e2f/a36/d9d/bc/g7010_1.jp
Session Number: 223
Session Number: 223
Pathogens
Abstract Title:
Zika Virus Genotype Causing Outbreaks in the Philippines, 2016-2017
A. Lee, K. Privaldos, M. Igoy; Res. Inst. for Tropical Med., Muntinlupa City, Philippines
Abstract Body:
Introduction: Zika virus (ZIKV) is an emerging mosquito-borne flavivirus known to circulate in Africa,
Asia, and Pacific. Transmitted by Aedes aegypti and Aedes albopictus, in 1966 the first case was reported
in Asia first detected by serology and later on, by reverse transcriptase (RT-PCR). In 2015, a massive
outbreak occurred in a province in Brazil causing the World Health Organization to declare this outbreak
as a Public Health Emergency of International Concern. In Philippines, Zika was last reported in 2012
despite the abundance of potential vectors and favorable environmental factors. This is the first report
of Zika in the Philippines following the outbreak in Brazil. Methods: Starting 2016, a Zika surveillance
was initiated, serum and urine samples from patients presenting with fever and or rash was sent to the
Research Institute for Tropical Medicine (RITM) to detect Zika RNA by Real-time PCR and IgM and IgG
testing. E gene was amplified using one-step RT-PCR and followed by direct Sanger sequencing.
Phylogenetic analysis was done using Maximum Likelihood and General Time-Reversible +G model with
1,000 replicates for boostrap (1,512nt) by MEGA 7.0. Results: A total of 3,365 samples received from
2016 until 2017, Zika RNA was detected in 60 (1.78%) samples while 14 samples were successfully
sequenced for the whole E gene. All of the Philippine isolates were clustered into the Asian genotype
with high similarity to the Micronesia strain reported in 2007 and other Southeast Asian countries from
2010 to 2016. Likewise the current Philippine strain has high homology with the 2012 isolate. Discussion
and Conclusion: Due to the mild symptoms seen in Zika patients, circulation of Zika virus in the
Philippines may remain under reported. With the 2017 Zika strain having high homology with the 2012
isolate, a case of a young boy without travel history, suggest that Zika virus may have been circulating in
the country but remains undetected due to its low levels and with the competitions from other
mosquito-borne viruses causing severe symptoms such as Dengue, Chikungunya, and Japanese
Encephalitis virus. Nevertheless, as what has been reported in Brazil, Zika remains to be a public health
threat due to its neonatal consequences such that proper attention should be given to its control
measures should be undertaken to prevent further transmission.
Session Number: 223
Session Number: 223
Pathogens
Abstract Title:
Emerging Cephalosporin Resistance in Salmonella Spp. is It Tip of Iceberg?
M. Shah; South City Hosp., Karachi, Pakistan
Abstract Body:
Introduction: Salmonella enterica infections are common causes of bloodstream infection in low-
resource areas[1] It remains an important public health problem in the developing countries with
inadequate sanitation and safe drinking water [2]. Method: Blood of patient with symptoms of fever has
been collected in Bectec bottle and stored in Bectec9240 .On Positive indication Sample has been
cultured on macConkey agar Sheep Blood agar , Chocolate agar And observed for Salmonella specie. Bio
chemicals reactions and Serology has been done for confirmation. Sensitivities has been done on Muller-
Hinton agar by Kirby baur’s disk diffusion method. 8744 blood samples From 01-01-2016 to 30-10-2017
collected , 775 blood cultures was positive in which 100 samples were found with Salmonella species.
S.typhi A was 40% S.typhi was 54% S.typhimurium was 2% , S. paratyphi 1% isolated. AMPICILLIN was 1%
Resistant AZITHROMYCIN was 1% Resistant CEFIXIME 5% Resistant CEFTRIAXONE 2% Resistant
CHLORAMPHENICOL 20% Resistant CIPROFLOXACIN 80 % Resistant CO TRIMOXAZOLE 16% Resistant.
Results: A human restricted pathogen transmitted from faeces to water and food or by contact and
fomites. Infection syndromes range from asymptomatic (including carriage) to severe disease with life-
threatening complications, including intestinal perforation, encephalopathy, and haemodynamic shock
[3] Conclusion: A human restricted pathogen transmitted from faeces to water and food or and fomites,
resistant to oral antibiotics embarks new era of injectables-only treatment option for centuries old
typhoid disease. References: 1 Crump, J. A., M. Sjolund-Karlsson, et al. "Epidemiology, Clinical
Presentation, Laboratory Diagnosis, Antimicrobial Resistance, and Antimicrobial Management of
Invasive Salmonella Infections." <u>Clin Microbiol Rev</u> 28(4): 901-37. 2 Khanal, P. R., D. Satyal, et al.
"Renaissance of Conventional First-Line Antibiotics in Salmonella enterica Clinical Isolates: Assessment
of MICs for Therapeutic Antimicrobials in Enteric Fever Cases from Nepal." <u>Biomed Res Int</u> 2017:
2868143. 3 Watson, C. H., S. Baker, et al. "A cross-sectional seroepidemiological survey of typhoid fever
in Fiji." <u>PLoS Negl Trop Dis</u> 11(7): e0005786.
Session Number: 223
Session Number: 223
Pathogens
Abstract Title:
Emergence of Multi Drug Resistant Salmonella Tyhpi As Epidemic among Lower Sindh Patients of
Pakistan
A. Naz; Liaquat Univ. Of Med. And Hlth.Sci., Jamshoro, Hyderabad, Pakistan
Abstract Body:
Background: Enteric fever is one of the most common bacteraemic illnesses in Pakistan, due to poor
sanitation, personal hygiene, lack of knowledge and misuse of frequently prescribed antibiotics without
proper cultures. This is causing emergence of multidrug resistance as well as has severely limited
therapeutic options in high disease burden countries such as Pakistan. This study was aimed to
determine the frequency of multidrug-resistant Salmonella Typhi (S.Typhi) among lower Sindh patients.
Methods: This cross-sectional study was conducted at diagnostic and research laboratory at Hyderabad
by evaluating cultures and sensitivity reports collected at different health care centers, during
November 2016 to November 2017. 92 patients were included in study most of patients were children
with mean age of 6.2 years , male population was predominant (63 %), detailed history and examination
was done and blood culture reports were followed for sensitivity and resistance . Results: This cross-
sectional study was conducted at diagnostic and research laboratory at Hyderabad by evaluating
cultures and sensitivity reports collected at different health care centers, during November 2016 to
November 2017. 92 patients were included in study most of patients were children with mean age of 6.2
years , male population was predominant (63 %), detailed history and examination was done and blood
culture reports were followed for sensitivity and resistance . Conclusions: MDR salmonella infection is
on the rise and we need to develop practice guidelines and expand treatment options to combat this
worldwide threat.
Session Number: 223
Session Number: 223
Pathogens
Abstract Title:
Chikungunya Fever: An Emerging Arboviral Disease in Karachi - Pakistan
G. Fatima1, S. Kainat2, S. U. Kazmi3; 1Civil Hosp., karachi, Pakistan, 2Civil Hosp., Karachi, Pakistan,
3Dadabhoy Inst. of Higher Ed., Karachi, Pakistan
Abstract Body:
Background: Chikungunya fever is an emerging arboviral disease transmitted by A. aegypti, which can
also transmit Zika and Dengue virus . Patients experience pain in their joints that persists for weeks or
months, or in some cases years . Symptomatically, it is challenging to distinguish chikungunya fever from
other arboviral fever including dengue, malaria and zika.. In November 2016, an outbreak of
chikungunya was reported in Karachi, Pakistan. This study was designed to determine the prevalence of
chikungunya virus infection among local population of Karachi as well as differential symptomatology to
aid in diagnosis and treatment. Methods: After Ethical Approval and written consent, blood samples
were collected from febrile patients (667) who reported to hospital during December 2016 to June 2017
with Chikungunia like symptoms,. Serum samples . T were first screened for Dengue virus antigen by ICT
and specific antibodies..Chikungunya specific IgM antibody was detected by Enzyme Linked Immuno
Sorbent Assay utilizing EUROIMMUN (UK) test pack. Statistical analysis was done by Statistical Package
for the Social Sciences (SPSS) v 22. Results: A total of 242 samples (36%) tested positive for Chikungunya
specific IgM antibodies, and all of these samples were negative for dengue antigen and antibodies. Most
of the Chikungunya positive patients were in age group of 61-80 years. The rate of infection was found
to be slightly higher in males (55%) than females (45%) and all positive cases had a history of high grade
fever, body aches and severe joint pain (arthralgia) and absence of body rash, nausea, vomiting .
Majority of our patients in the CHIKV-positive group had arthralgia in various joints, which was absent in
CHIKV negative group. These findings suggested that crippling joint pain due to increased inflammation
is an indicator symptom which was significantly associated with acute CHIKV infection but is not found in
Dengue and Zika virus infection . Conclusions: An alarmingly high prevalence of CHIKUNGUNYA infection
(36%) especially in patients with advanced age (61-80 years) is a matter of great public health concern ,
suggesting a role of weak immune status and inflammatory process - making patients susceptible to
such infection. Most prominent symptoms which helped in differential diagnosis to rule out false
positive cases and co-infection with dengue and zika virus were high grade fever, body aches and severe
joint pain (arthralgia) - an indicator symptom along with presence of Chikungunia specific antibodies.
Session Number: 223
Session Number: 223
Pathogens
Abstract Title:
Typhus Fever and Spotted Fevers Re-Emerging Infections in Remote Part of Central India
S. K. Rawat, A. Jain; Bundelkhand Med. Coll., Sagar, India, India
Abstract Body:
Background: Re-emergence of typhus and spotted fevers has been reported in south India and other
parts of the world recently in past few years; surprisingly no such studies have been done in other parts
of the country among patients of acute febrile illness. Our institute is surrounded by vast forest areas
and frequent outbreaks of unknown fatal encephalitis resembling rickettsial illnesses have been
reported from here in the recent past. An urgent need was felt to investigate clinical, epidemiological
and laboratory data to confirm presence and estimate prevalence of Typhus fevers and spotted fever in
our area. Our study attempts to use Weil Felix test which is a relatively simple and reliable serological
test for resource limited settings, along with clinical data to find their prevalence among patients of
acute febrile illnesses. Methods: Review based study was done for tests conducted on suspected
patients admitted from July 2013 to Dec 2017 at attached hospitals to Bundelkhand Medical College, in
Sagar. Patients admitted for acute febrile illness usually > 5 days, rash, unconsciousness, and signs of
meningo-encephalitis were included; these were tested on high suspicion of having rickettsial disease
and were mostly from adjoining forest area. Weil Felix test done on serum samples, titers of > 1:80 were
considered significant. Results: A total 106 patient records were available for the suspected patients
who were tested for Rickketsial fevers, 12 (11%) of these patients were admitted for coexisting signs of
Meningo-encephalitis, 35 showed a positive Weil felix test. Conclusion: It is substantiated that as
suspected, these infections are present in our area, sometimes with small outbreaks in adjoining forest
areas and prevalence high enough to pose a threat. The outbreaks showed peaks in 2013 and 2017
which might be related to the higher rainfall. Further population wide studies might be required in
adjoining forest areas to detect the actual prevalence in the community.<br /><p><a
href="http://files.abstractsonline.com/CTRL/c6/3/571/3fb/039/458/bb4/be5/cca/185/159/38/g4579_2.
src="http://files.abstractsonline.com/CTRL/c6/3/571/3fb/039/458/bb4/be5/cca/185/159/38/g4579_2.J
PG" alt="" border="0" /></a></p>
Session Number: 223
Session Number: 223
Pathogens
Abstract Title:
Molecular Epidemiological Investigation Revealed Serotype Switching Observed During Major Dengue
Outbreaks (2007-2015) in Nepal
S. Dumre1, R. Bhandari Dumre2, P. Chinnawirotpisan3, C. Klungthong3, G. Shakya4, B. Marasini5, S.
Shrestha6, P. Ghimire7, J. Karbwang8, K. Na-Bangchang9, S. Fernandez10, K. Hirayama8; 1Kantipur Coll.
of Med. Sci., Tribhuvan Univ., Kathmandu, Nepal, 2Sch.
Abstract Body:
Background: Global expansion of dengue virus (DENV) is a serious public health concern. Although DENV
was introduced in Nepal in 2006, its molecular epidemiology remains largely unclear. We uncovered it
through serotype/genotype analysis of circulating DENV strains. Methods: We employed the archived
serum samples (n = 972) collected during 2007-2015 from DENV infected individuals with linked clinical
and demographic data. Serotyping was performed by reverse transcription PCR. Complete envelope (E)
gene was amplified and sequenced for all four DENV serotypes from Nepal. Maximum-likelihood (M-L)
trees were constructed along with relevant global sequences from GenBank to understand the potential
origin of the Nepal viral strains. Results: During the study period, dengue affected at least 25 districts in
Nepal with an estimated case fatality rate of 1.5%. Majority were secondary infections (67%) and adults
(81%) with 13% severe manifestations. DENV was also detected in the hill districts suggesting their
expanding nature and possible adaptations towards higher altitudes (>1400 meters) in Nepal. All four
serotypes were confirmed; however, a clear serotype switching was observed during the major
outbreaks in the country. While DENV-3 was the major one in 2007-8, DENV-1 and -2 caused the large
outbreak in 2010-11 followed by a predominance of DENV-2 in 2013-14, and a re-emergence of DENV-1
in 2015. Nepal DENV 1 strains belonged to genotype-V and formed two distinct clades, which showed
spacio-temporal variation during these outbreaks. DENV 2, 3, and 4 strains were clustered into
cosmopolitan genotype, genotype-III, and genotypes-I/ IIB, respectively. M-L trees revealed that the vast
majority of Nepal DENV strains were closely related to India and contemporaneous Singapore strains.
India is the most plausible origin of Nepal DENV due to its physical proximity, extensive cross-border
activities (open border) and similar geo-climatic features supporting the phylogenetic relations.
Conclusions: DENV serotype switching occurred in Nepal during the major outbreaks. We have also
outlined genetic diversity of DENV serotypes in Nepal. This information is not only useful for Nepal in
epidemic preparedness but also aids in the understanding of evolutionary dynamics of DENV at regional
and global level. These findings also underscore the need of cross-border collaboration in dengue
control.
Session Number: 223
Session Number: 223
Pathogens
Abstract Title:
Burden and Trend of Measles in Nigeria: A Five-Year Review of Case-Base Surveillance Data, 2012-2016
B. S. Ibrahim1, R. Usman1, Y. Mohammed1, A. A. Abubakar2, P. M. Nguku1; 1Nigeria Field Epidemiology
and Lab. Training Program, Abuja, Nigeria, 2Ahmadu Belle Univ., Zaria, Zaria, Nigeria
Abstract Body:
Background: Measles is a vaccine preventable, highly transmissible viral infection that affects mostly
under-five year children. The disease is caused by a Morbillivirus; member of the Paramyxovirus family.
Measles surveillance in Nigeria is through the Integrated Disease Surveillance and Response (IDSR). We
reviewed surveillance data on measles from Nigeria over a five-year period to highlights its burden, and
make recommendations for improvements. Methods: We conducted a secondary data analysis of
measles specific IDSR records of all States in Nigeria from January 2012 to September 2016. The record
had reported measles cases with laboratory outcomes from all the States. IDSR weekly epidemiological
data were obtained from Surveillance Unit, Nigerian Centre for Disease Control (NCDC). Results: A total
of 131,732 cases were recorded within the period. Highest number of cases 57,892(43.95%) were
recorded in 2013 while the least number of cases 11,061(8.4%) were recorded in 2012. A total of 817
deaths were recorded, given a case fatality rate (CFR) of 0.62%. The CFR showed a decreasing trend over
the years with the highest CFR (1.43%) recorded in 2012 and the least CFR (0.44%) recorded in 2016.
Only 8,916 (6.7%) cases were confirmed by laboratory investigation. The Northwest region recorded the
highest attack rate (AR) of 149.7 cases per 100,000 population, followed by the Northeast region with
140.2 cases per 100,000 population, while the South-south region recorded the least AR of 15.8 cases
per 100,000 population. Case Fatality Rate per region followed similar pattern, with the Northcentral
region having the highest CFR of 4.38%. The trend of measles cases followed the same pattern. Cases
peaked at March, then gradually reduced to lowest level at June. Conclusion: Measles infection remains
a burden especially in the northern region of Nigeria. Though measles fatalities were on decline over the
years, laboratory diagnosis of cases has been suboptimal. We recommended improvement on routine
immunization and measles case management, and strengthening of regional laboratories capacity for
measles diagnosis.
Session Number: 223
Session Number: 223
Pathogens
Abstract Title:
Hepatitis C and Delta Viruses among Chronic Hepatitis B Surface Antigen Blood Donors in Abuja Nigeria
I. M. Ifeorah1, S. A. Bakarey2, N. F. Onyemelukwe1; 1Univ. of Nigeria Enugu Campus, Enugu, Nigeria,
2Univ. of Ibadan, Ibadan, Nigeria
Abstract Body:
Background: Hepatitis B virus (HBV) is a transfusion transmissible viral pathogen known to cause chronic
liver diseases associated with cirrhosis and hepatocellular carcinoma. The disease becomes more
aggressive and severe when co infected with Hepatitis D and C. Prevalence of Co-infection or Tri
infection of HBV with Hepatitis D and C in asymptomatic chronic carriers have been under reported
especially in developing world. Therefore, this study aimed to evaluate the burden of HCV and HDV
infections among intending blood donors who tested positive for HBsAg in selected hospitals in Federal
Capital Territory, Abuja, Nigeria. Methods: This cross sectional study was carried out among 193 (M=99;
F=94) consenting HBV infected population who were intending blood donors in four health facilities in
Abuja Nigeria (age ranged 18-57 years with mean age 31.6 (SD=12.4) years. The demographic and other
relevant data were captured by a structured questionnaire. Anti-HCV, HBsAg and HDV Ag/Ab were
detected by commercial quantitative enzyme linked immunosorbent assay following the manufacturer’s
instructions. Results: Overall rates of 5.2%, 5.7% and 0.5% were detected for anti-HCV, anti-HDV and
HDV-Ag among HBV infected population respectively. The rates were similar in male (7.1%) for HCV and
HDV but higher in male (7.1%) than in female (3.4% and 4.3%) counterparts (p>0.05) for both infections
respectively. The infection rate (7.7%) for HCV peaked at age group <u><</u>20 years while that for
anti-HDV Ab (10.3%) and Ag (3.8%) at age groups 41-50 years and <u><</u>20 years respectively.
However, HCV/HDV/HBV tri-infection rate (3.6%) was only found in male among age group 21-30 years.
Conclusions: This study has shown a high rate for HCV or HDV infection especially among chronic HBsAg
infected intending blood donors. We therefore, recommend screening for HCV and HDV among
intending blood donors in Nigeria.<u></u><strike></strike>
Session Number: 223
Session Number: 223
Pathogens
Abstract Title:
Prevalence of Leptospirosis among Acute Febrile Illness Cases Attending Publ. Hlth. Facilities in Chennai
B. Ganesh1, A. Sugunan2; 1ICMR-Natl. Inst. of Epidemiology, Chennai, India, 2ICMR-Regional Med. Res.
Ctr., Port Blair, India
Abstract Body:
Background: A large proportion of leptospirosis cases presents as uncomplicated febrile illness and is
difficult to distinguish from other endemic infectious diseases [Chrispal et al. 2010]. A study was
undertaken to estimate the proportion of leptospirosis among new fever cases attending health facilities
in Chennai during the period August 2016 - January 2017. Methods: Each day, 5 consecutive new fever
cases aged >2 years attending two health facilities in Chennai were recruited. The attending physician
recorded clinical information in a structured questionnaire. From each case, 5 ml of venous blood was
drawn on the day of reporting, and tested for the diagnosis of leptospirosis (PanBio Lepto IgM ELISA)
and other infectious diseases such as Dengue, Orientia tsutsugamushi and Salmonella enterica
(serology) and Malaria (microscopy). Results: A total of 918 fever cases were recruited. In 73% of the
cases, the duration of symptoms at the time of reporting was <5 days and in another 23.4% it was
between 5-10 days. Laboratory diagnostic testing showed that Leptospirosis (9.2%) as a leading cause
followed by Dengue (8.7%); O. tsutsugamushi (4.3%); S. enterica serovars Typhi & Paratyphi (2.7%) and
malaria (0.3%). A univariate analysis of partial data showed that body ache, headache, tiredness,
epistaxis, anorexia, bleeding gums, palpitation and joint pain were positively associated with
leptospirosis. Conclusions: The five infections studied accounted for about 25% of the new fever cases
and among these, leptospirosis was the leading cause. Further analysis of the data may help to improve
the predictive value of an etiological diagnosis for more targeted treatment and control. References:
Chrispal A, Boorugu H, Gopinath KG, Chandy S, Prakash JA, Thomas EM, et al. Acute undifferentiated
febrile illness in adult hospitalized patients: The disease spectrum and diagnostic predictors - An
experience from a tertiary care hospital in South India. Trop Doct. 40:230-234, 2010.
Session Number: 223
Session Number: 223
Pathogens
Abstract Title:
Prevalence and Antibiogram Profiles of Escherichia coli O157:H7 Isolates Recovered from Three Selected
Dairy Farms in the Eastern Cape Province, South Africa
L. Msolo; Univ. of Fort Hare, Alice, South Africa
Abstract Body:
E. coli O157:H7 is one of the most imperious foodborne pathogens predisposed for a number of
mortalities worldwide. To investigate the occurrence and antibiotics susceptibility of Escherichia coli (E.
coli) from three selected commercial dairy farms in the Amathole District Municipality, Eastern Cape
Province, South Africa, raw milk samples were collected from bulk storage tanks and swab samples were
month sampling regime between June and November 2014. A standard culture-based method was used
for the enumeration and isolation of E. coli O157:H7 using sorbitol MacConkey agar (supplemented with
cefixime (50 μg/L) and potassium tellurite (25 mg/L). A serological confirmation of the presumptive E.
coli O157:H7 isolates was conducted using the O157 latex agglutination test kit. A total of 252 E. coli
O157:H7 isolates were further subjected to PCR detection of rfbEO 157 and fliCH7 genes of which
27(11%) of the isolates were confirmed positive E. coli O157:H7. Our finding reveal that of the 27 E. coli
O157:H7 isolates from the dairy farms rate of resistance against penicillin was85% and resistance
against the other antibiotics follow the order: tetracycline (81%), erythromycin (70%), streptomycin
(52%) and chloramphenicol (45%). We conclude that the dairy farms are potential reservoirs of E. coli
O157:H7 serotype with multiple antibiotic resistance and consequently a concern to public and
environmental health.
Session Number: 223
Session Number: 223
Pathogens
Abstract Title:
Evaluation of Amsel Criteria against Lab. Diagnosis in Detection of Bacteria Vaginosis among Women in
Kakamega County Gen. Hosp. (Kcgh) Kenya
R. R. Okuku1, E. E. Makokha, supervisor2, J. J. Gikunju, supervisor1, C. C. Bii, supervisor2;
1Jomokenyatta Univ. of agriculture and technology, Nairobi, Kenya, 2Kenya Med. Inst. of
research(KEMRI), Nairobi, Kenya
Abstract Body:
Introduction: Bacteria Vaginosis (BV) is a condition characterized by changes in vaginal flora in which
normally predominant Lactobacillus species are replaced by potential pathogens, studies have
associated BV with a significant number of obstetric and gynecological complications and increases
women’s risk of risk of STD/HIV acquisition. BV is usually diagnosed using Amsel’s criteria or Nugent
scoring of Gram-stained vaginal smears both of which require microscopy. In countries like Kenya where
access to laboratory services is often limited, BV is typically managed using a syndromic approach for
vaginal discharge, a method with low sensitivity and specificity and hence the need of evaluating Point-
of-care testing method and determing the prevalence and risk factors associated with BV in this Study
population. Materials and Methods: A cross sectional study was conducted between Jan and Dec 2014
where 200 women were recruited in the study. A structured questionnaire was used to obtain
information on possible risk factors. Associations between categorical variables were assessed using
poisons regression. In evaluation of BV diagnostic methods smears were gram stained and read using
Nugent scoring system, in Amsel criteria the consistency and smell vaginal discharge including the
presence of clue cells was noted, The Quickvue advance PH and amines test was performed as per the
manufactures instructions. Results: The prevalence of BV by the Nugent criteria was 39%.The
concordance, sensitivity, specificity, positive and negative predictive values of these tests were: Amsel
criteria: 80.5%, 69.2%, 87.7%, 79.8% and 78.3% and Quickvue Advance pH and Amines test: 74.5%,
56.4%, 86.1%, 75.5% and 72.1%. Independent risk factors associated with BV were Women of ages 26 to
30 yrs (OR 1.9, 95% CI 1.01 to 4.03), business women (OR 1.7, 95% CI 1.05 to 2.8) and Douching (OR 1.4,
95% CI 1.2 to 1.98), condoms use was found to be protective against BV infection (OR 0.6, 95% CI 0.39 to
0.9) with PV <0.005. Conclusion: The prevalence of BV was high, risk factors associated with BV were
douching, age, women in business, condom use was found to be protective against BV The Amsel test
performed alone provided results that closely approximate those obtained by the Nugent criteria.
Recommendations: Provision of Health education on risk factors associated with the high prevalence of
BV in this study population. Amsel criteria for BV diagnosis should be used as the results closely
resembles those obtained by the Nugent criteria.
Session Number: 223
Session Number: 223
Pathogens
Abstract Title:
Molecular Epidemiology of Sexually Transmitted Infections among Females Attending Women Seeking
Gynecological Check-Ups, in Beirut, Lebanon
M. El Chaar1, R. Yassine1, J. Hanna1, C. Afif1, R. Skaf1, M. Hubeish2, S. Itani2, T. Jisr2, F. Hamzeh1, M.
Azar1, M. Curran3, A. Subaie4; 1Univ. of Balamand, Beirut, Lebanon, 2Makassed Gen. Hosp., Beirut,
Lebanon, 3Univ. of Cambridge, Cambridge, United K
Abstract Body:
Background: Sexually transmitted infections (STI) cause serious health, economic and social
consequences. They are responsible for serious morbidity in many developing countries where STI are
not always diagnosed or screened. Objectives: The aim of the study is to estimate the prevalence of STI
by detection of various microbes in women using three different nucleic acid detections assays.
Methods: A total of 505 vaginal and cervical specimens were collected from women above 18 years old.
Women were either asymptomatic or had signs and symptoms related to gynecological infection.
Nucleic acid was extracted from 200µl of samples using QIAamp DNA Mini Kit. Samples were tested for
the following microorganisms: Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium,
Mycoplasma hominis, Mycoplasma girerdii, Ureaplasma urealyticum, Urealplasma parvum, Trichomonas
vaginalis, Gardnerella vaginalis and Candida albican. Three assays were used for detection including in-
house multiplex assays, Anyplex™ II STI-7 and Euroassay STI. All HPV positive samples were amplified by
PCR and tested on the EUROarray according to the manufacturer’s instructions for genotyping. Results:
Two eighty-nine samples (57%) were positive for any pathogen: 184 (36%) for Gardnerella vaginalis, 176
(35%) for Ureaplasma parvum, 23 (4.5%) for Ureaplasma urealyticum, 18 (3.5%) for Mycoplasma
hominis, 1 for Mycoplasma genitalium (0.2%), 8 for Chlamydia trachomatis (1.6%), 9 for Neisseria
gonorrhea (1.8%) and 2 samples for mycoplasma gerardii (0.4%). Forty-one samples were positive for
Candida (8%), 18 (3.5%) for trichomonas vaginalis and 34 for HPV (6.7%) Among infected patients, 46%
(n= 134) were positive for a single microorganism. Dual microbial infections were observed in 40% (n=
116) of patients and multiple infection were observed in 8% of women. Different combinations of
microbial coinfections were observed: Ureaplasma parvum was often seen with gardenerella vaginalis
(n=133), with candida albican (n=30) or mycoplasma hominis<u> </u>(n=14). HPV was detected in 34
women, 84% were infected with cancerous type of HPV (High risk). Among the women infected with
HPV, 88% had more than one genotypes. Few discrepancies in positivity were observed among the three
commercial assays. Conclusions: This is the first report that provides data on the molecular
epidemiology of STI in the MENA word. This study demonstrated that the multiplex PCR system is highly
effective in the detection of multiple STI pathogens in women.
Session Number: 223
Session Number: 223
Pathogens
Abstract Title:
Sexually Transmitted Diseases: Time to Revisit the Syndromic Management Approach
H. Bah Camara1, E. Wright2, P. Kimmitt1; 1Univ. of Westminster, London, United Kingdom, 2Univ. of
Sussex, Brighton, United Kingdom
Abstract Body:
Sexually transmitted disease is a public health challenge especially in resource limited countries.
Approximately, 5 million new cases of curable STIs are reported each day, worldwide. The STI syndromic
management is widely used in resource limited countries for rapid and same day treatment. The Gambia
has adopted the STI syndromic management approach since 1991 and uses a combination treatment of
Ciprofloxacin, Doxycycline and Metronidazole for vaginal itching and discharges in females. However,
emerging antimicrobial resistance and new identified STI pathogens may pose challenges to the
syndromic management approach in the Gambian setting. To characterise Ureaplasma and other STI
pathogens in women attending a STI clinic who were being treated using the syndromic management
approach. Endocervical (ECS) and high vaginal (HVS) samples were collected from each participants prior
treatment (N = 115, Age 20 – 49). Microbiological analysis (Candida, Streptococcus agalactiae, Neisseria
gonorrhoea, T.vaginalis and bacterial vaginosis) and real time PCR (Chlamydia trachomatis, Neisseria
gonorrhoeae, Ureaplasma urealyticum/parvum and Mycoplasma genitalium) was carried out. No
organism was isolated from 23.5% (27/115) of the participants that reported urogenital symptoms.
However, 76.5% (88/115) were infected with either one or more pathogens. The majority 45% (52/115)
of participants were infected with urogenital Ureaplasma species either in the cervix, vagina or both.
Ureaplasma urealyticum accounts for 19.2 % (10/52), Ureaplasma parvum 80.7% (42/52) and 32.7%
(17/52) were co-infected with bacterial vaginosis. Other isolated organisms were: Bacterial vaginosis
29.6% (34/115), Candida albicans 14.8% (17/115), T.vaginalis 13.0% (15/115), Candida species 9.6%
(11/115), Streptococcus agalactiae and Neisseria gonorrhoeae each at 6.1% (7/115) were also isolated.
Antimicrobial susceptibility profile for Streptococcus agalactiae and Neisseria gonorrhoeae showed
resistance to one or more antibiotics used in the first and second line syndromic management treatment
Confirmation of STI by syndromic management alone is not enough as emerging drug resistance and
new STI organisms poses challenges to the health of the reproductive aged woman. There is a need to
review the STI syndromic management guidelines and to strengthen laboratory diagnosis to reduce the
burden of overuse antibiotic treatment in the Gambia. Treatment of sexual partner(s) should also be
encouraged to limit treatment failure and recurrent infections
Session Number: 223
Session Number: 223
Pathogens
Abstract Title:
Outstanding Abstract Award: Human Papillomavirus Genotypes Distribution in Cervical Cancer from
Nepalese Women
B. Gupta1, S. K. Sah2; 1Tribhuvan Univ., Kathmandu, Nepal, 2B. P. Koirala Mem. Cancer Hosp., Chitwan,
Nepal
Abstract Body:
Background: Cervical cancer is the leading cause of cancer morbidity and mortality among women in
Nepal. Data on HPV genotypes in cervical cancer (CC) in Nepalese women are sparse. We aimed to
determine the type- specific HPV distribution in CC from Nepalese women. Methods: This study included
248 archived paraffin-embedded specimens from CC cases (aged 30-99 years) from women attended
the BP Koirala Memorial Cancer Hospital, Nepal. DNA was extracted from the sections using the QIAamp
DNA Blood MiniKit (QIAGEN). HPV detection was performed using a PCR with generic biotinylated
primers BSGP 5+/6+, which amplify a 140 bp fragment of the L1 gene; the genotyping was carried out by
a reverse hybridization line which identifies 36 HPV types. Results: From 248 collected CC tissue blocks,
165 were determined to be adequate for PCR testing; most of samples corresponded to ssquamous cell
carcinoma (77.6%). All the analyzed specimens were positive for HPV. The identified HPV types belonged
to four of 15 species (α6, α7, α9, and α10) within the α-papillomavirus genus, most of them as single
infections (94.5%). The most common types, in decreasing order of frequency were 16, 18, 45, 33, 52, 56
and 31. Together, HPV types 16, 18, and 45 accounted for 92% of tumors. Women with CC related to
HPV types 16 and 18 presented at a younger mean age than did those with other HPV types Conclusions:
The present study strengthens the knowledge of HPV type distribution in cervical cancer cases from
Nepal. Since Nepal has not yet implemented any population prevention strategy, it is an opportune time
to begin evaluating this possibility by taking advantage of all available information and experience.
Considering the high level of detection of HPV 16 and 18 (83%) in the cervical cancers from Nepalese
women, it is expected that the introduction of vaccination with the first generation of vaccines
containing these two viruses in their formula, with sufficiently high coverage, sharply decreases the
incidence of this neoplasm in Nepal. This study may assist policy-makers in finding a more systematic
and tailored approaches to HPV prevention in Nepal
Session Number: 223
Session Number: 223
Pathogens
Abstract Title:
Assessment of External Quality Assurance Scheme Participation Level on Serogrouping and Antimicrobial
Susceptibility Testing of Salmonella And Shigella Species
F. A. Derra, B. Habtemariam, T. Biza, T. Legesse, R. Muzeyin, S. Girma, Y. Beyene, A. Gonfa; EPHI, AA,
Ethiopia
Abstract Body:
Background: External Quality assurance scheme (EQAS) is the system which allows every laboratory to
compare its overall performance with other internal and external existing laboratories, working in
similar disciplines. Significant improvements were reported in different laboratories and countries after
attending in one or more of such programs. Objective: To assess EQAS participation level on Salmonella
and Shigella species, that had been processed for six years under WHO-AFRO GSS EQAS program
Methods: Samples received for Salmonella and Shigella species sero-grouping and other unknown
enteric pathogens identification were directly inoculated to the suitable and selective media according
to the type of organisms. Sero-groups were reported using terms according to Kauffmann-White-Le
Minor procedures. For antimicrobial susceptibility testing drug diffusion method and CLSI interpretation
guideline were used. Results: From the overall participation (2008-2013), sero-grouping results were
correctly reported as 62/ 70 (88.6%). None of the deviation was recorded for Shigella species.
Participation for Campylobacter species were done only twice per six years, at 2009 and 2010; which the
results of agreement from expected values were ½ (50%) and 2/2 (100%) respectively. In line with this,
the antimicrobial susceptibility participation was correctly reported as 320/356 (89.9%). Conclusion:
Even though, everyone has got a knowledge and awareness about the benefits of EQAS by default, its
acceptance and implementation in developing countries is less communicated and exercised. The final
recommendation will be, all higher officials and policy makers in the field have to give attention and
allocate adequate budget in a continuous basis.<br /><p><a
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Session Number: 223
Session Number: 223
Pathogens
Abstract Title:
Molecular Epidemiology of Pneumococcal Infections in Aged Patients At the Far East of Russia
A. V. Martynova; Far Eastern Federal Univ., Vladivostok, Russian Federation
Abstract Body:
Background: Pneumococcal infections remain major public health problem worldwide for aged
population.Aims: was to perform multilocus sequence typing (MLST) in strains of S.pneumoniae gained
in patients elder of 60 years at the territory of Primorsky region as the most southern part of the Russian
Far East. The strains were taken since Jan 2014 till Dec 2015. Methods: MLST was conducted with
housekeeping genes on standard method on recommendations of MC Enright (1998) et al. Results:there
were taken 50 isolates ( group 1, from patients with community-acquired pneumonia), 30 isolates (
group 2, from patients with invasive pneumococcal infections such as bacteriemia and menigitidis).
Isolates form 1st group included 24 serotypes ( 26 of non-typable) and 22 sequence types ( ST) according
to MLST of which 7 were novel, 8 were of Taiwanese clones (TW-28,19,26) of 19F serotype, 3 were of
R6, EU38, SP95. In group 2 of 20 strains there was revealed 8 serotypes ( 12 of non-typable) and 6 ST of
Taiwanese clones.The pattern of antimicrobial agents resistance from the strains of 2nd group was 35%
to erythromycin, 15% to tetracycline, 5% to fluoroquinolones. Conclusions:multilocus sequence typing
allows us to suggest the epidemiological significance of Taiwanese isolates of S.pneumoniae in our
region
Session Number: 223
Session Number: 223
Pathogens
Abstract Title:
Molecular Detection of Common Respiratory Viruses among Acute Respiratory Illness Cases in Ethiopia,
2015-2016
A. Mekonnen, Mefin Mengesha, Wondatir Nigatu, Melese Hailu, Berhan Beyene, Desalegn Belay,
Woubayehu Kassa; Ethiopian Publ. Hlth.Inst., Addis Ababa, Ethiopia
Abstract Body:
Background: Acute respiratory illnesses(ARI) are among the major public health problems both in the
world and viruses are recognized as the major etiological agent. There was no any previous study about
the types and magnitude of viral etiologies responsible for ARI in both hospitalized and outpatients in
Ethiopia except influenza viruses. Improved etiological insight is needed to improve prevention, control
and clinical management of ARIs. Method: Multiplex RT-PCR based Laboratory test was conducted at
National Influenza Reference Laboratory (NIL) of Ethiopian Public Health Institute (EPHI) to identify the
respiratory viruses and to assess circulation level of these viruses including Rhinovirus(RV), Respiratory
syncitialvirus(HRSV A/B),Human adenovirus( HAdV), human Parainfluenzavirus(HPIV1-4), Human
coronavirus(HCoV-NL63, 229E, OC43 and HKU1), Human metapneumoviruse(HMPV A/B), Human
parechoviruses(HPeV), Enteroviruses( HEV), , Human bocavirus(HBoV) and Influenza C(FLU C) among
cases with ARI from natioal influenza sentinel surveillance sites 2015-2016. Result: A total of 422
samples were tested and 55.5% (234) were positive for at least one respiratory virus from 2015-2016.
From these, 20.8 %(88) samples were positive for more than one respiratory virus. Respiratory viruses
were more frequently detected among under five children 69.1%(139/201) and hospitalized (SARI) cases
67.3%(142/211) compared with other age groups 43.0%(95/221) and outpatients cases(ILI)
43.6%(92/422) respectively. The positivity became peak at month of March and lower in September
without a clear pattern for specific respiratory viruses activity. RV, 94(22.2%) was the most frequently
detected Virus followed by RSV 54(12.8%), AdV 48(11.4%). The proportion of positive samples for the
other viruses were 31(7.3%)HPIV1-4, 6.2%(26)HCoV, 6.2%(26)HEV, 5.7%(24)HPeV, 4.5%(19)HMPV A/B,
3.8%(16)HBoV and 0.2%(1) FLUC. Conclusion and Recommendation: This is the first study of its kind
which identified a wide range of viral etiologies of ARI and assessed their level of circulation among both
ILI and SARI cases using multiplex RT-PCR.Many types of Respiratory viruses circulated in Ethiopia with a
high prevalence rate. The study also demonstrated the crucial importance of multiplex PCR for the
simultaneous detection of many possible pathogens from a small sample and Large-scale study has to be
done to better understand the seasonal variation, spectrum of illness and severity of ARI due to the
different respiratory viruses.
Session Number: 223
Session Number: 223
Pathogens
Abstract Title:
Implementing A Multi-Pathogen Model for Forecasting New York Flu Season Using A Cloud Based
Epidemiological Network
J. Nawrocki, B. Galvin, J. Jones, A. Hoffee, L. Meyers; BioFire Diagnostics, LLC, Salt Lake City, UT
Abstract Body:
Background: Predicting onset and severity of influenza is of interest to healthcare professionals. Two
major factors in implementing effective flu season forecasting models include sourcing reliable data and
selecting the appropriated forecasting method. We seek to solve these problems by sourcing data from
a real-time epidemiological web tool, BioFire FilmArray® Trend, and use machine learning techniques to
build robust, multivariate time series models. FilmArray Trend is an epidemiologic surveillance system
that receives FilmArray® Respiratory Panel (RP) results from more than 20 clinical sites across the US,
with over 350,000 tests dating back to 2012. Our model, built to predict influenza A positivity is a novel
approach in that it uses multiple respiratory pathogens to make the predictions. We seek to apply our
model to forecast the 2017-2018 flu season in New York, and potentially improve the forecasts by
including environmental factors as model inputs. Methods: We used a long short term memory (LSTM)
neural network for predicting influenza A seasonality, 28 days in advance, in the United States using
FilmArray Trend data. The model utilized percent positivity of multiple pathogens since 2012 as inputs.
The 2016-2017 flu season was held out for validation. The most predicative combination of pathogens
were influenza A and human rhinovirus (HRV), while coronavirus contributed the least to predicting flu
seasonality. We will include regional environmental factors, such as weather and humidity, in order to
potentially increase the predictive power of the model. Results: We will apply our model to the 2017-
2018 flu season in New York as a regional validation, making predictions throughout the season. A
comparison of the predicted and actual flu season will be reported in spring, 2018 and compared to the
Center for Disease Control (CDC) influenza like illness (ILI). Conclusion: Our demonstration that influenza
A and HRV positivity rates can be used to forecast the 2016-2017 influenza A seasonality is promising. By
applying this model to an active flu season, we can better understand the accuracy and utility of the
model as well as potentially increase the effectiveness by including environmental factors.
Session Number: 223
Session Number: 223
Pathogens
Abstract Title:
Microbial Etiology of Periodontal Diseases in Enugu, Nigeria
U. C. Maduakor1, S. N. Maduakor2, N. F. Onyemelukwe1, D. Ukaji1, O. C. Dozie-Nwakile1, E. A.
Onyebueke1, N. C. Azubuike1, A. O. Onyemelukwe1; 1Univ. of Nigeria Nsukka (Enugu Campus), Enugu,
Nigeria, 2Coll. of medicine, Enugu, Nigeria
Abstract Body:
Background: Periodontal disease is plaque induced inflammation of surrounding tooth structures. It is a
major factor in adult tooth loss. There is paucity of information on microbial etiology of periodontal
diseases in Enugu. The study focused on the isolation and identification of bacteria in periodontitis
among patients in Enugu and comparing same with healthy subjects. Materials and Methods: This was a
case controlled study that spanned over 8 years. A total of 433 samples were randomly collected
comprising of 140 patients with chronic periodontitis, 173 with gingivitis and 125 healthy subjects.
Sterile paper points were used for the sample collection. Standard culture and biochemical techniques
were used for the isolation and Identification (Saini et al., 2003 and Baron et al., 1994) Structured
questionnaires were used to record demographic variables and other risk factors. Analysis was done
with Graph pad prism Version 5 Results: A total of 1,053 microorganisms were isolated. Gingivitis
accounted for 425 isolates, chronic periodontitis 359, and healthy subjects 269 isolates. Anaerobic
microorganisms predominated in gingivitis with Actinomyces spp ranking highest and the least was
Capnocytophaga spp. Predominant microorganisms in chronic periodontitis were anaerobic Gram
negative rods with Fusobacterium nucleatum ranking highest followed by Porphyromonas gingivalis.
Aerobes predominated in healthy subjects. Gender difference was significant in gingivitis but not in
periodontitis with female preponderance. Prevalence of periodontitis increased with age. Conclusion:
This study is novel in Enugu. Mixed bacteria growth was evident in the study. The factors significantly
associated with periodontitis were age, level of education, method of oral hygiene, history of toothache,
age at which odontalgia and bleeding gums occurred. High quality oral care and diet are advocated.
Session Number: 223
Session Number: 223
Pathogens
Abstract Title:
Association of Toxigenic Clostridium Difficile, Animal Feces, Soil and Tree Leaves Contamination
M. Alam, J. Miranda, J. K. McPherson, M. Al Ameri, M. Davis, K. Begum, K. W. Garey; Univ. of Houston
Coll. of Pharmacy, Houston, TX
Abstract Body:
Background: Spores of C. difficile fecal sources can survive and disseminate in any environs and act as a
reservoir for human and animal enteric colonization/infection. Although likely ubiquitous worldwide,
the ecology and prevalence of C. difficile spores in soil and animal feces as related to human health is
poorly understood. The objective of this study was to isolate and characterize C. difficile from top- and
sub-soil, tree leaves contaminated with bird/ or animal feces. Methods: As part of a Texas state-wide
surveillance effort, we collected paired samples of top-soil (1 square foot of soil surface swab) and sub-
soil (1.0 gram of soil collected from 1 inch below the surface) samples (top n=117; sub n=117), tree
leaves with bird feces (n=164) from parks in and around Houston, Texas. Samples were assessed for C.
difficile using anaerobic enrichment culture and molecular methods. Suspected colonies of C. difficile
from cycloserine cefoxitin fructose agar (CCFA) plates were isolated and then characterized by multiplex
PCR (tcdA, tcdB, cdtA, cdtB, and tpi genes). PCR ribotyping was then performed to determine C. difficile
strain genotypes. Results: We found that 47.0% (55/117) of top-soil samples contained C. difficile, of
which 29.9% (35/117) of top-soil samples contained toxigenic C. difficile. Sub-soil samples contained
20.5% (24/117) C. difficile, and 14.5% (17/117) of sub-soil samples contained toxigenic C. difficile. We
successfully ribotyped 71 isolates of C. difficile and found 16 different ribotypes from the top-soil and 11
ribotypes from the sub-soil. Ribotypes F106, F014-020, and FP310 were the three (out of 16) dominating
ribotypes in top-soil samples. F106, F002, and FP310 were the three (out of 11) dominating ribotypes of
C. difficile in sub-soil samples. A total 54 of 164 (32.9%) leaf samples were culture positive for C. difficile
of which 44 of 164 (26.8%) samples were toxigenic C. difficile. A total of 7 distinct ribotypes (F002, F106,
F014-020, F015, FP310, FP415, and F027) were identified from 51 C. difficile isolates tested. Conclusions:
We identified a high prevalence of toxigenic C. difficile with diverse ribotypes from soil, bird or animal
fecal contaminated tree-leaf sources and such toxigenic strains can be the source of human- or animal-
gut colonization/infection.
Session Number: 223
Session Number: 223
Pathogens
Abstract Title:
Microbial Risks Associated with Open Defecation in Atlanta
D. Capone, J. Brown; Georgia Inst. of Technology, Atlanta, GA
Abstract Body:
Homeless persons (HPs) in the United States (US) who sleep in unsheltered locations experience limited
access to sanitation facilities which can force some HPs to resort to open defecation. The association
between open defecation (OD) and a wide range of health problems is well documented in the
developing world. Despite recent disease outbreaks – notably, Hepatitis A virus – associated with poor
sanitation in HP communities in the USA, microbial risks of OD have not been characterized in this
context. In response to anecdotal reports of OD by homeless persons in Atlanta, we conducted a pilot
study to measure the spatial distribution and enteric pathogen profile of discarded human feces in the
city. We identified and mapped 43 OD sites containing 120 stools. Of these, we analyzed 28 stool
samples via multiplex RT-PCR (Luminex Gastrointestinal Pathogen Panel), a stool-based diagnostic assay
identifying presence of 15 common parasitic, bacterial, and viral enteric pathogens. 25% of the 28
collected OD stools tested positive for one or more pathogen. We estimated point prevalences to be
10.7% for enterotoxigenic E. coli (ETEC), 10.7% for Giardia, 3.6% for norovirus, and 3.6% for Salmonella.
Mapping OD sites in Atlanta reveals that 88% (38/43) of documented OD sites exist within ¼ mile of a HP
service center, suggesting that existing facilities are not meeting sanitary needs of the HP population.
Though preliminary, these results suggest that OD in Atlanta is common and may pose risks to public
health. Further work is needed to characterize risks and identify appropriate hygiene and sanitation
interventions for HP populations.
Session Number: 223
Session Number: 223
Pathogens
Abstract Title:
Characterization of Skin, Oral, and Gut Microbiome among A Cohort of U.S. Military Veterans
L. A. Brenner1, A. J. Hoisington2, K. A. Stearns-Yoder1, C. Hoffman1, J. D. Heinze3, C. E. Stamper3, T. T.
Postolache4, C. A. Lowry3; 1Denver VA Med. Ctr., Denver, CO, 2Air Force Inst. of Technology, Wright-
Patterson AFB, OH, 3Univ. of Colorado Boulder,
Abstract Body:
Background: In 2007, the United States National Institute of Health launched the Human Microbiome
Project (HMP) on 300 "healthy" human subjects that were lacking evidence of any disease. Aided by that
landmark study, researchers have started investigated the human microbiome, most often the gut
microbiome, and its relation to clinical conditions ranging from obesity to Alzheimer’s disease. However,
there are currently few studies that establish a baseline condition on individuals that have a mental
health diagnosis. Here we present the results of 16S rRNA gene sequencing of the skin, oral, and gut
microbiomes of 189 U.S Veterans. This population is unique in terms of exposures (e.g., combat, higher
percentage mental health diagnosis/PTSD/suicide rate) and has yet to be studied through microbial
conditions. Methods: The oral, skin, and gut microbiome were sampled from 189 U.S. Veterans.
Bacterial DNA was retrieved from 567 samples and 16S DNA was amplified, sequenced using bi-
directional Illumina MiSeq, and analyzed in QIIME and R. Health information was obtained through
electronic medical records for extensive longitudinal data and a clinic visit that included surveys and a
multitude of psychometrically sound measures. Results: The Veterans in this study ranged from 24-77
years old with 47% experiencing at least one combat duty. They displayed many conditions that would
not be found in healthy individuals as defined by the HMP to include 44% with significant mental health
issues (OQ-45 survey), 46% with insomnia (ISI survey), and 15 % with severe depression (BDI survey).
Additionally, 71% reported a history of traumatic brain injury and 9% were currently homeless. In terms
of the microbial community, the results show similar overall community composition of the Veterans to
the HMP participants but the diversity of the Veterans samples are significantly higher. Certain species,
including those related to stress such as Akkermansia, are observed in higher percentage in the
Veterans. Conclusions: This study begins the process of investigating the microbiome of U.S Veterans.
The foundations of the results may be used for a treatment of unique conditions in that cohort such as
chronic symptoms associated with PTSD that may be mediated by a immunoregulatory/anti-
inflammatory probiotic.
Session Number: 223
Session Number: 223
Pathogens
Abstract Title:
Microbiological Study of Clin. Samples from Skin Sources in Gen. Hosp. Ikare-Akoko Community, Nigeria
O. A. Ajayi; Adekunle Ajasin Univ., Akungba-Akoko, Nigeria, Akungba-Akoko, Nigeria
Abstract Body:
Background: Skin are congenial sources of many nosocomial infections which are notable causes of
mobility and mortality based on epidemiological investigations. Methods: Skin samples of hospitalized
patients in General hospital Ikare-Akoko, Ondo state were examined for the purpose of this study. The
bacterial isolates were identified by standard microbiological methods and grouped primarily by colonial
characteristics on the plates. Morphological, physiological and biochemical tests were carried out on all
the selected isolates. Results: Fourteen samples (56%) were from male sources while eleven (44%)
were from female sources out of the twenty-five samples collected. Twenty isolates were analyzed, out
of which eleven (55%) were Gram positive bacteria, while nine (45%) were Gram negative bacteria.
Thirteen (65%) isolates were cocci, while seven (35%) were rods. The bacterial species generally
encountered were Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa and Escherichia
coli. The significance of this study is to know the causes, mode of transmission, frequency, prevention
and treatment of hospital acquired infection also known as nosocomial infection. Conclusions: The
samples collected for analysis from hospitalized patients of this project vary with age groups.
Statistically, age range 3months-7yrs accounted for 6(24%) males and 2(8%) females. Similarly, age
range 20-30yrs had 2(8%) males and 6(24%) females. Nevertheless, in age range 31-40yrs we have 2(8%)
males and 2(8%) females. In, age range 41-50 we have 2(8%) males and no female was sampled.
Whereas, age range 51-60yrs had 2(8%) males and 1(4%) female recorded. Further studies showed
multiple antibiotic resistance among this study groups which is significant epidemiologically. Some of
the data obtained in this study would be helpful for our health management systems in control of skin
related nosocomial infections
Session Number: 223
Session Number: 223
Pathogens
Abstract Title:
Genetic Profiling for Anaplasma, Ehrlichia and Rickettsia Species in Ticks Collected in the Eastern Cape
Province of South Africa
B. C. Iweriebor, L. C. Obi; Univ. of Fort Hare, Alice, South Africa
Abstract Body:
Introduction: Studies of microorganisms carried in ticks in non-disease-endemic areas might provide
information about potentially pathogenic organisms, their vectors, and reservoirs. These data might also
provide an opportunity to examine the ecology of emerging zoonoses for which different ecologic
determinants for disease transmission may be present. Anaplasma, Ehrlichia and Rickettsia spp. are tick-
borne pathogens that cause severe diseases in humans and other animals worldwide. Infections caused
by these pathogens are deadly if left untreated. There has been relatively few systematic survey of these
pathogens among ticks in South Africa, thus necessitating this study. Methodology: The presence of
Anaplasma, Ehrlichia, and Rickettsia species were demonstrated by PCR in ticks collected from domestic
ruminants and vegetation at some selected communities in the Eastern Cape of South Africa. The ticks
were identified by morphological characteristics and thereafter processed to extract bacterial DNA,
which was analyzed for the presence of genetic materials of Anaplasma, Ehrlichia and Rickettsia spp.
Results: Three genera of ticks comprising five species were identified. The screening yielded 31
sequences of which 16 of them were phylogenetically related to Ehrlichia sequences obtained from
GenBank, 15 sequences clustered phylogenetically with Rickettsia sequences from GenBank while no
positive result was obtained for Anaplasma. The generated Ehrlichia sequences were closely related to
E. chaffeensis, E. canis, E. muris and the incompletely described Ehrlichia sp. UFMG-EV and Ehrlichia sp.
UFMT while those of Rickettsia sequences showed that five were phylogenetically related to members
of the spotted fever group (SFG) rickettsia and the remaining ten sequences were Candidatus Rickettsia
spp. whose pathogenic potentials are not known. The SFG rickettsia were of the genera Rickettsia akari
and Rickettsia raoultii both being reported in South Africa for the first time and also in Rhipicephalus
spp. as a vector. Conclusion: The findings showed that ticks in the studied areas were infected with
Ehrlichia and Rickettsia spp. and that the possibility of transmission to humans who might be tick
infested is high. Keywords:- Tick-borne, Anaplasma, Ehrlichia, Rickettsia, spotted fever group, zoonoses
Session Number: 223
Session Number: 223
Pathogens
Abstract Title:
Mycological and Bacteriological Quality and Safety of Bottled Water in Ethiopia
T. L. Bedada1, F. A. Dera1, R. M. Edicho1, S. G. Gebre12, Y. B. Asefa3, W. G. Sima1, R. F. Maheder1, T. Y.
Yohannis1, A. G. Biegna1; 1Ethiopian Publ. Hlth.Inst., addis Ababa, Ethiopia, 2Ethiopian Publ. Hlth.Inst.,
America, American Samoa, 3Ethiopian Publ
Abstract Body:
Background: Safe water supply is vital and can result in significant benefits to health. However,
contaminated bottled water poses a great health risk due to poor microbiological quality of water.
Methods and Materials: A retrospective study was conducted on 222 Bottled water samples collected
from bottled water manufacturers of various regions of Ethiopia from January 2008 to December 2015,
tested and recorded in Ethiopian Public Health Institute to determine heterotrophic plate count and
Staphylococcus aureus by the help of pour plate method; for coliforms using multiple tubes
fermentation techniques; for mould and yeast count using spread method, and for Salmonellae and
Shigella spp. using ES ISO 6579. The data was analyzed using SPSS 20 statistical package. Results: Among
the total samples examined from 44 brands, detections of heterotrophic plate count, mould, yeast, total
and thermotolerant coliforms, Escherichia coli and Staphylococcus aureus were observed in 114 (51.4%),
33 (14.9%). 5(2.3%), 2(0.9%), 1(0.5%), 1(0.5%) and 1(0.5%) samples respectively, but there were no
detections of Salmonellae nor Shigellae species. Conclusion and Recommendation: More than one-third
of bottled water samples were mycologically and bacteriologically unsafe for human consumption. To
prevent public health hazards, regular monitoring of bottled water using quality and safety indicators
should be a priority agenda.
Session Number: 223
Session Number: 223
Pathogens
Abstract Title:
Determination of Endotoxin in Tattoo Inks
M. Moon1, M. J. Huang1, J. Misock1, G. Periz1, K. Dewan1, A. Alavi2, J. Du2, J. Gogley2, H. Hills2, M.
Kawalek2, L. Phan2, S. Ruelle2, S. Torres2, D. Williams-Hill2, N. Sadrieh1; 1Office of Cosmetics and
Colors, Ctr. for Food Safety and Applied Nutrition
Abstract Body:
Background: The use of tattoo inks which are contaminated with microorganisms and endotoxins may
lead to adverse effects, including irritation, infection and permanent scarring of skin.1 Even though
cosmetic product are not required to be sterile, they must be free of harmful pathogens and the total
microbial counts must be low, there are no specific recommendations for endotoxin testing or
endotoxin limits for tattoo inks. The goals of this study were: 1) to assess the reliability and robustness
of the gel clot assay for measuring endotoxin in tattoo inks, and 2) to understand potential sources of
endotoxin in tattoo inks. Methods: Microbial burdens and endotoxin were assessed in 31 tattoo inks
purchased for surveys from 2015 to 2017 or collected from a manufacturing site inspection in 2017.
Using the Food and Drug Administration’s (FDA) Bacteriological Analytical Manual (BAM), Chapter 23, as
our reference for microbial contamination, we determined for each sample, the aerobic plate counts
(APC) for bacteria and fungi. For identification of isolates from plates and a 7-day enrichment culture,
VITEK 2 system or 16S rRNA sequencing was employed. To avoid interference from the colors present in
tattoo inks, endotoxin levels were measured by a modified gel clot method based on the United States
Pharmacopeia (USP) <85>. Results: Of 31 tattoo ink samples tested, 10 samples had detectable microbial
loads ranging from 20 to 1,820,000 colony forming unit (CFU)/mL; whereas 21 samples found no growth
on plates (<10 CFU/mL). For endotoxin, 16 out of 31 ink samples had wide ranges of endotoxin levels
from 3.125 to 6,250 endotoxin unit (EU)/mL. The remaining 15 samples showed an inherently strong
inhibitory effect on the gel clot assay due to the unique matrix of tattoo pigments and inks. There was
no correlation between endotoxin levels and total microbial burdens. Of 21 ink samples which had no
bacterial and fungal growth on plates, endotoxin was detected in 9 samples, with the three highest
levels being 312.5, 625, and 6,250 EU/mL. Conclusions: The data show the gel clot assay is a reliable and
robust method for determining endotoxin levels in tattoo inks, although some product matrices had a
strong inhibitory effect on the assay. The results indicate that not only may inks be adulterated with
microorganisms, but that endotoxin may be present in tattoo inks, from both live bacteria and remnants
of dead bacteria. Further, this study suggests the need for establishing an endotoxin limit to ensure
microbiological safety for tattoo inks.
Session Number: 223
Session Number: 223
Pathogens
Abstract Title:
Prevalence and Distribution of 46 Antibiotic Resistance Genes in Pseudomonas Aeruginosa Across Three
Tertiary Care Hospitals
S. E. Condon, R. Suwal, R. Khare; Northwell Hlth., Lake Success, NY
Abstract Body:
Background: Pseudomonas aeruginosa is notable for its intrinsic resistance to many antibiotics.
Carbapenems have typically been used as agents of last resort, however, resistance to these drugs is
increasing and is associated with higher morbidity and mortality. At our institution, carbapenem
resistant P. aeruginosa represent 50% of all carbapenem resistant organisms (CROs). Multiple types of
mechanisms may be responsible for carbapenemase resistance in this organism including
carbapenemase genes on mobile genetic elements, efflux pumps, and porin loss. In this study we
determined the prevalence of 46 antibiotic resistance genes across three geographically separated
hospitals using the CarbaR and Acuitas Lighthouse assays. Methods: We assessed the prevalence and
distribution of antibiotic resistance genes in imipenem and/or meropenem resistant isolates of P.
aeruginosa. Isolates were randomly selected from all isolates submitted to our laboratory for routine
clinical testing by three tertiary care hospitals in the greater New York area. Testing for 5 key
carbapenemase genes was performed using the using GeneXpert CarbaR assay (Cepheid). All isolates
were also tested with the Acuitas Resistome test (Opgen) for confirmation and identification of
additional carbapenemase, ESBL and AmpC related genes. Results: A total of 51 P. aeruginosa isolates
were evaluated by the CarbaR and Acuitas Resistome assays. Of these, only 1 (2%) was positive for KPC
by both assays. OXA-50 was detected in 39 (76.4%) isolates, and OXA-2, SHV-G156, and SHV-
G238S/E240K were detected in 2 isolates each (3.9% each). OXA-50 isolates were, unexpectedly,
resistant to imipenem more often than meropenem (84.6% compared to 66.7%). Conclusions: Despite
the increasing prevalence of meropenem and/or imipenem resistant P. aeruginosa, extrinsic resistance
genes such as KPC remain exceedingly rare. However, intrinsic resistance genes such as OXA-50 were
highly prevalent in our patient population, even though they are infrequently reported in the literature.
Session Number: 223
Session Number: 223
Pathogens
Abstract Title:
Molecular Epidemiology of Legionella pneumophila Isolates in Michigan
S. Altamimi1, M. Perri1, H. Misikir1, D. Vager1, S. McElmurry2, P. Kilgore2, M. Zervos1; 1Henry Ford
Hlth.System, Detroit, MI, 2Wayne State Univ., Detroit, MI
Abstract Body:
Background: Legionella pneumophila is a water borne pathogen that mainly affects the lungs among
other organs, causing a severe form of pneumonia. Little is known about molecular epidemiology.
Methods: Environmental Legionella samples were collected from home water throughout Flint and
Genesee County, Michigan (year 2016) and human samples were obtained from Michigan Department
of Health and Human Services (MDHHS) from patients in Oakland, Wayne and Genesee County,
Michigan. Water samples were originally cultured for Legionella using buffered charcoal yeast extract
agar (BCYE) and BCYE with polymyxin B, anisomycin and vancomycin (PAV), and all of the resulting
Legionella isolates were sub-cultured onto BCYE, and incubated as the water samples, for 48 hours in
humidified ambient conditions at 35 degrees °C. Technical assistance was obtained from the CDC for
processing water cultures. Pulsed-Field Gel Electrophoresis (PFGE) was performed to determine the
relatedness of the Legionella isolates. Genomic DNA was prepared and digested with SfiI (New England
BioLabs, Beverly, MA). Isolates were placed in the same PFGE strain group if their SfiI restriction patterns
were ≥ 80% similar by Dice co-efficient Results: There were 6 strain types that had more than one
isolate from different patients or the environment and patients. Among 18 environmental strains there
were 6 strain types and among 33 human isolates there were 14 strain types. Common strains were
seen in 8 strain types, the strains groups had up to 40 strains including 25 from humans and 15 from
environmental isolates. Three isolates were common to water and humans. Table 1 shows a summary of
findings. <table class="AbstractTable" id="{33FD0BAF-F065-4DE8-AE94-7BEA66005A8F}"><caption
colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1">Strain
Type</td><td rowspan="1" colspan="1">Total no strains</td><td rowspan="1" colspan="1">Sample
source</td></tr><tr><td rowspan="1" colspan="1">1</td><td rowspan="1" colspan="1">12</td><td
rowspan="1" colspan="1">Environmental</td></tr><tr><td rowspan="1" colspan="1">7</td><td
rowspan="1" colspan="1">14</td><td rowspan="1" colspan="1">Human</td></tr><tr><td
colspan="1">Human, Environmental</td></tr><tr><td rowspan="1" colspan="1">18</td><td
rowspan="1" colspan="1">3</td><td rowspan="1" colspan="1">Human</td></tr><tr><td rowspan="1"
colspan="1">Environmental</td></tr><tr><td rowspan="1" colspan="1">8</td><td rowspan="1"
colspan="1">2</td><td rowspan="1" colspan="1">Human</td></tr><tr><td rowspan="1"
colspan="1">Human</td></tr><tr><td rowspan="1" colspan="1">19</td><td rowspan="1"
colspan="1">2,3,4</td><td rowspan="1" colspan="1">3</td><td rowspan="1"
colspan="1">Environmental</td></tr><tr><td rowspan="1"
colspan="1">10,11,12,13,14,15,16,17</td><td rowspan="1" colspan="1">8</td><td rowspan="1"
colspan="1">Human</td></tr></table> Conclusion: Common banding patterns between human and
the environmental isolates was rare, however did occur among the human strains and could be of use to
assist in epidemiologic investigation. Common strains among humans suggest a common source
including potentially from the environment. This study supports the need for obtaining culture for
Legionella from water sources and patients with clinical illness to better understand epidemiology of
this infection.
Session Number: 223
Session Number: 223
Pathogens
Abstract Title:
Evaluating of Bruce-Ladder Pcr Assay and Multiple Locus Variable Number Tandem Repeat Analysis
(Mlva) for Genotyping of Egyptian Brucella Isolates
A. M. El Sawah, W. S. Shell, A. M. Ali; Central Lab. for Evaluation of Veternary Biologics, Cairo, Egypt
Abstract Body:
Brucellosis is a major bacterial zoonosis of global importance. In animals, the disease primarily affects
their reproductive system also, lack of vaccines suitable for use in man has led to the investigation of
Brucella as agents for bioterrorism. Eradication of human brucellosis largely depends on accurate
diagnosis program in animals. This study aimed to evaluate and establish the use of a and Multiple Locus
Variable Number Tandem Repeat Analysis(MLVA) assays and multiplex PCR (Bruce-ladder) as
epidemiological tracing tools for diagnosis of brucellosis in Egypt. Five Brucella reference strains along
(control positive) with, three Brucella vaccinal seed strains in addition to 30 local isolates. BRUCE-
LADDER PCR and MLVA assay were used to identify and differentiate between Brucella field isolates in
comparison with traditional methods (Serological and Biochemical tests). MLVA (Photo 1) and Bruce-
ladder PCR (Photo 2) were identical to those obtained by the conventional methods. The sensitivity of
both methods were 100% by the traditional methods as the gold standard test. All Brucella field isolates
were identified as Brucella melitensis biovar 3 by traditional methods and MLVA assay (biovar level) and
as brucella melitensis (species level) using BRUCE LADDER PCR assay. Vaccinal seed strains and their
roughness; Brucella abortus RB51 and S19 and Brucella melitensis Rev-1 could be identified by BRUCE-
LADDER assay but cannot by MLVA assay. However, the molecular typing of the Brucella strains by
Bruce-ladder PCR and MLVA had several advantages over the use of the conventional methods being
precise, fast, sensitive, easy and economic with the same sensitivity. So, Bruce-ladder PCR and MLVA are
reliable epidemiological tools for brucellosis diagnosis and testing of the seed culture of living Brucella
vaccines. The results confirmed the high discriminatory power of MLVA as an epidemiological tracing
tool to identify Brucella isolates to biovar level.<br /><p><a
href="http://files.abstractsonline.com/CTRL/6d/d/236/992/16a/46e/caf/cd9/789/c60/433/e5/g5563_2.
src="http://files.abstractsonline.com/CTRL/6d/d/236/992/16a/46e/caf/cd9/789/c60/433/e5/g5563_2.J
Session Number: 223
Session Number: 223
Pathogens
Abstract Title:
Virulence Gene Prevalence and Outbreak Investigation of Enterohemorrhagic Escherichia coli Strains in
Japan
H. Baba1, H. Kudo2, K. Oka2, M. Takahashi2, Y. Makino1, Y. Fujikawa1, C. Oe1, H. Kanamori1, K.
Oshima1, T. Aoyagi1, M. Yoshida1, K. Tokuda1, M. Kaku1; 1Tohoku Univ. Graduate Sch. of Med., Sendai,
Japan, 2Miyarisan Pharmaceutical Co., Ltd., Saitama, Japan
Abstract Body:
Background: Enterohemorrhagic Escherichia coli (EHEC) has several some virulence factors other than
Shiga toxin (Stx), such as intimin, which is coded by eaeA and is associated with adhesion of bacteria to
intestinal epithelial cell. Although these virulence factors have been reported, regional distribution of
these virulence genes in Japan remains unclear. We investigated the prevalence and characteristics of
EHEC strains from symptomatic patients and asymptomatic carriers, and investigated the presence of
familial outbreaks by comparing the genotypes of strains from index patient with that from their
contacts. Methods: A total of 78 EHEC isolates were collected between 2016 and 2017 in 15 different
cities within the same region in Japan (22 strains from symptomatic patients, 30 strains were from
contacts in 13 cases of familial outbreaks, and 25 strains were detected by routine examination of
asymptomatic food handlers, and 1 strain was from contact with asymptomatic carrier). We performed
serotyping, detection of 4 virulence factor genes (stx1, stx2, eaeA and hlyA), and BOX-A1R-based
repetitive extragenic palindromic-PCR (BOX-PCR). Results: The patient group was younger (mean age =
24.2 years, n = 22) than the asymptomatic carrier group (mean age = 49.8 years, n = 25) (P = 0.0011).
O157 was dominant (10/22, 45.5%, P = 0.00013) in patient group, while all strains isolated from carriers
were non-O157. There was no difference in the prevalence of stx1 and stx2 between patient group and
carrier group (stx1: 60.0% (15/25) vs 59.1% (13/22), stx2: 44.0% (11/25) vs 63.6% (14/22). Although both
of eaeA and hlyA genes were detected from all strains in patient group, the prevalence of those were
32.0% (8/25) and 64.0% (16/25) in carrier, respectively (P < 0.001 in eaeA and P = 0.0015 in hylA). BOX-
PCR revealed total 78 strains were categorized into 32 genotypes and all strains from contacts were
same types as that from their corresponding index patient, though the contacts with a same index
patient included both patients and carriers. Conclusions: The presence of eaeA and hlyA are likely
contributed to EHEC pathogenesis. EHEC strains causing symptom do not necessarily contain both stx1
and stx2 genes. Even in our small region, there are several EHEC outbreaks caused by different types of
strains. As indicated in BOX-PCR analysis, whether causing infection or not were depending on host
protective factors, which block the colonization of EHEC. More detailed studies are needed to determine
the dissemination of EHEC and their unknown virulence factors.
Session Number: 223
Session Number: 223
Pathogens
Abstract Title:
Rapid Discovery of An Emerging Contamination Event in Nut Butter Using Whole Genome Sequencing
M. Allard, E. Strain, J. Pettengill, D. Melka, W. Correll, L. Hintz, A. Ottesen, D. Macarisin, R. Bell, J. Zheng,
M. Hoffmann, N. Gonzalez-Escalona, E. Stevens, R. Timme, S. Tallent, E. Brown; US FDA, College Park,
MD
Abstract Body:
Background: Next-generation whole genome sequencing (WGS) is now used regularly by government
and state food safety investigators as a high-resolution molecular epidemiologic tool for delimiting
scope of foodborne outbreak events and for the tracking of complicated and genetically homogenous
bacterial strains during foodborne contamination events. Making a genomic link between possible food
sources and clinical isolates is essential for effective traceback to a contamination source. Here, WGS
was applied to Salmonella enterica serovar Braenderup (S. Braenderup) to examine the relationship of
several environmental swab samples previously associated with a nut butter facility and several
reported cases of Salmonellosis in 2014. Methods: To this end, we relied upon the GenomeTrakr
foodborne pathogen WGS network and database and downloaded and analyzed the genomes of 26
strains of S. Braenderup from disparate clinical and environmental sources, the latter of which included
several that were isolated from a nut butter facility in the US. These genomes were combined with other
ecologically diverse S. Braenderup strains and subjected to comparative genomic analysis using a
maximum likelihood approach. Results: Surprisingly, analysis of all 292 variable hqSNP positions
revealed a single monophyletic and highly clonal lineage (separated by ≤ 5 SNPs) of S. Braenderup was
identified that contained both environmental swab isolates from the nut butter facility as well as clinical
isolates several food contamination events. As expected, unrelated environmental reference isolates of
S. Braenderup were phylogenetically disparate to the nut butter clade, falling at least 32 SNPs away. It is
noteworthy that the link between illnesses and the processing facility was established by 15 diagnostic
SNPs from WGS even though a common food vehicle was not identified at this point. Conclusions: Taken
together, these findings support applicability of WGS in delimiting contamination sources even in rare
cases where a food vehicle remains elusive. Moreover, these data underscore extensive utility of this
technology in revealing otherwise undetected genotypic differences essential to the tracing of bacterial
pathogens as they emerge in the food supply.
Session Number: 223
Session Number: 223
Pathogens
Abstract Title:
A Model for Detection of Ev-D68 Outbreaks Through A Cloud Based Epidemiology Network
B. Galvin, J. Nawrocki, J. Jones, L. Meyers; Biofire Diagnostics, Salt Lake City, UT
Abstract Body:
Background: Real time surveillance of pathogens can facilitate management of outbreaks and enrich
understanding of disease dynamics. The BioFire FilmArray® Respiratory Panel (RP) can detect 20
respiratory pathogens including rhinovirus/enterovirus (HRV/EV). FilmArray® Trend is an epidemiology
tool that automatically receives de-identified test results from the FilmArray® Systems. In 2014,
enterovirus (EV) D68 was associated with an outbreak of severe respiratory illness in children worldwide
resulting in some cases with Acute Flaccid Myelitis. Although HRV/EV is not subtyped by the FilmArray
RP, we have developed a model for EV-D68 identification using the (masked) PCR data exported to the
database from 2014-2016. Methods: Over 90,000 HRV/EV positive test results were queried from the
FilmArray Trend dataset. Additionally, one European site and five US sites contributed FilmArray® data
files (N=772) from samples that were independently characterized by an EV-D68 specific PCR test or by
sequencing. We employ a semi supervised, support vector machine algorithm to identify EV-D68
signatures within the real time PCR data from the six HRV/EV assays. This algorithm, henceforth referred
to as the Pathogen Extended Resolution (PER) test, will then be evaluated using a large set (N=1120) of
independently verified results to be provided by a western United States site. Results: The percent
positivity for the PER test, trained to identify EV-D68, demonstrated an overall sensitivity of 91% and
specificity of 91% when evaluated against existing clinical US samples. This algorithm demonstrated 94%
sensitivity and 86% specificity on 59 samples from the Netherlands collected in 2014 and 2016. When
plotted over time, the percentages of predicted EV-D68 per regional RP tests were generally low with
occasional regional outbreaks predicted in 2014 and 2016. Conclusions: We have demonstrated that the
PCR amplification data available to BioFire through the FilmArray Trend database can be used to identify
signatures associated with EV-D68 in both the US and EU over a 3-year period. The addition of new
verified data, as well as further algorithmic developments will add to the PER Test’s capability to identify
patterns within the FilmArray Trend database. This type of test has the potential to provide alerts to
network participants of anomalous patterns in pathogen trends. Further validation, development and
refinement of these alert algorithms must be explored before broad implementation.
Session Number: 224
Session Number: 224
Session Title: CPHM12 - Molecular Diagnostic Microbiology: Pre-Analytical Considerations
Abstract Title:
Proof of Concept: Preservation of Cerebrospinal Fluid for Delayed Molecular Testing
N. L. LePage, T. W. Cullen, T. E. Davis; Indiana Univ. Sch. of Med., Indianapolis, IN
Abstract Body:
Background: Rapid detection and identification of infectious organisms within the cerebrospinal fluid
(CSF) may prove vital in the proper diagnosis and treatment of meningitis. In facilities with PCR
diagnostic tools available, these diagnoses can be made the same day as the lumbar puncture and
without concern for degradation of the sample during transportation. In facilities without access to
these diagnostic tools an educated guess on the infectious organism can be made from chemical CSF
analysis or the organism can be identified through time-consuming traditional methods. Methods: CSF
samples from patients with positive meningitis/encephalitis (ME) BioFire FilmArray Panel results,
bacterial and viral sources, were separated into four 87µL samples stored at room temperature (two
aliquoted into vials, two aliquoted onto two separate HemaSpot™-HF devices). After 24 hours of storage
one room temperature portion and one portion on the absorbent paper within the HemaSpot™-HF
device were transferred to 300µL of BioFire buffer solution. Both samples were vortexed to ensure
proper mixing. The samples were loaded and run on the BioFire FilmArray system in accordance with
manufacture guidelines. Due to sample availability after elution of the absorbent paper only 150µL of all
samples was loaded into the panel instead of the recommended 200µL. This process was repeated at 72
hours. Results: When preserved on the HemaSpot™-HF device, the infectious organism was detected in
7 out of 12 samples at 24 hours and in 6 out of 12 samples at 72 hours. In comparison, the non-
preserved samples detected the infectious organism in 9 out of 12 samples at 24 hours and in 8 out of
12 samples. Conclusions: The positive results from HemaSpot™-HF preserved samples is a promising
concept for facilities without access to molecular testing. Our preliminary data indicates that this
method of preservation is viable for delayed PCR diagnostics allowing samples to be shipped safely at
room temperature. Continued testing will allow for further analysis including the quantitative limitations
of this method. Molecular testing even delayed 24 hours will be faster than traditional identification
methods allowing for timely and more accurate treatment plans. This expanded application of the
HemaSpot™-HF proves interesting for future exploration of fluid preservation.
Session Number: 224
Session Number: 224
Abstract Title:
Evaluation of Sample Transportation Solutions and 16s Rrna Gene Regions for Microbial Study
Z. Chen, M. Hui, P. Hui, P. Wong, P. Chan; The Chinese Univ. of Hong Kong, Hong Kong, Hong Kong
Abstract Body:
Background: The amplicon profiling of 16S ribosomal RNA (rRNA) gene is widely used to investigate the
microbial diversity. One of fundamental challenge has been the choice of sample collection methods and
16S rRNA gene primers for profiling accuracy of microbial community. Methods: We compared six DNA
preservation solutions without cold chain for stool sample collection, and compared 16S rRNA gene
regions of V1-V2 and V3-V4 for gut and oral microbial communities. The stool samples in different DNA
preservation solutions were cultured for bacteria growth in aerobic and anaerobic conditions. Results:
Different to the stool samples in PBS and the culture medium, all collections in six DNA preservation
solutions showed high identical microbial communities when compared to the freshly frozen samples,
with three most common taxa of Bacteria (mean abundance of 24%), Faecalibacterium (18%) and
Escherichia-Shigella (14%). The microbial community was inter-individual diversified (R2=0.80, p<0.001)
irrespective of preservations (R2=0.10, p=1.000). Both aerobic and anaerobic cultures indicated four
commercial available preservations could inhibit the growth of bacteria within minutes. When 16S rRNA
gene regions of V1-V2 and V3-V4 were compared, the weighted UniFrac distances showed similar
clustering for stool samples (mantel test 0.9676, p<0.001), oral rinse samples (mantel test 0.9047,
p<0.001), and the head and neck squamous cell carcinoma tissues (mantel test 0.9419, p<0.001).
However, the 16S V1-V2 primer pair failed to amplify Bifidobacteriales due to in part the high rate of
mismatch of 27f primer to bifidobacteria. Conclusions: We provide solutions for stool sample collection
without cold chain and the promising 16S rRNA gene primers. The data may be useful as a guideline to
obtain microbial samples with stable bacteria DNA and reduce PCR-based bias in oral and gut microbial
diversity studies.
Session Number: 224
Session Number: 224
Abstract Title:
Elucidation of A Non-Thermal Mechanism for Dna and Protein Degradation Using Lyse-ItÒ
T. Santaus, C. Geddes; UMBC-IoF, Baltimore, MD
Abstract Body:
Background: The central hypothesis being addresses is: as you increase microwave irradiation power,
there is an increase in singlet oxygen, hydroxyl radical, and superoxide generation or diffusion. There
has been significant literature reported on the effects of Reactive Oxygen Species (ROS) on DNA.
However, the exact mechanism of ROS generation from microwave irradiation is under debate.
Excessive amounts of ROS are known as oxidative stress and can lead to toxic and carcinogenic effects
due to oxidation of proteins and nucleic acids. Singlet oxygen is able to react with DNA and is thought to
induce guanine to thymine transversions. Hydroxyl radicals mediate H-atom abstraction from the 2-
deoxyribose backbone primarily at C3, C4, and C5 of DNA and leads to DNA strand breaks in cells.
Superoxide can accelerate DNA damage by increasing free-iron levels in cells and can promote the
increase in hydroxyl radicals. Method: The method of detecting these three ROS species include the use
of a Frigidaire 900W microwave, Lyse-IT® technology, and ROS specific fluorescent probes. Singlet
Oxygen Sensor GreenTM is used to detect singlet oxygen, Hydroxyphenyl fluoresceinTM for hydroxyl
detection, and DihydroethidiumTM for superoxide detection. Each probe is independently concentrated
in de-ionized water. The solution is microwaved with and without the Lyse-IT® technology and the
absorption and fluorescence is taken post microwave irradiation. Results and Conclusions: In this study,
we demonstrate that ROS detected is increased with the use of the Lyse-IT® technology as compared to
standard conventional heating. This implies that singlet oxygen, hydroxyl radicals, and superoxide all
serve a purpose in DNA damage. Also, using the Lyse-IT® technology allows for more ROS generation
and therefore more DNA and protein fragmentation in a simple, low-cost manner.
Session Number: 224
Session Number: 224
Abstract Title:
Design and Validation of A Synthetic Dna Molecule, Nuversa, to Be Used As A Standard in Quantitative
Real-Time Pcr for Molecular Detection and Serotyping of Pneumococcus
F. Sakai, G. Sonaty, D. Watson, K. P. Klugman, J. E. Vidal; Emory Univ., Atlanta, GA
Abstract Body:
Background: Identification of Streptococcus pneumoniae (Spn) and its more than 90 serotypes is
routinely conducted by culture and Quellung reactions. To improve detection of Spn and its serotypes
with molecular technology, quantitative (q)PCRs have been used. However, assays for several Spn
serotypes have not yet been developed. Moreover, qPCR assays require genomic DNA from targets to
prepare standards, which is time consuming. In this study novel qPCR assays and a synthetic DNA
molecule, to be used as a pan-serotype standard, were developed and validated. Methods: New single-
plex qPCR reactions (N=11) for individual serotypes/serogroups were developed and optimized.
Specificity of reactions for target serotypes/serogroups was investigated using a collection of strains. A
synthetic DNA molecule (NUversa, ~8.2 kb) was then engineered to contain all available qPCR targets
(N=67) for serotyping and lytA. NUversa was cloned into pUC57-Amp-modified to generate pNUversa
(~10.2 kb). Standards prepared from NUversa were compared to those prepared from genomic DNA.
Results: Specificity of these new reactions was confirmed and, after optimization, the limit of detection
of new reactions fell between 2 to 20 genome equivalents/reaction. Molecular studies demonstrated
that linearity [NUversa (R2>0.982); pNUversa (R2>0.991)] and efficiency of qPCR reactions using the
synthetic DNA were similar to those that were obtained utilizing chromosomal DNA (R2>0.981).
Quantification with plasmid pNUversa was affected, whereas quantification using synthetic NUversa was
comparable to genomic DNA. Conclusions: Novel single-plex reactions, together with published qPCR
reactions, make it possible to detect and quantify 94 pneumococcal serotypes/serogroups. Furthermore,
NUversa was validated as a control for the detection and quantification of pneumococcal serotypes
using most, if not all, published single-plex qPCR reactions.
Session Number: 224
Session Number: 224
Abstract Title:
Eliminating Cold-Chain Transport of Gastrointestinal Specimens
E. T. Villazana1, B. C. Connors1, A. E. Ostrow2, J. R. Meyer1, A. K. Javorina1, G. Kovachich3, A. A. Fisher2,
B. Goder-Reiser2, C. R. Starr1; 1USAF Sch. of Aerospace Med., Wright-Patterson AFB, OH, 2Oak Ridge
Inst. for Sci. and Engineering, Oak Ridge, TN
Abstract Body:
Complications of transporting clinical specimens include the need for cold-chain custody, which requires
access to a continuous supply of dry ice and reliable shippers to ensure samples are received at the
appropriate temperatures for diagnostic testing. As molecular and genetic diagnostic testing becomes
more common, another growing concern of long-distance shipment is preserving RNA for analysis due
to the numerous RNases in the environment. While diagnostic testing may require a “live” sample for
confirmatory testing, molecular evaluation does not necessarily require a live specimen; nucleic acids
just have to be stabilized for successful amplification. The purpose of this study was to evaluate the
ability of two commercial stabilization buffers to kill or inactivate bacteria (diarrheagenic E.
coli/Shigella), parasites, and viruses in fecal or bile medium while preserving the nucleic acids for
downstream amplification for polymerase chain reaction (PCR), recovery, culturing, and next-generation
sequencing (NGS). Qiagen RNAlater and Zymo Research DNA/RNA Shield stabilizing buffers were added
to a sample with either phosphate buffered saline, fecal, or bile sample combined with a 106
concentration of the FilmArray gastrointestinal (GI) panel targets, incubated at 25°C or 42°C for 9 days,
and analyzed on the FilmArray. Additionally, each sample was plated onto brain heart infusion media
and bacterial growth was documented post 24 hours and up to 5 days post incubation. All PCR-based
testing was successful in all buffer and sample conditions, indicating the nucleic acid was stable enough
for amplification despite long-term incubation. Zymo was a bactericidal agent, completely killing all
bacteria incubated in the matrix. RNAlater was bacteriostatic, allowing regrowth of all incubated
bacteria when placed on enriched media. Initial NGS analysis showed positive hits for the GI panel
tested. The results revealed confirmation of one transport stabilization buffer that completely killed
bacteria but preserved the DNA and RNA, which could greatly reduce shipping costs. We also identified
RNAlater as a preservation reagent that can be used as a method for downstream culturing if necessary
for confirmatory testing by the Centers for Disease Control and Prevention or any lab.
Session Number: 224
Session Number: 224
Abstract Title:
Discrepant Result Analysis for Emerging Technologies in Microbiology
T. Stanford1, L. Brenton2, S. Giglio1; 1LBT Innovations, Adelaide, Australia, 2St. Vincent's Hosp.
Melbourne, Melbourne, Australia
Abstract Body:
Background: To evaluate the effectiveness of a newly introduced test or system, the truth status of a
sample result needs to be established, typically by the use of a gold standard. This is problematic where
the gold standard is imperfect, resulting in difficulties to understand the true performance of the test or
system under investigation. This is especially relevant now in microbiology where a number of plate
imaging systems, some with the ability to interpret microbial growth using artificial intelligence, are
evolving. The gold standard in this case is the microbiologist assessment (or panel of microbiologists).
Methods: Discrepant analysis traditionally involves investigating only those results where the test and
the imperfect truth (microbiologist assessment) do not agree. These samples have their truth status re-
assessed with the expectation the new truth status and the test’s classification agree. This method
ignores the possibility of samples previously in agreement having the truth status re-assessed in
disagreement with the test’s classification, biases interpretation and statistical analysis, and therefore
alternative methods are required. Using data from an evaluation of the APAS Independence at St.
Vincent’s Hospital Melbourne screening urine samples, we present three unbiased methods to
undertake discrepant analysis: a novel simulation method using re-sampling, the composite reference
standard method (Alonzo & Pepe, 1999), and latent class analysis (Walter & Irwig, 1988). The APAS
Independence is a stand-alone plate reading and interpretation system that utilizes artificial intelligence
algorithms to provide a detailed screening result for the sample. Results: In addition to the St. Vincent’s
case study data, simulated data are used to illustrate characteristics of the modelling processes and
detail the limitations of the proposed methods. We also demonstrate that the novel re-sampling
simulation method produced an estimate of significant growth sensitivity close to that of discrepant
resolution while, importantly, using defensible methodology. Conclusions: This presentation is timely
given the advances in microbiology imaging automation and will provide information to laboratories to
objectively resolve samples that are discrepant.
Session Number: 225
Session Number: 225
Session Title: CPHM12 - Molecular Diagnostic Microbiology: The Future of Diagnostics is Here
Abstract Title:
Identification of 11 Std Pathogens in Semen Using Polymerase Chain Reaction (Pcr) and “Flow-Through”
Hybridization Technology
M. Uzair1, M. F. Ali1, R. Ghani2; 1Dadabhoy Inst. for Higher Ed., Karachi, Pakistan, 2Rubina Ghani
Molecular and Pathological Labratory, Karachi, Pakistan
Abstract Body:
Background: The prevalence of sexually transmitted Disease (STDs) among hotel-based sex workers
(HBSWs) in Karachi, Pakistan, was studied. These hotel workers are considered as high risk group
because of their age, economic independence, low education and residence in a place away from their
family. Aim: The aim of this study was to access in health care facilities for diagnosis and common
pathogens of STDs, those causing infertility and Chlamydia trachomatis, Neisseria gonorrhoeae and
Mycoplasma hominis. Material and Methods: Semen samples were obtained by masturbation into
sterile containers after sexual abstinence of 48 to 72 hours. Samples were subjected to semen analysis
within one hour of collection and processed for freezing within two hours of collection. The
concentrations of sperm as well as sperm motility were also determined. DNA extraction was extracted
of all the samples and the PCR assay was performed. The amplicons are subsequently hybridized to
pathogen-specific capturing probes via “Flow-through” hybridization. Result: During our study we came
across with the STI pathogens present in our population and the reason for infertility was the main
cause. When Chlamydia trachomatis and Neisseria gonorrhoeae were detected in their wife’s were also
screened and these STI pathogens were identified. The main route for the transfer of STI pathogens
were the men special those who visited commercial sex workers or hotel-based sex workers as they
were working in other cities and the complained for infertility. Discussion: Screening for these
pathogens should be performed using the most sensitive methods, such as nucleic acid amplified tests.
Screening for bacterial STI pathogens, Mycoplasma hominis, Chlamydia trachomatis and Neisseria
gonorrhoeae is strongly recommended because these pathogens can cause serious reproductive
complications in the recipients of semen donations and infection in their offspring.
Session Number: 225
Session Number: 225
Abstract Title:
Molecular Detection of Bacteria by Real-Time Pcr in Human Cardiac Valves
F. Tuon, V. Ribeiro, L. Kraft, L. Wollmann, M. Pilonetto, F. Costa, P. Suss; Pontificia Univ.e Catolica do
Paraná, Curitiba, Brazil
Abstract Body:
Background: The Human Tissue Bank (HTB) of Pontifícia Universidade Católica do Paraná is in charge of
collecting, transporting, processing, storing and distributing cardiovascular tissues for therapeutic use.
The microbiological control is essential to all steps, ensuring safety and quality of the tissues and
minimizing the risks of disease transmission. This study aimed to determine the sensitivity of a protocol
for bacterial detection in liquid media during all steps of tissue processing. Methods: Routine samples
from HTB without previous bacterial detection and samples artificially contaminated with Escherichia
coli (ATCC 25922) in concentrations from 10-1 up to 104 CFU/mL were collected from all the tissue
processing steps: Transport Solution (TS), in which the heart is transported (NaCl 0,9%), Pre-Antibiotics
Treatment Solution (PRE), Post-Antibiotics Treatment Solution (POS) and Washing Solution (WS). Ten
microliters from all the solutions were spiked in Tryptic Soy Broth, Thioglycollate Broth and Sabouraud
Broth and 1.5 mL of each broth was immediately aliquoted for DNA extraction, which was performed
using a sequence of lysis buffers and QIAamp DNA Blood Mini Kit® (QIAGEN), according to a protocol
under development by the Carlos Chagas Institute (CCI) / Institute of Molecular Biology of Paraná
(IMBP). DNA was amplified using real time PCR targeting the 16S rDNA (7500 Real Time PCR System,
Applied Biosystems®) using a pair of universal primers for the 16S rDNA, a specific probe (FAM) and 16S
PCR Master Mix - IMBP® under development by CCI / IMBP. An amplification control (E. coli DNA at
1ng/μL, Microseq 500, Applied Biosystems®) in ultrapure water was used to generate a standard curve,
with a detection limit of 0.0001 ng/μl (R2 of 0.977), with a Ct (threshold cycle) between >16.71 and <
33.08. Results: Artificially contaminated Transport and Pre-Antibiotics Treatment solutions in
Thioglycollate broth showed a Ct between >25.31±0.05 < 30.39 ±0.03 and >23.72±0.03 < 29.03±0.06,
respectively. It was possible to detect 16S rDNA in all routine samples, observing a Ct between
>23.57±0.13 < 30.32±0.15 for ST, >24.48±0.28 < 26.10±0.22 for PRE, >23.89±0.04 < 25.68±0.10 at POS
and >24.49±0.06 < 31.05±0.21 at SL. Conclusions: These partial results demonstrated that this method
has a superior sensitivity in comparison to manual culturing methods described in the literature, a faster
turnaround time, and it could be applied for the detection of other microorganisms groups and in other
contexts.
Session Number: 225
Session Number: 225
Abstract Title:
A Gene Chip for Detecting Tuberculosisdrug Resistance Based on Armspolymerasechain Reaction(Pcr)-
Magnetic Bead Tag Array Platform
W. Wu, J. Xu, Z. Zhang; Southwest Hosp., Third Military Med. Univ., Chongqing, China
Abstract Body:
Background: There remains a large gap for the detection and treatment of global MDR-TB. We
developed the second generation of detection chip system for tuberculosis drug resistance based on
ARMS-PCR-magnetic bead Tag Array platform. The chip was used to detect plasmids of different
template concentration, and then analyzed for sensitivity and specificity. Drug resistance genes from
bone and joint tuberculosis clinical isolates were detected, giving comparative phenotypic resistance
results to explore the feasibility and value of clinical applications. Methods: Twenty-four strains of
Mycobacterium tuberculosis (MTB) having sequence differences in extracted plasmids of mutant strains
(each contains only one mutant strain per sample). The plasmid was diluted into different
concentrations, and then multiplex PCR amplification was performed to analyze the sensitivity and
specificity of the chip system. A total of 187 clinical isolates were collected from patients. Among them,
there were 50 strains of sensitive bacteria, and 137 strains of drug-resistant (including drug sensitivity
and sequencing results). Design, optimization and preparation of the chip detection system, sequencing
and phenotypic drug susceptibility results were analyzed to evaluate the sensitivity and specificity of the
gene chip. Results: In the template, concentrations of 1×103 copies/μL and above were consistent with
sequencing results in the mutant, but 29 mutant types gave false negative results in the 1x102 copies/μL
group. The detection of rifampin (RFP), Isoniazid (INH) fluoroquinolones (FQs), streptomycin (SM)
resistance genes was highly sensitive and specific: however, for detection of amikacin (AMK),
capreomycin (CPM), kanamycin (KM), specificity was higher, but sensitivity was lower. Sensitivity for the
detection of a mutation in the eis promoter region could be improved. The detection sensitivity of the
EMB resistance gene was low, therefore it is easy to miss a diagnosis of ethambutol (EMB) drug
resistance, but its specificity was high. Conclusions: Tag Array chip can achieve rapid, accurate and high-
throughput detection of tuberculosis resistance, which has important clinical significance and feasibility.
Session Number: 225
Session Number: 225
Abstract Title:
Tacking the Challenges to Translating the Use of Molecular Techniques Into the Diagnosis of Blood
Stream Infection
N. Trung; 108 Military Central Hosp., Hanoi, Viet Nam
Abstract Body:
Purpose: Blood culture based methods are widely employed to establish diagnostics of bloodstream
infection but it takes long time with low sensitivity. The molecular method possesses capacibility to
provide a quick result with better sensitivity. However, the presence of human DNA in huge junks can
hamper the PCR sensitivity in detecting microbes. Material & Methods: We used serial dilutions of E. coli
spiked pseudo-blood-sepsis samples to develop a simple method that combines the use of a polar
detergent solvent and adjustment of the basic pH to remove human DNA. Additionally, we optimized
the uses of dual-priming oligos into species specific and groups specific primers/probes of fourteen most
common sepsis causative pathogens. The developed method was further validated by real-time PCR on
blood samples from 110 “Sepsis-3 consensus” confirmed patients. Results: This newly developed
approach allows to remove up to 99% of human DNA from patients’ peripheral blood. The inhibitory
effect of human DNA is efficiently prevented that raises detection limit of RT-PCR to 1CFUs/ml. This cut-
off is 10 times better compared to conventional RT-PCR assays. The classical blood culture detected
35/110 (32%) of study cohort; whereas, the newly developed approach was able to provide correct
diagnoses in 62/110 (56.3%) of collected samples. The combination of two methods brings positive
diagnostics rate for our recruited patients of “Sepsis-3 consensus” up to 62%. Of the blood culture
confirmed positive, 32/35 (88.5%) cases showed fully overlap with result gained by our novel RT-PCR
assay. Conclusion: We report a simple optimized protocol for removal of human DNA from blood sepsis
samples as a pre-analytical tool to prepare DNA for subsequent optimized PCRs with optimized species
specific and groups specific primers/probes that helps to increase detection limits of PCR to 1 E. coli
CFUs/ml and significantly improve the diagnostic rate for patients of “Sepsis-3 consensus” up to 62%.
Session Number: 225
Session Number: 225
Abstract Title:
High Frequencies of Corynebacterium Striatum and Finegoldia Magna Detected in the Chronic Wounds
by Using A Quantitative 16s Rrna Metagenomics Test
S. Yang1, S. Melanson2, T. Mittleman3, M. Vaughn4; 1Univ. of New Mexico, Albuquerque, NM, 2TriCore
Reference Lab., Albuquerque, NM, 3NextGen Lab. Inc., Newport Beach, CA, 4American Med.
Technologies Inc., Irvine, CA
Abstract Body:
Background: Chronic wounds, characterized by a polymicrobial microflora causing continuous
inflammation and tissue damage, and biofilm formation that delays healing, are a tremendous challenge
for testing and treatment. Microbiological culture is limited in providing useful information, due to a low
sensitivity and difficulties to isolate and quantify species in mixed culture. In this study, we utilized a
quantitative 16S rRNA metagenomics test to characterize and quantify the bacteria in chronic wounds.
Methods: Twenty four chronic wound samples were collected from several long-term-care facilities from
patients with leg ulcers, foot ulcers and pressure ulcers by using the Levine method. The total DNA was
extracted and the V1-V2 region of the 16S rRNA gene was amplified by PCR and sequenced by Ion
Torrent PGM. The sequences are then analyzed by a commercial software RipSeq. Results: A total of 55
bacterial species (29 anaerobes and 26 aerobes/facultative anaerobes) were detected from the 24
wounds with an average of 6.3 species in each wound. However, only 17 species were found from more
than 2 samples, with Corynebacterium Striatum (19/24, 79.2%) and Finegoldia magna (10/24, 41.7%)
found most frequently. Other frequent species detected were Anaerococcus spp. (33.3%), Pseudomonas
aeruginosa (29.2%), Staphylococcus aureus (29.2%), Bacteroides spp. (25.0%), Porphyromonas spp.
(25.0%), and Proteus mirabilis (25.0%). Anaerobes were found in 70.8% (17/24) of the wounds. The
combined abundance of anaerobes was high (mean = 41.3%, 86.1% - 1.2%), and was positively
correlated with the number of the anaerobic species detected in a sample (R² = 0.7533). The other 2
most abundant bacteria were also the 2 most pathogenic species: Pseudomonas aeruginosa (mean
abundance = 45.3%, 97.7% - 4.3%) and Staphylococcus aureus (mean abundance = 35.9%, 72.9% - 2.5%).
Corynebacterium Striatum (mean abundance = 23.7%, 75.8%- 1%) and Finegoldia magna (mean
abundance = 14.5%, 37.4% - 2.5%) were also found with relatively higher abundance. Discussion: We
discovered very high frequencies of Corynebacterium Striatum and Finegoldia magna, both of which had
rarely been reported in wound infections. The pathogenicity of these two species, and their capability of
forming biofilm in the chronic wound are unknown and require further investigation. But their high
frequencies and abundance suggest they may be important players in chronic wounds, and could
potentially be used as microbiological markers for chronic wound testing.
Session Number: 225
Session Number: 225
Abstract Title:
Evaluation of Analytical Reactivity and Limit of Detection of the Filmarray® Pneumonia Panel Plus for
Identification of Viruses, Bacteria, and Antimicrobial Resistance Genes from Lower Respiratory Tract
Samples
J. S. Arce1, M. Buccambuso1, T. Edwards1, M. Hockin1, C. Alberti-Segui2, C. Weber2, C. Dubost2, R.
Lems1, L. E. O'Connor1, J. Larsen1, J. Manwaring1, A. J. Fratto1, D. Abbott1, J. P. Southwick1, A. Judd1,
M. Brooks1, E. Amiott1; 1BioFire Diagnostics, Sal
Abstract Body:
Background: FilmArray Pneumonia Panel plus identifies viral and bacterial analytes as well as genetic
markers of antimicrobial resistance from lower respiratory tract specimens (bronchoalveolar lavage
(BAL) and sputum). Viruses and atypical bacteria are identified qualitatively, while potentially colonizing
bacteria are reported with estimates of relative abundance over a clinically relevant range, 10^4 to
≥10^7 copies/mL (1-log bins). Antimicrobial resistance genes are reported only when an applicable
bacterium is detected. Analytical studies were completed to establish performance characteristics,
including determination of the limit of detection (LoD) for viruses and atypical bacteria in BAL and
sputum matrices, and an assessment of analytical reactivity (inclusivity) for panel assays. Methods: LoD
was estimated via serial dilution and confirmed as the lowest concentration where reproducible
detection was observed (≥95% detection in at least 20 replicates per FilmArray® System) in both sample
types. For evaluation of analytical reactivity, approximately 350 isolates were tested at a concentration
near LoD or lowest reported bin level (10^4 copies/mL). Isolates tested were temporally and
geographically diverse, and included multiple species, serotypes, genotypes, and variants. All testing was
performed with Investigational Use Only pouches. Results: The confirmed LoD for viruses and atypical
bacteria ranges from 0.01 to 500 TCID50/mL (~100 to 10,000 copies/mL) and LoD is equivalent between
BAL and sputum matrices. Of the >150 viral and atypical bacterial isolates tested, all were detected
within 10× LoD, with >98% detected within 1-3× LoD. Similarly, bacterial detection and reported bin
levels were consistent with input concentrations and equivalent in both BAL and sputum matrices. For
the >195 bacterial isolates tested at 10^4 copies/mL, 99% were detected and >97% were reported with
accurate bin levels (within 1-log of the input concentration). Conclusions: The FilmArray Pneumonia
Panel plus is a sensitive multiplex sample-to-answer test for detection of viral and bacterial analytes
(with several markers of antimicrobial resistance) from complex lower respiratory specimens. Panel
assays are reactive with the currently known genetic diversity of potential pathogens and are capable of
reporting accurate estimates of nucleic acid abundance for a diverse collection of bacteria.
Session Number: 225
Session Number: 225
Abstract Title:
Clinical Evaluation of the Biofire Filmarray® Pneumonia Panel Plus
S. Kerr1, C. Graue1, K. Broadbent1, J-M. Balada-Llasat2, A. Carroll2, H. Stone2, O. Akerele2, B. Buchan3,
S. Windham3, A. Hopp3, S. Ronen3, R. Relich4, R. Buckner4, D. Warren4, R. Humphries5, S. Miller5, H.
Huse5, S. Chandrasekaran5, A. Leber6, K. Everhar
Abstract Body:
Background: The FilmArray Pneumonia Panel (BioFire Diagnostics, LLC) is a molecular diagnostic test for
rapid identification of bacterial and viral causes of lower respiratory tract infections (LRTI) using native
(unprocessed) sputum and bronchoalveolar lavage (BAL) specimens. The test detects 18 bacteria (15
with semi-quantitation), 9 viruses, and 7 antibiotic resistance markers. The FilmArray Pneumonia Panel
integrates nucleic acid extraction, reverse transcription, nested multiplex PCR amplification, and
automated analysis into one closed system that provides results in about one hour. Typical bacteria
associated with LRTI are reported semi-quantitatively over four 1-log bins centered on 10^4, 10^5, 10^6,
and ≥10^7 copies/mL. Here, we summarize the results of a multi-center evaluation conducted to
establish the clinical performance of an Investigational Use Only version of the assay. Methods: The
prospective clinical evaluation was performed at 8 U.S. sites. Residual specimens from standard of care
(SOC) testing were enrolled between October 2016 and July 2017. A total of 847 BAL (including mini-
BAL) and 836 sputum (including endotracheal aspirate) specimens were evaluated. Bacteria reported
with semi-quantitation were compared to quantitative bacterial culture as well as quantitative Next
Generation Sequencing (qNGS). Analytes reported qualitatively were compared to PCR and bi-
directional sequencing. Results: In a preliminary analysis of the results, the FilmArray Pneumonia Panel
demonstrated an overall positive and negative percent agreement (PPA/NPA) of 93.6% and 99.2%,
respectively, compared to the reference methods. For analytes reported with semi-quantitation, the
FilmArray Pneumonia Panel demonstrated essential agreement with the correct log bin value +/- 1 bin
97.7% of the time as compared to qNGS. For polymicrobial specimens, organisms were ranked in the
correct order by quantity 96.1% of the time compared to qNGS. Conclusions: The FilmArray Pneumonia
Panel is a sensitive and specific test for detection of pathogens in lower respiratory tract specimens. In
addition, the FilmArray Pneumonia Panel provides accurate semi-quantitative measure of 15 bacterial
analytes. Data presented are from assays that have not been cleared or approved for diagnostic use.
Session Number: 225
Session Number: 225
Abstract Title:
Molecular Prevalence of Pneumocystis Jirovecii in A Multi-Site Prospective Evaluation of the Filmarray
Pneumonia Panel
S. N. Naccache1, S. Kerr2, C. Graue2, J. Dien Bard1, K. Broadbent3, J-M. Balada-Llasat4, A. Carroll4, H.
Stone4, O. Akerele4, B. Buchan5, D. Mahmutoglu5, A. Hopp5, S. Ronen5, R. Relich6, R. Buckner6, D.
Warren6, R. Humphries7, S. Campeau7, H. Huse7, S. Ch
Abstract Body:
Background: Pneumocystis jirovecii (Pj) can cause severe respiratory disease in immunocompromised
patients that may be fatal if untreated. Conventional methods for Pj identification include microscopic
examination of cysts visualized with stains or immunofluorescent markers. Detection by molecular
methods offers increased sensitivity and may facilitate testing if provided in a commercially available
and easy-to-use system. Therefore, an assay to detect Pj was included in an Investigational Use Only
version of the FilmArray® Pneumonia Panel (Pneumo Panel; BioFire Diagnostics LLC), a multiplexed
molecular diagnostic test intended to aid lower respiratory tract infections (LRTI) diagnosis. We explore
Pj prevalence from a clinical evaluation of the panel and the implications of testing for Pj in a general
population of subjects with LRTI. Methods: A multi-center prospective clinical evaluation of the Pneumo
Panel was performed at 8 U.S. medical centers from Oct 2016 to July 2017, excluding patients with cystic
fibrosis or tuberculosis. 847 bronchoalveolar lavage and 836 sputum samples (including endotracheal
aspirates) were evaluated. Chart review was conducted for patient demographics, standard of care
(SOC) testing within 24 hours of specimen collection, diagnosis and treatment. Statistical analysis was
conducted using Spearman’s correlations and Fisher’s exact test. Results: The Pneumo Panel was
positive for Pj in 5.7% (96/1683) of subjects evaluated. 131 samples were tested for Pj by SOC methods
(31 by PCR; 100 by DFA). 10/96 Pneumo panel Pj positives were negative by SOC, and 1/96 was positive
by SOC PCR. The majority of Pj positives by Pneumo Panel (88.5%; 85/96) were from patients with no Pj
SOC testing. Pj prevalence did not correlate with study site, age, specimen type, season, underlying
chronic conditions, types of immunosuppression, antibiotic/steroid treatment, associated viral positivity
or bacterial burden (measured by the Pneumo Panel). There was no significant difference in Pj
prevalence based on immune status. Pj suspicion, or development of fungal pneumonia. Conclusions:
Rates of Pj detection in this study were lower than the 10-20% previously reported in the healthy
population. Since Pj was as prevalent in immunocompetent and immunocomprised patients, inclusion of
Pj on a multiplexed panel with indication for LRTI could result in positive detection lacking clinical
relevance. Therefore this analyte will not be included in further iterations of the Pneumo Panel.
Session Number: 225
Session Number: 225
Abstract Title:
Clinical Comparison of Multiplex Tem-Qpcr to Histopathology for Detection of Helicobacter Pylori
S. Brzezinski1, J. Noteboom2, D. Lazas3, E. Grigorenko1; 1Diatherix Eurofins, Huntsville, AL, 2AIG Res.
Services, Hermitage, TN, 3Associates in Gastroenterology, Hermitage, TN
Abstract Body:
Background: Helicobacter pylori is a fastidious human pathogen causing peptic ulcers and gastric cancer.
Early detection and eradication can relieve symptoms, and current diagnostic methods do not offer
antibiotic susceptibilities. Clarithromycin is an effective and commonly prescribed antibiotic for H. pylori
eradication, but resistance to clarithromycin is increasing worldwide. A multiplex PCR method was
developed to identify H. pylori and the presence of genetic determinants of clarithromycin resistance in
gastric biopsies. This study evaluated the clinical accuracy of a target enriched multiplex-qPCR
Helicobacter pylori panel (TEM-qPCR) compared to conventional histopathology. All biopsies were also
tested for presence of Epstein-Barr virus (EBV) by qPCR to assess correlation of this potentially
oncogenic virus with co-detection of H. pylori. Methods: Gastric biopsies were collected from
symptomatic patients (n=110, mean age 53, 57% female, 51% on acid lowering medications) and
submitted for histopathology and TEM-qPCR. The Diatherix TEM-qPCR panel tests for the urease gene
and point mutations conferring clarithromycin resistance. The limit of detection for the TEM-qPCR H.
pylori panel was determined to be less than 10 cfu/mL with no cross-reactivity observed when tested
with 90 off-target organisms. Results: H. pylori was detected by histopathology in 13/110 specimens and
by TEM-qPCR in 15/110 with 100% sensitivity and 98% specificity before discrepancy resolution.
Discrepancies were resolved as true positives using end-point PCR with a separate gene target and a
hybridization based detection. Of all samples with H. pylori detected, one was predicted to be a mixed
clarithromycin susceptible and resistant H. pylori population and three samples were predicted to be
clarithromycin resistant. EBV was detected in 25% of tested samples with 73% prevalence of EBV co-
detected with H. pylori compared to 20% prevalence of EBV in samples without H. pylori detected (P<
0.0001). Conclusions: In conclusion, the novel TEM-qPCR HP panel is more sensitive than histopathology
for detecting H. pylori in gastric biopsies and provides detection of the genetic determinants of
clarithromycin resistance. EBV detection in gastric mucosa was highly correlated to H. pylori presence.
This finding is significant as both pathogens have been shown to cause gastric cancer and may
potentiate each other’s carcinogenic effects.
Session Number: 225
Session Number: 225
Abstract Title:
Evaluation of An Algorithm for the Detection of Bacterial Vaginosis from Vaginal Swabs
M-H. Tremblay, S. Morasse; BD Diagnostic systems, Québec, QC, Canada
Abstract Body:
Introduction: Despite the increasing knowledge on the impact of a highly diverse vaginal flora and the
demonstrated advantages of the use of molecular techniques for the diagnosis of Bacterial Vaginosis
(BV), the number of accurate molecular diagnostic tools for BV is limited. The molecular-based algorithm
for the detection of BV embedded in the BD MAX™ Vaginal Panel (MVP), a new automated in vitro
diagnostic test based on real-time polymerase chain reaction (PCR) for the rapid detection of
Vagnitis/vagniosis, was validated using the results from symptomatic women in an effort to answer the
need for developing more accurate and efficient approaches. Methods: The performance of the BV
algorithm embedded in the MVP was validated with 1559 eligible symptomatic women of different
ethnicities. A performance comparison of the AmC versus the MVP results in women with NS of 0-3 or 7-
10 was made. Discrepant samples analysis was performed by comparison of Nugent sub scores for
Lactobacillus spp and G vaginalis morphotypes with the corresponding CT values obtained by the
multiplex PCR reaction. Results: Sensitivity and specificity were 90.5% (797/881) and 85.8% (582/678)
respectively relative to a combined AmC/NS reference method. When stratified, differences were
observed across ethno-racial groups. The lowest specificity 79.1% (223/282) was observed in Black
women who represented >50% of the studied population and had the highest BV prevalence (65.2%).
When compared to with NS of 0-3 or 7-10, MVP outperformed AmC in sensitivity (92.7% vs 82.0%) and
specificity (91.5% vs 90.6%). Ct values obtained for detection of G vaginalis and Lactobacillus spp
demonstrated a higher sensitivity of the molecular assay than the NS. Conclusions: The performance of
the molecular MVP was determined to be of clinical value as an aid in the diagnosis of BV. The
performance and differences observed among ethnicities were primarily attributable to the intrinsically
low sensitivity of AmC and NS combined with the likelihood of false positive results when considering
the NS sub-scores. These conclusions were supported by a discrepant analysis. The inaccuracy of these
methods became apparent when compared to a molecular approach that uses the current emerging
microbiome information regarding the pathophysiology of BV.
Session Number: 225
Session Number: 225
Abstract Title:
Development of An Algorithm for the Detection of Bacterial Vaginosis from Vaginal Swabs
S. Morasse, M-H. Tremblay; BD Diagnostic Systems, Quebec, QC, Canada
Abstract Body:
Introduction: Current methods for diagnosing bacterial vaginosis (BV), vulvovaginal candidiasis (VVC)
and trichomoniasis (TV) mostly rely on subjective assessments. Clinical Amsel criteria (AmC) and direct
Gram stain of vaginal discharges (Nugent Score (NS)) represent the currently established references for
diagnosing BV. There is a need for developing more accurate and efficient approaches. Materials and
Methods: A quantitative PCR-based algorithm for the detection of BV was developed using the results
from 828 symptomatic women with agreeing NS and AmC results. The agreement between both clinical
references was assessed with the simple concordance fraction and the Cohen's kappa statistic. Several
logistic regression models were then compared for their ability to correctly predict concurrent NS and
AmC results. The calculated scores along with the reference results were then used as inputs for receiver
operating characteristic (ROC) curve analyses. That allowed to determine the most predictive models as
well as liable threshold scores for the diagnosis of BV. Results: After excluding all samples yielding
indeterminate AmC results, the agreement between both references was found to be satisfactory as the
simple concordance fraction and the Cohen's kappa statistic were 83.7% and 0.668 respectively. It was
found that the most predictive models had to include the cycle threshold (CT) of each preselected BV
markers, i.e. Lactobacillus crispatus, Lactobacillus jensenii, Atopobium vaginae, Gardnerella vaginalis,
bacterial vaginosis-associated bacterium-2 (BVAB-2) and Megasphaera-1. To reach maximal
performance, it was also discovered that the final algorithm had to take into account whether several
markers were simultaneously detected or not. The ROC curve analyses established that the expected
clinical sensitivity and specificity of the best performing models should reach between 88.2-93% and
89.4-97.2%, depending on the composition of the vaginal microbiota, i.e. presence or absence of some
markers. Conclusions: The results demonstrated that the developed molecular-based algorithm for the
detection of BV should demonstrate good sensitivity and specificity for the detection of BV. This was
later confirmed during a multicenter, prospective clinical investigation across the US during which the
PCR algorithm (BD MAX™ MVP) was compared to the AmC and NS.
Session Number: 225
Session Number: 225
Abstract Title:
Comparison of the Xpert Xpress Strep A Assay with the Cobas Strep A Test for the Detection of Group A
Streptococcus (Gas)
J. L. Cimino, N. W. Dresser, A. J. Castro, S. A. Butt, K. A. Stellrecht; Albany Med. Ctr., Albany, NY
Abstract Body:
The bacteria Streptococcus pyogenes, or Group A strep (GAS), is a beta-hemolytic coccus that is also one
of the most common causes of acute pharyngitis. GAS pharyngitis effects patients of all ages but is most
common in children between ages 5 and 15, and it is primarily transmitted person-to-person. This
means that a rapid clinical diagnosis is essential not only to identify the organism and begin treating the
patient, but also to have the best chance at preventing the spread of the illness at home and at school. A
quick diagnosis can also prevent unnecessary antibiotic prescriptions, but these aforementioned
benefits of rapid testing only become a reality if this faster testing is also accurate in its result.
Advancements in molecular diagnostics have made it possible to identify this pathogen in under a half
hour with a high level of sensitivity, and this gives clinicians the ample time to help the patient and to
make the spread of the bacteria more difficult. In this study we compared the performance of two rapid
molecular test systems to identify GAS: Roche’s cobas Strep A on the cobas Liat system and Cepheid’s
Xpert Xpress Strep A on the GeneXpert Dx system. E-swab specimens in Liquid Amies were collected
from 195 patients with symptoms of pharyngitis at BayCare Laboratories and sent to our laboratory for
testing on both molecular systems. Both assays encountered invalid results. Although the GeneXpert
assay failed at a higher rate (3.6% vs 0.5%) it obtained results on all samples after retesting, as opposed
to the one invalid sample on the Roche system, which remained invalid after retesting. Of the remaining
194 specimens, both assays identified 35 positive GAS specimens. There were two discrepant
specimens, with one being positive on the GeneXpert only and the other positive on the Liat only.
Discordant samples were sequenced by Cepheid R&D which determined the sensitivity and specificity of
the Xpert system to be 100%, whereas the sensitivity and specificity of the Liat were 97% and 99%,
respectively. Both systems performed quite comparably, with one notable caveat being the Xpert’s run
time is nearly 10 minutes longer for a negative sample. However, for positive samples the Xpert’s Early
Assay Termination (EAT) can give results as soon as 18 minutes which is comparable to the cobas Liat’s
run time of 15 minutes. Overall, each system’s assay provides a rapid and highly sensitive test for the
identification of GAS, and this aspect of these assays can adequately provide clinicians with a tool for
quicker and better patient care.
Session Number: 225
Session Number: 225
Abstract Title:
Development of A Molecular Diagnostic Assay for Staphylococcus Epidermidis Detection in Skin and Soft
Tissue Infection
Y. WANG1, E. Baum-Jones1, Y. Li1, R. Schechter2, T. Moreno1; 1Millennium Hlth., San Diego, CA,
2Palomar Hlth., Escondido, CA
Abstract Body:
Background: A member of normal skin microbiota, Staphylococcus epidermidis, can be an important
opportunistic pathogen in skin and soft tissue infection (SSTI) or surgical site infection (SSI). It also serves
as a reservoir of staphylococcal cassette chromosomal mec (SCCmec) for horizontal transfer of the
methicillin resistance gene to S. aureus. Commercially available Taqman assays targeting the 16s rRNA
gene have failed to differentiate S. epidermidis from S. aureus, posing difficulties in determining the
actual infectious agent or carriage of the SCCmec. This prompted our efforts to develop a S. epidermidis-
specific assay for rapid and accurate detection of the organism. Methods: House-keeping genes, such as
rpoB or dnaJ, have historically been targets for genomic typing studies on Coagulase Negative
Staphylococcus (CoNS) and S. aureus. We designed a dnaJ-targeted Taqman assay and assessed its
performances with genomic DNA from 5 S. epidermidis isolates, 91 challenging isolates of various
species including 10 S. aureus and 8 other CoNS. Extracted microbial DNA of 112 clinical specimens from
54 subjects was also tested. Assay performance was further evaluated for concordance with results from
traditional culture approach and Sanger sequencing Results: The dnaJ-targeted assay demonstrated
100% accuracy, specificity and sensitivity when amplified with genomic DNA from 96 pure microbial
isolates of multiple sources. Remarkably, it had no cross-reactivity with any S. aureus or non-S.
epidermidis CoNS isolates tested. In clinical SSTI specimens, the assay showed 99% accuracy. The assay
successfully detected S. epidermidis from subjects whose culture results reported rare or moderate
presence of the organism. S. epidermidis was also identified in additional clinical specimens for which
culture results were negative; in these specimens, detection of S. epidermidis was also confirmed by
Sanger sequencing. Conclusions: We have developed a sensitive and specific assay for detection of S.
epidermidis from pure microbial isolates and natural polymicrobial infections. It is highly specific against
S. aureus and other CoNS. Its application can assist in accurate bacterial speciation in SSTI and provide
informative assignment on carriage of the mec cassette. The identification of S. epidermidis in SSTI
specimens which had negative culture results suggest that treatment may be enhanced by utilizing
targeted molecular testing.
Session Number: 225
Session Number: 225
Abstract Title:
Performance of the Verigene Gram-Positive Blood Culture Panel after Implementation in A Pediatric
Institution
C. Vareechon, J. Mestas, C. M. Polanco, J. Dien Bard; Children's Hosp. Los Angeles, Los Angeles, CA
Abstract Body:
Background: High accuracy of multiplexed molecular panels in organism identification and detection of
resistance markers directly from blood cultures (BC) is imperative since it allows for prompt
antimicrobial optimization prior to culture results. In this study, we describe our 5-year experience with
the Verigene Gram-positive blood culture panel (BC-GP, Luminex) in pediatric patients. Methods:
Patients with positive BCs for Gram-positive organisms (GPO) were tested on the BC-GP panel between
February 1st, 2013 and August 2nd, 2017. All positive BCs were subcultures to agar plates and colonial
morphology were examined with reflex to matrix-assisted laser desorption ionization-time of flight mass
spectrometry (MALDI-TOF MS). Antimicrobial susceptibility was performed on the BD Phoenix system.
Discordant results between mecA and oxacillin or cefoxitin MIC were confirmed by cefoxitin disk
diffusion method. Results: Over a five year period, 61,422 blood cultures were processed at CHLA with
2.7% (1634/61422) of positive BCs analyzed using the BC-GP panel. The BC-GP panel detected 1520 GPO
targets in 1516/1634 (92.8%) positive blood cultures tested. The assay yielded an average turnaround
time of 3 h for identification and detection of resistance markers. Staphylococcus epidermidis (485, SE)
was the most common GPO detected followed by Staphylococcus spp. (411), Staphylococcus aureus
(273, SA), Streptococcus spp. (205) and Enterococcus faecalis (85). The BC-GP failed to identify
organisms included in the panel in 38/1634 (2.3%) positive BCs with SE and S. salivarius as the top two
organisms not detected. 4.9% (80/1634) BCs tested were positive for GPOs not included in the panel.
For monomicrobial BCs, we observed 100% (494/494) and 99.5% (492/494) concordance to the genus
and species level, respectively. Compared with BD Phoenix susceptibility testing, the BC-GP had a 98.0%
(49/50) positive percent agreement (PPA) and 99.1% (224/226) negative percent agreement (NPA) for
methicillin-resistant SA (MRSA). PPA and NPA for methicillin-resistant SE (MRSE) was 99.4% (365/367)
and 98.4% (120/122), and vancomycin-resistant Enterococcus faecium (VRE) was 100%. No vancomycin-
resistant Enterococcus faecalis was identified during this time period. Conclusions: In our institution, the
BC-GP panel demonstrated excellent performance in the identification of GPOs from positive BCs.
Moreover, the assay accurately predicted antimicrobial resistance phenotypes for SA, SE, E. faecium,
and E. faecalis.
Session Number: 225
Session Number: 225
Abstract Title:
Performance of the Verigene® Gram-Negative Blood Culture Assay for the Detection Of P. Aeruginosa,
Acinetobacter Spp And Carbapenem Resistance
D. Smith, P. Pancholi, J. Balada Llasat; The Ohio State Univ. Wexner Med. Ctr., Columbus, OH
Abstract Body:
Background: Pseudomonas aeruginosa and Acinetobacter baumannii bacteremia are associated with
poor prognosis. Patients presenting with pneumonia, hematologic malignancy, solid tumor or health-
care-associated infections are at a higher risk. Molecular tests that supplement Gram stain results from
positive blood cultures (BC) provide rapid and specific organism information and antimicrobial
resistance data to guide therapy. Methods: We retrospectively evaluated cases of P. aeruginosa and
Acinetobacter spp. bacteremia over a 32-month period after implementing a microarray-based early
identification and resistance marker detection system (Verigene® BC-GN; Luminex). Identification of the
organism (P. aeruginosa, Acinetobacter spp) and carbapenem resistance markers (KPC, OXA, NDM, IMP
and VIM β-lactamases) by Verigene BC-GN were compared to culture with identification by MALDI-TOF
Mass Spectrometer (Bruker) and meropenem susceptibility (MicroScan, Beckman Coulter). Results: A
total of 179,294 BC were tested during this time with a positive rate of 6.44%. Of these 165 BC were
positive for P. aeruginosa and 67 for Acinetobacter spp. For P. aeruginosa, 20% (34/165) of the BC were
polymicrobial. The Verigene BC-GN detected P. aeruginosa in 88% of the BC, missing 20 BC that were
polymicrobial. Only 4 % (7/165) of the P. aeruginosa were resistant to meropenem, but were negative
for the resistance genes included in the Verigene BC-GN. Regarding Acinetobacter spp. 7.5% (5/67) of
the BC were polymicrobial. The Verigene BC-GN detected 95.5% (64/67) of the Acinetobacter spp (47 A.
baumannii-calcoaceticus, 17 Acinetobacter spp), but missed 3 BC that were polymicrobial. The Verigene
BC-GN detected the OXA gene in 85% (17/20) of meropenem Acinetobacter spp resistant strains.
Conclusions: The Verigene BC-GN detected 100% of monomicrobial P. aeruginosa and Acinetobacter spp
in the BC and 60% of polymicrobial BC. The OXA gene was the predominant marker of carbapenem
resistance in Acinetobacter spp and was useful for treatment management.
Session Number: 225
Session Number: 225
Abstract Title:
Evaluation of the Minion Nanopore Sequencing Device for Whole Genome Sequencing of Yersinia Pestis
A. S. Gargis1, B. Cherney1, A. Conley2, H. P. McLaughlin1, D. Sue1; 1CDC, Atlanta, GA, 2Applied
Bioinformatics Lab. (ABiL) IHRC Inc., Atlanta, GA
Abstract Body:
In the event of a plague outbreak, the detection of genetic engineering, virulence factors, and plasmids
in the implicated Yersinia pestis strain(s) will rely on high quality, whole genome sequencing (WGS). The
MinION (Oxford Nanopore Technologies) nanopore sequencing instrument produces long read WGS
data in minutes and allows for real-time data analysis. The portable instrument is deployable to public
health laboratories and on-site nanopore sequencing may reduce the time to results during a public
health emergency. DNA isolation kits (e.g. Epicentre MasterPure Complete DNA, QIAGEN QIAamp DNA
Blood Mini) are available to purify DNA from Y.pestis, but have not been evaluated for nanopore
sequencing. Purified DNA must first be prepared into sequencing libraries. The Rapid Sequencing Kit is
commercially available and requires a two-step, 10 minute protocol. A prototype Field Sequencing Kit
that does not require cold chain storage was pre-released to our laboratory for testing. We evaluated
DNA isolation and library preparation methods for Y.pestis to determine (1) DNA quantity and quality,
(2) if the purified DNA can be used for library preparation by the rapid and field kits, (3) the sequence
quality of MinION versus Illumina data, and (4) whether known virulence factors and plasmids can be
identified from Y.pestis strains. Both extraction methods yielded DNA of sufficient quantity and quality
for nanopore sequencing. WGS data generated by the Rapid and the Field Sequencing libraries were
comparable in quality (average base-call quality scores of 12, and read lengths from 5 to 11kb per run).
De novo assemblies (using Miniasm, Racon, and Nanopolish software) resulted in >99% genome
coverage per run. Large Y.pestis plasmids (>100 kb) were assembled, but plasmids smaller than 10 kb
were not. Mapping the MinION reads to a reference (A1122) sequence showed extensive insertions and
deletions (avg. 9,000 indels) per run, compared to an Illumina assembly (20 indels). Increased depth of
coverage did not improve the assembly quality. Indels were predominantly found in homopolymer
regions and open reading frame predictions could not be completed. While the nanopore sequencing
system could be rapidly deployed for long read sequencing, a comprehensive assessment of Y.pestis
virulence factors and plasmids from an unknown strain currently relies on mapping WGS data to a
known reference sequence. The nanopore sequencing system and WGS data quality must be improved
to produce WGS data with fewer errors to be useful for public health laboratory testing of Y.pestis.
Session Number: 225
Session Number: 225
Abstract Title:
Metagenomic Detection of Viral, Bacterial, and Parasitic Infections from Dried Blood Spots
L. M. Filkins1, K. Edes1, M. R. Couturier2, D. T. Leung1, R. Schlaberg2; 1Univ. of Utah, Salt Lake City, UT,
2ARUP Lab., Salt Lake City, UT
Abstract Body:
Background: Febrile illness in travelers and patients in remote areas may be due to diverse pathogens,
often with overlapping clinical presentation, requiring timely diagnoses and pathogen-specific
treatments. Medical care is frequently delayed by days or weeks in this population due to limited access
to quality health care facilities abroad, further challenging infectious disease diagnoses. Next generation
RNA sequencing (RNA-seq) from peripheral blood dried blood spots (DBS) enables hypothesis-free,
broad-spectrum testing for diverse pathogens from a single, low volume sample collected during the
acute stage of illness when sensitivity is generally greatest. We evaluated the analytic performance of
metagenomic RNA-seq for the detection of viral, parasitic, and bacterial infections from DBS.
Materials/Methods: Cultured Zika virus, P. falciparum, and Salmonella enterica were spiked into venous
blood from healthy human donors, serially diluted, spotted on Whatman 903 filter paper, and stored for
up to 10 weeks under dry conditions and room temperature. DBS were disrupted by bead beating and
total RNA extracted using the miRNeasy Micro Kit (Qiagen). cDNA libraries were prepared using KAPA
Stranded RNA-Seq Library Preparation Kit and sequenced on an Illumina NextSeq 500 instrument (5-14
million sequences/sample). Sequences were mapped to pathogen and human reference sequences
using Geneious software (Biomatters). Limit of detection was defined as the pathogen load required for
detection of at least one pathogen-derived read. Results: Equivalent pathogen detection was achieved
from DBS stored in ambient, dry temperatures for 3 days and 10 weeks demonstrating stability of total
RNA in these conditions. Zika virus, P. falciparum, and S. enterica were detected at a wide range of
concentrations from spiked DBS samples using RNA-seq. Preliminary limits of detection were
determined to be 2670 copies/ml for Zika virus, 4-110 copies/ml for P. falciparum, and 30-170 colony-
forming units/ml for S. enterica. Conclusions: DBS can be used for long-term preservation of RNA in dry,
ambient conditions, enabling simple patient self-collection and sample storage while traveling for
metagenomic pathogen detection. The impact of storage in heat and humidity on DBS stability is being
evaluated. These analytic performance studies demonstrate detection of representative parasitic and
viral pathogens at or below expected loads during acute illness. Additional optimization of library
preparation and sequencing may improve pathogen sensitivity.
Session Number: 225
Session Number: 225
Abstract Title:
Evaluation of A Rapid Highly Multiplexed Molecular Diagnostic Lower Respiratory Panel for Clin. Impact
and Antibiotic Stewardship
H. Mopuru, K. Powell, M. SIMS; Beaumont Hlth., Royal Oak, MI
Abstract Body:
Background: The Unyvero system is a rapid molecular diagnostics platform currently being reviewed for
IVD clearance by the FDA for a cartridge designed to detect lower respiratory tract (LRT) pathogens from
endotracheal aspirates and bronchoalveolar lavages (BAL). While there are clear benefits to a rapid and
sensitive diagnostic system, there is debate as to whether such a system would lead to overuse of broad
spectrum antibiotics versus improving stewardship. Methods: A retrospective chart review was
performed for the 442 patients included in the clinical trial of the Unyvero LRT cartridge. Culture results,
antibiotic treatment given, and outcomes were analyzed and a determination was made as to whether
knowing the results of the Unyvero test, which detects 20 pathogens and 16 antibiotic resistance genes,
would have led to no change in the antibiotics, a change favoring expanding the antibiotic coverage, a
change favoring stewardship by narrowing the antibiotic coverage, or a change that potentially missed a
pathogen detected on culture. Results: Of the 442 patient specimens the majority, 163 (37%) had a
negative result which agreed with culture results. 65 (15%) additional specimens had results favoring no
change in treatment, 91 (21%) had results favoring expanding the antibiotic treatment, 111 (25%) had
results favoring stewardship by narrowing the antibiotic treatment, 7 (1%) would have missed a
potential pathogen found on culture, and 5 (1%) did not have sufficient data to make a determination.
The difference in time to results from Unyvero to standard culture is >2 days (5 hours vs. 2.7 days).
Conclusions: The Unyvero system can significantly impact the time to optimization of antibiotics in
patients with lower respiratory tract infections in which an endotracheal aspirate or BAL is obtained. The
system had excellent agreement for negative cultures and could lead to both expansion and narrowing
to more appropriate antibiotics and the frequency in which a potential pathogen was missed was low
(1%). Thus the Unyvero platform and the LRT cartridge has significant potential to improve the
management of lower respiratory tract infections and can improve antibiotic stewardship at the same
time.
Session Number: 225
Session Number: 225
Abstract Title:
Implementation of the Abbott RealtiME Cmv Ivd Viral Load Assay At A Large Tertiary Care Med. Center
L. Rigali1, D. Lucic2, P. Pancholi1; 1The Ohio Univ. Wexner Med. Ctr., Columbus, OH, 2Abbott Molecular,
Des Plaines, IL
Abstract Body:
Background: Cytomegalovirus (CMV) viral load assays are the cornerstone for diagnosis and monitoring
of patients at risk for CMV disease. This study compared the performance characteristics and workflow
analysis of the Abbott RealTime CMV IVD (IVD) assay with analyte-specific reagents (ASR) manufactured
by artus Qiagen. Methods: IVD assay was ran using the m2000sp/rt platform while the ASR reagents
were used with EasyMag and ABI7500 instruments. RealTime CMV IVD viral load testing was carried out
per manufacturer’s package insert recommendations. Samples processed using EasyMag/ABI7500 were
extracted with 200ul of sample input volume with 11ul of eluate and 15ul of master mix reagents.
Acrometrix CMV linearity panel is used as the calibration material. IVD assay has a limit of detection of
31.2IU/ml and limit of quantitation of 50IU/ml. Validated limit of detection/quantitation for the ASR
assay was 69IU/ml. 219 clinical samples across the dynamic range were evaluated as part of the study
(52 negative samples, 93 samples <69IU/ml, 74 samples >69IU/ml). Agreement, bias, coefficient of
correlation were evaluated. Workflow analysis consisted of analysis of hands on time, walk away time
and saved labor days. Results: CMV was not detected by both assays in 90.4% (47/52) of negative
specimens. Additionally, the IVD assay detected 9.6% (5/52) of specimens of which 2/5 specimens were
quantitated by the IVD assay at log 1.99 and 2.14. This quantitation was above the LOQ of the ASR assay.
IVD assay quantitated 48% (45/93) of previously <69IU/ml specimens by the ASR assay. Quantitation
ranged between 1.72-2.42 log IU/ml. CMV viral load was quantitated by both assays in 74 specimens
(>69IU/ml) with a mean bias (Abbott-artus) of 0.3 log IU/ml (95% CI of mean 0.26, 0.33) with the
maximum bias not exceeding 0.62 log IU/ml and R2=0.981. Workflow comparison demonstrated that
the IVD assay had 70% fewer steps and 54% increase in walk away time and a savings of 15 labor days
per year when hands on time between methods was compared. Conclusion: The results between the
two methods were highly correlated and presented no challenge in implementation. Workflow
improvements have allowed us to reallocate resources and focus on other projects within the lab.
Session Number: 225
Session Number: 225
Abstract Title:
Semi-Quantitative Molecular Detection of 5 Bacterial Pathogens and 47 Antibiotic Resistance Genes in
Urine Specimens and Culture Isolates
G. Walker1, B. Dews1, K. Pitzer1, J. Quan1, S. G. Higgins1, N. Toraskar1, N. Whitfield1, B. K. Lopansri2, S.
Riedel3; 1OpGen Inc., Gaithersburg, MD, 2Intermountain Hlth.care, Murray, UT, 3Beth Israel Deaconess
Med. Ctr., Boston, MA
Abstract Body:
Background: Molecular tests rapidly detect bacteria in patient specimens but do not comprehensively
predict antibiotic resistance compared with identification and antibiotic susceptibility testing (ID/AST).
Materials/methods: We developed the Acuitas® AMR Gene Panel u5.47 for detection of Escherichia coli,
Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis and Enterococcus faecalis plus 47
antibiotic resistance genes. The molecular test can be used with statistical algorithms from the Acuitas
Lighthouse® Knowledgebase to predict phenotypic resistance to 17 antibiotics. The test uses automated
DNA extraction and real-time multiplex PCR to provide results in 2.5 hours from urine specimens or
bacterial isolates. We used the test to evaluate 50 remnant clinical urine specimens and 60 bacterial
isolates from the FDA-CDC Antimicrobial Resistance Isolate Bank. Results: E. coli, E. faecalis, K.
pneumoniae, P. mirabilis and P. aeruginosa were detected with decreasing prevalence in the urine
specimens with 30% exhibiting mixed infections. The molecular test identified urine pathogens with 96%
semi-quantitative consistency compared with urine culture. The test detected two resistance genes on
average per urine specimen including genes for aminoglycosides, cephalosporins, sulfonamides,
fluoroquinolones and trimethoprim/sulfamethoxazole. The Acuitas Lighthouse® Knowledgebase
accurately predicted phenotypic resistance for molecular results from urine (e.g., 95% accuracy for
ciprofloxacin resistance). Several resistance genes were detected per FDA/CDC isolate on average: E. coli
(8), K. pneumoniae (8), P. aeruginosa (4) and P. mirabilis (3) with decreasing prevalence of genes for
aminoglycosides, cephalosporins, sulfonamides, carbapenems, fluoroquinolones and
trimethoprim/sulfamethoxazole. The Acuitas Lighthouse® Knowledgebase predicted phenotypic
resistance with average accuracy of E. coli (94%), K. pneumoniae (86%) and P. aeruginosa (82%) across
gentamicin, tobramycin, ciprofloxacin, levofloxacin, trimethoprim/sulfamethoxazole, imipenem,
cefazolin, cefepime, cefotaxime, ceftazidime, ceftriaxone, ampicillin and aztreonam. Conclusions:
Acuitas AMR Gene Panel u5.47 rapidly detects 5 pathogens and 47 resistance genes in urine specimens
and culture isolates. The Acuitas Lighthouse® Knowledgebase predicts phenotypic resistance. Rapid and
accurate detection of antibiotic resistance can improve antibiotic therapy and outcomes in patients with
bacterial infections.
Session Number: 225
Session Number: 225
Abstract Title:
Development and Implementation of A Multiplex Vaginitis Ldt Panel Using the User Defined Workflow
(Udf) Software on the Cobas 4800 System
S. Salyers1, T. Boyle1, M. Sanford1, E. Polo2, E. Thuroff3, L. R. Buckner4, D. V. Baewer1, L. Picklesimer1;
1P and C Lab., Lexington, KY, 2TIB Mol Biol, Howell, NJ, 3TIB Mol Biol, Adelphia, NJ, 4LSU Hlth.Sci. Ctr.,
New Orleans, LA
Abstract Body:
Disease syndromes characterized by vaginal discharge, often associated with itching and odor, are
exceedingly common in both high- and low-risk US populations. Unfortunately, the accurate diagnosis of
vaginitis is complicated by the subjectivity of symptoms, non-specific clinical signs linked to specific
etiologies, and the lack of accurate and rapid diagnostic tools. We sought to develop a molecular
diagnostic test that targets the most common and clinically-actionable etiologies/biomarkers of
vaginitis, which included Candida albicans, Trichomonas vaginalis, and Gardnerella vaginalis.
Oligonucleotides for the detection of C. albicans, T. vaginalis, and G. vaginalis were developed by TIB
Mol Biol. Initial verification and testing was performed per the manufacturer’s instructions on the
cobas® 4800 System using the User Defined Workflow (UDF) software. With optimized thermal
parameters, all three analytes were run in parallel on the same amplification/detection plate. Our in-
house validation was conducted using both an analytical and method comparison approach, and
included ThinPrep PreservCyt and UniSwab specimens in order to integrate efficiently into our current
specimen workflow. Compared to a national reference lab, and with empirically-determined Cp cutoff
values, we observed overall percent agreements (OPA) of 89.0, 100.0, and 85.0 for Candida albicans,
Trichomonas vaginalis, and Gardnerella vaginalis, respectively. Kappa correlation coefficients yielded
qualitative agreements of ‘good’ or ‘very good’ for each analyte. Precision values were 100% for all
three analytes in 20 tests performed daily over a ten days span. Excellent analytical specificity was
observed as ascertained using ProBit analysis, and no interference was observed from a panel of
common interfering substances. Analytical specificity was confirmed by a lack of cross-reactivity to a
panel of organisms found in the lower urogenital tract. Using the cobas® 4800 UDF software, the newly-
implemented molecular panel is a rapid and reproducible solution to aid in the accurate diagnosis of
vaginitis.
Session Number: 225
Session Number: 225
Abstract Title:
Dried Blood Spot (Dbs) Sampling Technique for Diagnosis of Hbv and Hcv Through Serological and
Molecular Technique
S. Bibi1, T. R. Siddiqui2, S. E. Alam2; 1Pakistan Hlth.Res. Council, Karachi, Palau, 2Pakistan Hlth.Res.
Council, Karachi, Pakistan
Abstract Body:
Background: Hepatitis B&C are major health concerns in Pakistan. Being a developing country there is a
crucial need for affordable and yet reliable sampling method for Hepatitis B&C testing for
epidemiological studies. This study evaluated dried blood spot (DBS) sampling using conventional blood
sampling as gold standard. Methodology: Blood samples of 100 participants were collected by both
conventional and DBS method. These included twenty samples positive for each HBsAg, HBV DNA,
antiHCV and HCV RNA and 20 controls. Respective tests were run for each sample. ROC curve was
plotted to determine the ideal cut off points, sensitivity and specificity. Tests were repeated after 3 and
6-months to evaluate the effect of storage. Results: Sensitivity of DBS for antiHCV, HBsAg, HCV RNA and
HBV DNA was 95.2%, 95%, 80% and 70% respectively with 100% specificity suggesting a significant
correlation of DBS and conventional sampling (p-value: <0.01). Sensitivity of tests dropped with the
length of storage time which was more significant for HCV RNA with a drop in sensitivity to 30%.
Conclusion: DBS sampling correlates significantly with conventional blood sampling for serological and
molecular testing hence it may be considered for serological testing of HBV and HCV for epidemiological
surveys.<br /><p><a
href="http://files.abstractsonline.com/CTRL/ed/0/2b4/ab0/9e8/4ef/b8e/549/846/b41/db2/ae/g5400_1
src="http://files.abstractsonline.com/CTRL/ed/0/2b4/ab0/9e8/4ef/b8e/549/846/b41/db2/ae/g5400_1.j
pg" alt="" border="0" width="600" height="450" /></a></p><p><a
href="http://files.abstractsonline.com/CTRL/ed/0/2b4/ab0/9e8/4ef/b8e/549/846/b41/db2/ae/g5400_2
src="http://files.abstractsonline.com/CTRL/ed/0/2b4/ab0/9e8/4ef/b8e/549/846/b41/db2/ae/g5400_2.j
Session Number: 225
Session Number: 225
Abstract Title:
New Molecular Approach to Identify the Adherent-Invasive Escherichia coli (Aiec) Pathotype
C. Camprubí-Font, M. Lopez-Siles, M. Ferrer-Guixeras, L. Niubo-Carulla, C. Abellà-Ametller, L. Garcia-Gil,
M. Martinez-Medina; Univ. of Girona, Girona, Spain
Abstract Body:
Background: To date, no molecular tools are available to identify the adherent-invasive Escherichia coli
(AIEC) pathotype, which is associated to Crohn’s disease. Current techniques, based on phenotypic
screening of isolates, are non-standardisable and time consuming. We aimed to find new biomarkers for
AIEC identification by (i) searching for genes and Single Nucleotide Polymorphisms (SNPs) specific for
AIEC and (ii) determining their distribution in a diverse E. coli collection to assess their usefulness in AIEC
classification. Methods: The genomes of three AIEC/non-AIEC strain pairs displaying different phenotype
but identical pulsotype, and each pair belonging to a different phylogroup, were sequenced. By
comparative genomics, gene content and SNPs were analysed. Only non-synonymous SNPs present in
coding regions of an AIEC reference genome were selected. We performed Sanger sequencing to
confirm the presence of SNPs and to evaluate the distribution of the SNPs in a collection of 22 AIEC and
28 non-AIEC isolates. Nucleotides for each SNP were analysed considering AIEC phenotype, adhesion
and invasion indices, and suitability for AIEC screening was assessed. In this collection, point mutations
in, or prevalence of, previously AIEC-associated genes has also been examined. Results: Comparative
genomics described similar genome structures for the strains of the same pair. No differences in
previously reported AIEC-related genes (presence of genes and sequence variants) were observed
between AIEC/non-AIEC strain pairs. Amongst the six strains, 3327 gene clusters were detected and no
gene was found specifically in all AIEC strains. However, differential SNPs were found between strain
pairs; in total, 20 SNPs in D-phylogroup pair, 10 in the B2-pair, and 30 in the B1-pair met the selection
criteria. Of those, only 20 were confirmed by Sanger and were further analysed on the strain collection.
Differential nucleotide distribution between AIEC and non-AIEC strains (p<0.012) and also association
with adhesion and invasion strain capacities (p<0.006) was obtained for three SNPs. We designed a
classification algorithm based on the identification of the nucleotides present in three SNPs which
displayed 82.1% specificity, 86.4% sensitivity and 84.0% accuracy within our strain collection.
Conclusions: Our study corroborates the absence of AIEC-specific genetic markers widely distributed
across all AIEC strains. Nonetheless, SNPs putatively involved in the AIEC phenotype can be used for the
molecular identification of the AIEC pathotype.
Session Number: 225
Session Number: 225
Abstract Title:
A Molecular Diagnostic Approach for Pathogen Identification of Culture-Negative Cardiovascular
Implantable Electronic Device (Cied) Infection and Analysis of Methicillin Resistance in Staphylococcal
Cases
Z. Esquer Garrigos, J. Uhl, K. Greenwood, S. Cunningham, P. Vijayvargiya, R. Sohail, R. Patel; Mayo Clinic,
Rochester, Rochester, MN
Abstract Body:
Background: Cardiovascular implantable electronic device infections (CIEDI) carry significant morbidity,
mortality, and expense. In addition to device removal, management involves pathogen-directed therapy,
rendering identification of the associated organism(s) and determination of susceptibility important.
Conventional cultures fail to identify a pathogen in 50% of cases. We are developing a molecular
diagnostic approach using 16S ribosomal RNA (rRNA) gene PCR and sequencing of materials dislodged
from resected devices for pathogen identification of culture-negative CIEDI. Methods:
Vortexing/sonication of resected CIEDs in a salt solution was performed to dislodge bacterial biofilms,
yielding a specimen referred to as sonicate fluid. We tested 33 sonicate fluids stored at -80°C collected
from 01/2012 through 07/2017. CIEDI was ascertained using standardized clinical criteria. The infected
group was sub-classified into “culture-negative” and “culture-positive” based on conventional
laboratory results. To minimize the risk of exogenous DNA contamination and false positives, a DNA
extraction method using autoclaved reagents, and thermal and mechanical sample lysis was
performed.16S rRNA gene PCR targeting the V3-V4 hypervariable regions was employed. A positive
result corresponded to crossing points (Cps) <30 cycles, and a negative result to Cps >32 cycles. Cps of
30 to 32 were considered weakly positive. Positive specimens underwent Sanger sequencing, with
results analyzed using SmartGene Inc. software. mecA gene PCR was performed on samples in which
staphylococci were detected by 16S rRNA gene PCR and culture was positive for staphylococci, with
results compared those of phenotypic susceptibility testing. Results: We tested sonicate fluids from 11
CIEDI culture-positive and 22 CIEDI culture-negative cases. All 11 culture-positive samples were PCR
positive, with sequencing results recapitulating culture data. PCR was additionally positive or weakly
positive in 6/22 and 8/22 culture-negative CIEDI specimens, respectively. Sequencing of the 6 positive
specimens identified Mycobacterium abscessus, Finegoldia magna, Cutibacterium acnes (2), and
Staphylococcus species (2). mecA PCR was performed on 8 samples, 4 of which were positive, with 7/8
(87.5%) yielding results concordant with phenotypic susceptibility testing. Conclusions: Our results
demonstrate that molecular methods may be useful for detecting bacteria and resistance genes in
biofilms dislodged from the surfaces of resected CIEDs.
Session Number: 225
Session Number: 225
Abstract Title:
Comparison of Microbial Dna Enrichment and Extraction Kits Preparatory to Metagenomic Shotgun
Sequencing of Blood
P. Vijayvargiya, K. Greenwood Quaintance, S. Cunningham, M. Thoendel, Z. Esquer, A. Tande, R. Patel;
Mayo Clinic, Rochester, MN
Abstract Body:
Shotgun metagenomic sequencing is an agnostic approach for detection of microbial nucleic acids (NAs)
in clinical specimens. When NAs are extracted from human whole blood, the majority of sequencing
reads are of human origin, even in infected specimens. Human reads can be removed bioinformatically;
however, this requires deep sequencing to find microbial reads, and thereby engenders cost. Here, we
compared four commercial NA extraction and two microbial NA enrichment kits, selected based on their
compatibility with downstream next-generation sequencing, to determine which would provide the
highest yield of microbial NAs from human blood. MolYsis Complete (MC), AllPrep PowerViral DNA/RNA
(AP), ZymoBIOMICS DNA/RNA Mini (ZD) and Qiagen UCP Pathogen (QU) nucleic acid extraction kits were
tested. MC includes a built-in protocol for removal of human NAs. The other three kits were used in
combination with MolYsis Basic (MB) or NEBNext Microbiome DNA Enrichment (NN) kits for removal of
human NAs. Pseudomonas aeruginosa and Candida albicans were spiked together into 15ml EDTA blood
to final concentrations of 105 CFU/ml each. Aliquots of spiked and unspiked blood were stored at -80°C.
NAs were extracted using various combinations of extraction and enrichment kits (table).The quantity of
total extracted NAs was determined by Qubit HS DNA assay. Microbial NAs were quantitated using
quantitative PCR (qPCR), targeting the 16S rRNA gene for P. aeruginosa and ITS for C. albicans. PCR was
performed on a Roche LightCycler™ 1.0. Overall, MC, which provides both DNA extraction and
enrichment, provided the highest yield of microbial NAs of the seven extraction/enrichment protocols
studied for both organism-types (table).<table class="AbstractTable" id="{32D36E96-84F7-4F5B-A413-
8A10E47B764B}"><caption class="AbstractTableCaption"></caption><tr><td rowspan="1"
rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1">Kits used</td><td rowspan="1"
colspan="1">16S rDNA qPCR (crossing point)</td><td rowspan="1" colspan="1">ITS qPCR<br
/>(crossing point)</td><td rowspan="1" colspan="1">Qubit HS DNA amount (ng/mL)</td></tr><tr><td
rowspan="1" colspan="1">MC</td><td rowspan="1" colspan="1">16.76</td><td rowspan="1"
colspan="1">AP/MB</td><td rowspan="1" colspan="1">28.75</td><td rowspan="1"
colspan="1">ZD/MC</td><td rowspan="1" colspan="1">27.01</td><td rowspan="1"
colspan="1">16.71</td><td rowspan="1" colspan="1">148</td></tr><tr><td rowspan="1"
colspan="1">QU/MC</td><td rowspan="1" colspan="1">28.93</td><td rowspan="1"
colspan="1">AP/NN</td><td rowspan="1" colspan="1">18.28</td><td rowspan="1"
colspan="1">ZD/NN</td><td rowspan="1" colspan="1">19.57</td><td rowspan="1"
colspan="1">QU/NN</td><td rowspan="1" colspan="1">17.55</td><td rowspan="1"
colspan="1">18.75</td><td rowspan="1" colspan="1">12,580</td></tr></table>
Session Number: 225
Session Number: 225
Abstract Title:
Outstanding Abstract Award: Application of Next-Generation Sequencing in Identification of Pathogens
During Clin. Infectious Diseases, A Prospective Study
J-W. Ai, P. Cui, Y. Zhu, Y. Qian, Y. Qian, X. Zhou, Y. Ying, W. Zhang; Huashan Hosp. affiliated to Fudan
Univ., Shanghai, China
Abstract Body:
Background: The implementation of the NGS enabled a fast microbiological diagnostic method without
requiring a predefined range of suspicious pathogens [1-2]. This study aims to evaluate the diagnostic
efficacy of NGS during clinical approach to patients with suspected infections. Methods: Patients with
suspected infections were enrolled and high-throughput sequencing was performed on the collected
samples using BGISEQ-100 platform. Data was processed and mapped to the Microbial Genome
Databases after filtering low quality data and human reads. Results: We prospectively enrolled 1048
patients, including 234 suspected blood stream infections, 284 suspected respiratory infections, 312
suspected central nervous infections and 218 focal infections. The overall NGS sensitivity is 46.5%, while
blood stream, respiratory, focal, and central nervous system infection had a sensitivity of
78.72%,89.4%,96.4% and 19.4% respectively. Among which, NGS had a significantly higher sensitivity
than traditional laboratory method (46.5% vs. 32.4%) and the diagnostic sensitivity of CNS infection is
significantly lower than others (P<0.05). When combined with traditional laboratory methods, the
overall diagnostic sensitivity further increased to 54.3%. Among different pathogens, the diagnostic
sensitivity of protozoa and virus were the highest (100%, 11/11), followed by virus (69.5%, 168/242) and
bacteria( 74.29%, 351/473), while fungi had a lowest sensitivity of 33.3% (P value<0.05). Furthermore, in
blood stream infection, antibiotics application was found to associate with a decreased accumulative
sensitivity in both NGS and traditional culture (16.1% vs 15.2%, P>0.05)(Figure 1). Conclusions: This
study highlights the promising potential of NGS in rapid etiological diagnosis during clinical approach of
suspected infectious diseases.<br /><p><a
href="http://files.abstractsonline.com/CTRL/a2/5/a1d/ff0/a19/473/1a2/401/320/1d7/e57/49/g6817_1.
src="http://files.abstractsonline.com/CTRL/a2/5/a1d/ff0/a19/473/1a2/401/320/1d7/e57/49/g6817_1.j
Session Number: 225
Session Number: 225
Abstract Title:
High Volume Workflow and Performance Comparisons for Chlamydia Trachomatis and Neisseria
Gonorrhoeae Testing on the Cobas® 6800 Sys. and the Hologic Panther System
A. Frontzek1, G. Areztweiler1, D. Winkens1, D. Duncan2, E. Marino2, E. M. Marlowe2; 1Limbach Group,
Labor Mönchengladbach, Mönchengladbach, Germany, 2Roche Molecular Solutions, Pleasanton, CA
Abstract Body:
Background: High volume centralized testing has become the cornerstone of effective sexually
transmitted infection screening programs. Automated solutions, using a variety of urogenital specimen
types, can facilitate high throughput testing in the clinical laboratory, reduce workflow complexity,
address staffing needs, and improve turn-around-time. The purpose of this study was to evaluate the
performance and high volume workflow of Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG)
with the cobas® CT/NG assay for use on the cobas® 6800/8800 systems with the cobas p480 for pre-
analytics compared to the APTIMA Combo 2 assay on the Hologic Panther system. Methods:
Performance and workflow were evaluated using remnant de-identified urine specimens tested on both
platforms. Workflow was evaluated through interviews with laboratory personnel, observations and
detailed time studies using a protocol that simulated high volume testing with 376 specimens. Hands-on
time, number of manual interventions, time to first results and time to last results were determined. An
additional 230 specimens were tested for the performance evaluation and the results of 556 specimens
were eligible for correlation analysis. Results: Pre-analytical preparations and system start up on the
cobas® 6800 required 00:27:38 (hrs:min:sec) hands on time and the Panther required 00:30:43 hands on
time. After initial loading of specimens the cobas® 6800 required 8 interactions with 00:43:59 hands on
time to process the 376 samples and the Panther required 6 interactions with 00:39:10 hands on time.
Time to first results was 2:53:00 on cobas® 6800 for 96 samples (94 results, 2 controls) and 3:28:29 on
Panther for 5 samples (3 results, 2 controls). The cobas® 6800 delivered all 376 results 3 hours faster
than the Hologic Panther (7:43:31 and 10:47:30, respectively). The performance correlation between
both assays was comparable with an overall percent agreement of > 99% observed for both CT and NG.
Conclusion: For high volume automated CT/NG testing, the cobas® 6800 System with the cobas p480 for
pre-analytics provided accurate results with improved turn-around-time compared to the Panther. The
cobas® Systems would be able to provide 33% more results in the same time needed for Panther to
complete testing for 376 specimens. The additional testing capacity on the cobas® 6800 System would
allow a growing laboratory service to deliver more results in a single shift.
Session Number: 225
Session Number: 225
Abstract Title:
Use of Whole Genome Sequencing to Accurately Serotype Salmonella Ser. Thompson
E. A. Arnold, IV, C. G. Lane, A. C. Lauer, M. S. Van Duyne, P. I. Fields; Ctr. for Disease Control and
Prevention, Atlanta, GA
Abstract Body:
Salmonella serotyping continues to be an important subtyping method for epidemiological
characterization of strains in disease outbreaks. Identification of Salmonella serotypes is performed by
determining the somatic O and flagellar H antigens presented by the specific isolate. The different
serotypes are then named using the Kauffmann-White Scheme, which defines serotypes by their
subspecies and O and H antigens. Most Salmonella serotypes express two different flagellar antigens;
when one of these antigens is absent, the isolate is classified as monophasic and named using its
antigenic formula for surveillance purposes. For example, isolates belonging to serotype Thompson (I
6,7:k:1,5) could manifest as one of two distinct monophasic variants I 6,7:k:- or I 6,7:-:1,5. However, I
6,7:k:- could also be a monophasic variant of serotype Daytona (I 6,7:k:1,6) and I 6,7:-:1,5 could be a
monophasic variant of the common serotypes Infantis (I 6,7:r:1,5) and Bareilly (I 6,7:y:1,5), or rare but
clinically relevant serotypes Paratyphi C, Cholerasuis, and Typhisuis (I 6,7:c:1,5). Thus, when either of
those two variants are identified phenotypically they cannot be classified as a named serotype. It is also
a common problem for serotype Thompson to switch flagellar antigens rather slowly, making it difficult
to identify both phases phenotypically. This issue may cause the isolate to be misidentified as
monophasic. We investigated if advancements in genetic testing of isolates can help characterize
isolates initially identified as being monophasic. To do this we used eleven isolates in our collection that
had been sequenced and phenotypically identified with antigenic formulas of I 6,7:k:1,5; I 6,7:k:-; I 6,7:-
:1,5; or I Rough:k:1,5. All eleven of these sequences were analyzed using SeqSero and ten were
identified as serotype Thompson with detection of genes of both H-phases, and the one rough isolate
was identified as -:k:1,5. Also, eight of the eleven sequences, along with various other sequences not
identified as serotype Thompson or one of the monophasic variants, were analyzed in a whole genome
alignment tree produced using Harvest, and those eight isolates of interest clustered together. These
findings suggest that new genetic methods of testing may have more discriminatory power than
phenotypic testing alone.
Session Number: 225
Session Number: 225
Abstract Title:
Comparison of Cdna Library Construction Kits for Metatranscriptomic Analysis of Prosthetic Joint
Infection
T. L. Masters, C. Hilker, P. Jeraldo, A. Bhagwate, K. Greenwood-Quaintance, B. Eckloff, N. Chia, A.
Hanssen, M. Abdel, J. Yao, J. Jen, R. Patel; Mayo Clinic, Rochester, MN
Abstract Body:
Background: We are applying metatranscriptomics to materials dislodged from the surfaces of explanted
hip and knee arthroplasties (sonicate fluid) and to synovial fluid to study in vivo RNA expression of both
microbes and humans concomitantly to understand how they impact each other in driving pathogenesis
of prosthetic joint infection. An obstacle to this approach is the limited yield of total RNA in these
specimens, which hinders preparation of sufficient high quality cDNA libraries for sequencing. Here, we
present our experience optimizing cDNA library construction from sonicate and synovial fluids, to yield
informative sequencing data for both microbial and human RNAs. Methods: We compared two
approaches for cDNA library preparation; the Illumina TruSeq stranded total RNA (Illumina) and the
NuGEN Ovation SoLo RNA-Seq (NuGEN) systems. Sonicate fluid derived from the resected knee
arthroplasty and synovial fluid of the same subject who had Streptococcus mitis prosthetic joint
infection, were studied. Total RNA, including human and bacterial RNA, was extracted using the Qiagen
miRNeasy Serum/Plasma system and DNase treated prior to reverse transcription. The Illumina kit
requires 100 ng- 1 µg of total RNA which undergoes an rRNA depletion process prior to cDNA synthesis.
The NuGEN kit allows input RNA as low as 10 pg- 10 ng and incorporates an rRNA reduction step after
cDNA library preparation. The NuGEN SoLo cDNA libraries were sequenced on an Illumina HiSeq 2500
with 2 x 250 base reads in one lane and the Illumina TruSeq cDNA libraries were sequenced on an
Illumina HiSeq 4000 sequencer with 2 x 250 base reads in one lane. Rockhopper and MAP-RSeq were
utilized for RNA-Seq analysis of S. mitis and human RNAs, respectively. Results: Results are shown in the
table:<br /><table class="AbstractTable" id="{9BC081CD-DEC4-4E0C-AE06-6E2D3513FBFC}"><caption
colspan="1"></td></tr><tr><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="3">Sonicate
fluid</td><td rowspan="1" colspan="3">Synovial fluid</td></tr><tr><td rowspan="1"
colspan="2"></td><td rowspan="1" colspan="1">Illumina TruSeq stranded total RNA</td><td
rowspan="1" colspan="2">NuGEN Ovation SoLo RNA-Seq</td><td rowspan="1" colspan="1">Illumina
TruSeq stranded total RNA</td><td rowspan="1" colspan="1">NuGEN Ovation SoLo RNA-
Seq</td></tr><tr><td rowspan="1" colspan="2">cDNA library average concentration (ng/µl)</td><td
colspan="1">6</td><td rowspan="1" colspan="1">5</td></tr><tr><td rowspan="1" colspan="2">cDNA
library average size (bp)</td><td rowspan="1" colspan="1">302</td><td rowspan="1"
colspan="1">292</td></tr><tr><td rowspan="1" colspan="2">% Reads mapped to S. mitis</td><td
rowspan="1" colspan="1">0.78%</td><td rowspan="1" colspan="2">1.57%</td><td rowspan="1"
colspan="1">0.69%</td><td rowspan="1" colspan="1">1.98%</td></tr><tr><td rowspan="1"
colspan="2">Number of S. mitis non-rRNAs</td><td rowspan="1" colspan="1">6</td><td rowspan="1"
colspan="1">454</td></tr><tr><td rowspan="1" colspan="2">% Reads mapped to human
genome</td><td rowspan="1" colspan="1">86.6%</td><td rowspan="1" colspan="2">44.4%</td><td
rowspan="1" colspan="1">88.9%</td><td rowspan="1" colspan="1">43.7%</td></tr><tr><td
rowspan="1" colspan="2">% Human genes detected</td><td rowspan="1" colspan="1">15.1%</td><td
rowspan="1" colspan="2">10.2%</td><td rowspan="1" colspan="1">9.7%</td><td rowspan="1"
colspan="1">7.6%</td></tr></table><br />Conclusions: Our results suggested that to generate
informative sequencing data for both microbial and human RNAs, different strategies may need to be
carried out for cDNA library preparation from sonicate and synovial fluid. The NuGEN SoLo approach
produced more informative expressed bacterial RNA data whereas the Illumina stranded total was more
useful for analyzing human RNA expression.
Session Number: 225
Session Number: 225
Abstract Title:
Development of 16s Ribosomal Rna Reverse Transcriptase Real-Time Pcr for Salmonella Detection in
Blood Stored in Paxgene Blood Rna Tubes
J. Rutanga1, S. Van Puyvelde2, T. de Block2, J. Jacobs2, C. Muvunyi1, S. Deborggraeve2; 1Univ. of
Rwanda, Kigali, Rwanda, 2Inst. of Tropical Med., Antwerp, Belgium
Abstract Body:
Background: Bacterial bloodstream infections (BSIs) present a huge global burden. Molecular diagnostics
based on the polymerase chain reaction (PCR) have been developed for the diagnosis of BSIs but
generally show low sensitivity and reproducibility. Targeting the highly abundant 16S ribosomal RNA
(rRNA) molecules can increase the sensitivity of molecular detection of bacteria in blood and improve
the diagnosis of BSIs. However, there is no standardized protocol for field applicable blood collection for
subsequent bacterial RNA analysis. The PAXgene Blood collection system (PreAnalytix) is currently used
for storing up to 2.5 ml blood at -80°C for human RNA analysis, but has never been applied to bacteria.
Our aim was to investigate the efficiency and biosafety of storing Salmonella cells in PAXgene® Blood
RNA tubes and to develop a Salmonella specific 16S rRNA reverse transcriptase real-time PCR (RT-PCR)
for the detection of Salmonella in blood samples stored in PAXgene® Blood RNA tubes. Methods:
Experimental samples were prepared in PAXgene® Blood RNA tubes and PAXgene® Blood DNA tubes
containing 2.4 ml healthy human blood spiked with 100µl PBS containing known numbers of Salmonella
Typhimurium SL1344 cells, ranging from 10,000,000 to 1 cell per blood sample. PAXgene tubes were
stored overnight at -80°C and processed on the next day. The viability of the Salmonella bacteria after
storage in PAXgene tubes was assessed by microbiological viability assays. Total RNA and DNA were
extracted with the in house adapted protocols of the PAXgene® Blood RNA Kit and PAXgene® Blood DNA
Kit (PreAnalytix/Qiagen) respectively. A Salmonella specific 16S rRNA RT-PCR was developed with the
SensiFAST™ SYBR® No-ROX One-Step Kit (Bioline) on a Lightcycler 480 instrument (Roche). Results:
Resuspended cell pellets after storage in PAXgene tubes did not show growth on nutrient agar plates
indicating deactivation of Salmonella by the PAXgene tubes. Salmonella 16S rRNA could efficiently be
detected at lower detection limit of 1 Salmonella cells per 2.5 ml blood compared to 10,000 bacteria per
2.5 ml blood for rDNA detection. Conclusion: We showed that PAXgene® Blood RNA tubes are an
efficient and safe system to store Salmonella for subsequent extraction of total RNA, and we developed
a sensitive 16S rRNA RT-PCR assay for the detection of Salmonella in blood.
Session Number: 225
Session Number: 225
Abstract Title:
Comparison of A Novel Pcr Assay for Bordetella pertussis Detection with A Helicase-Based Assay
I. Harrold, W. Greene; PennState Hershey Med. Ctr., Hershey, PA
Abstract Body:
Background: Bordetella pertussis and parapertussis are gram negative coccobacilli that are responsible
for pertussis or whooping cough. Early detection of Bordetella is vital for proper treatment. Culture is
the gold standard, but can take 5-7 days for a result. Methods: Our institution currently uses the Quidel
AmpliVue Bordetella Assay to identify B. pertussis, and we compared AmpliVue results with the DiaSorin
Simplexa Bordetella Direct PCR based method. We ran 100 frozen specimens that were sent for B.
pertussis analysis over the past year. Each specimen was originally sent as a nasal swab in universal
transport media. Results: Of the 100 samples, 5 had originally been positive by the Quidel AmpliVue
Bordetella Assay method. These 5 samples were also positive on the Simplexa PCR assay. Three
additional specimens that were originally identified as negative by AmpliVue were identified as positive
by Simplexa. Two of specimens positive by PCR-only had Ct values of 29 and 33. The remaining positive
specimens had Ct values above 38 and did not repeat as positive on 2 additional runs. The remaining 92
specimens had negative results in both assays. For Simplexa, the hands-on time per sample run of eight
specimens was <10 minutes, and the turn-around-time from sample setup to run completion was
roughly 70 minutes. Conclusions: The new Simplexa Bordetella Direct PCR based assay had sensitivity
equal to or greater than the AmpliVue Bordetella assay for detection of B. pertussis. Of note but not
tested in this study, the Simplexa assay is also able to detect B. parapertussis. The hands-on time for the
new assay is less than AmpliVue, and the Simplexa assay can be set up and run in less than 10 minutes
hands-on-time. The previous assay took multiple steps with 10 minute incubation periods in between
steps. Overall the new Simplexa PCR assay demonstrated improved performance compared to the
Amplivue assay.
Session Number: 225
Session Number: 225
Abstract Title:
Comparison If Core- and Whole-Genome Multilocus Sequence Typing (Cgmlst and Wgmlst) of Legionella
pneumophila
S. A. Cunningham1, N. Zinsmaster1, D. Boxrud2, A. J. Taylor2, B. H. Raphael3, M. P. Murphy1, R. Patel1;
1Mayo Clinic, Rochester, MN, 2Minnesota Dept. of Hlth., St. Paul, MN, 3CDC, Atlanta, GA
Abstract Body:
Background: Whole-genome multilocus sequence typing has been previously reported to provide
advanced resolution in the investigation of L. pneumophila (LP) epidemic events. We studied a collection
of LP isolates, which had been characterized by wgMLST using Bionumerics v. 7.5 software at the
Centers for Disease Control and Prevention (CDC) and compared isolate clustering to that provided by
Ridom SeqSphere+ cgMLST. Methods: Twenty-seven isolates were studied, including eight from a recent
outbreak in the Twin Cities metro area, all provided by the Minnesota Department of Health (MDH).
Isolates were analyzed using a previously-described clinical microbiology laboratory developed
workflow. Sequencing was performed on the MiSeq platform utilizing 2 X 250 bp paired-end chemistry
with a maximum pooling capacity of 14 libraries per flow cell for targeted coverage of 150X. Reads were
processed for adapter and index removal, and imported into SeqSphere+ software. Velvet de novo
assembly and LP cgMLST were exercised within the software suite using default settings. Minimum
spanning trees were generated from the typing data table. Results of cgMLST typing were compared to
those of wgMLST typing. Results: wgMLST and cgMLST demonstrated topographically similar results
(Figure). Using a cut-off of 98%, wgMLST clustered 15 isolates into 3 genetically related groups, with the
remaining isolates being unrelated to the groups and differing from each other. cgMLST likewise
demonstrated the identical 3 groupings for the same 15 isolates. 13 of the 15 isolates grouped together
in their clusters with 0 to 3 allelic differences between members of their group, with two isolates
(C2015010756 and C2016008544) differing from members of their group by 15 and 24 allelic
differences, respectively. The remaining 12 isolates differed from each other and the clustered isolates
by ≥50 allelic differences. Conclusions: Clustering results of cgMLST typing of LP were similar to those of
wgMLST.<p><a
href="http://files.abstractsonline.com/CTRL/eb/7/9ad/e0d/9b4/4b4/087/bcd/29c/8e4/0d5/8b/g6812_1
src="http://files.abstractsonline.com/CTRL/eb/7/9ad/e0d/9b4/4b4/087/bcd/29c/8e4/0d5/8b/g6812_1.j
Session Number: 225
Session Number: 225
Abstract Title:
Validation of A Microbial Cell-Free Dna Sequencing Test for Infectious Disease
T. A. Blauwkamp1, S. A. Thair2, S. Yang2, M. Rosen1, L. Blair1, M. Linder1, I. Vilfan1, F. Christians1, T.
Kawli1, D. D. Hollemon1, H. Seng1, M. Vaughn1, S. Bercovici1, D. K. Hong1, J. C. Wilber1, M. Kertesz1;
1Karius, Redwood City, CA, 2Stanford Univ.,
Abstract Body:
Background: Sequencing microbial cell-free DNA from plasma offers a comprehensive, non-invasive
approach to identifying disease-causing pathogens. However, the validation of an open-ended
sequencing assay entails unique challenges. Here we describe the analytical and clinical validation of a
quantitative next-generation sequencing (NGS) test that detects microbial cell-free DNA (cfDNA) in
blood plasma from patients suspected of infectious disease. Methods: Analytical validation employed a
panel of thirteen representative microbes to characterize performance across a spectrum of potential
performance determinants, including GC-content, super-kingdom, presence as a commensal on the skin
or in the gut, extent of background contamination in reagents, genome length, and high genetic
similarity to other organisms. More than 500 samples containing sheared microbial DNA at various
concentrations and combinations, 1000 in silico generated data sets, and 120 asymptomatic volunteers
were analyzed. In addition, plasma from 350 patients meeting sepsis criteria in the emergency room
were collected, and the results of microbial cfDNA sequencing was compared to all microbiological
testing performed during the first week after enrollment. 30 samples from individuals with confirmed
CMV infection were also compared to quantitative CMV PCR results. Results: Analysis of the first 100
samples meeting SIRS criteria showed that microbial cfDNA sequencing of blood drawn at enrollment
identified 96% of microbes identified by blood culture (25/26), and 95% of all microbes identified
through all microbiological testing during the first week after enrollment (38/40). Microbial cfDNA
sequencing also showed 100% sensitivity compared to quantitative CMV PCR testing, with linear
regression of quantities detected by the two methods showing an R2 value of 0.977. Analytical
specificity determined using fifty well-characterized plasma aliquots was 96% on a per sample basis, and
>99.99% on a per analyte basis. The limit of detection and limit of quantitation of the assay were both
determined to be approximately 50 microbial cfDNA fragments/microliter plasma at average sequencing
depth, with the test exhibiting an average within-laboratory quantitative precision of 19.9% CV.
Conclusions: Analytical and Clinical validation demonstrates that this non-invasive microbial cfDNA
sequencing test is an accurate and precise way of identifying and quantifying pathogens.
Session Number: 225
Session Number: 225
Abstract Title:
Transition of A Manually-Performed Laboratory-Developed Test (Ldt) to the Fully Automated Cobas®
6800/8800 Sys. Using the Cobas® Omni Utility Channel
R. Hein, C. L. McGowin, S. Cagas, S. McCune, P. Rodriguez, S. Moseley, J. Osiecki, J. Engstrom-Melnyk;
Roche Diagnostic Corp., Indianapolis, IN
Abstract Body:
Background: Laboratory-developed tests (LDTs) remain an integral component of patient management
within US clinical laboratories. LDTs often afford rapid responses to outbreaks and emerging threats,
and therefore are important elements of laboratory medicine and public health. Unfortunately, LDTs are
often labor intensive and typically require several instruments to complete the testing workflow. As
diagnostic testing menus expand and complexity of laboratory workflow increases, the need for LDT
automation is unequivocal. The cobas® 6800/8800 Systems are fully automated, sample-to-result
platforms for routine or high-volume molecular testing that relies on TaqMan®-based PCR detection.
The cobas® omni Utility Channel (UC) facilitates the automation of user-defined PCR tests, including
sample pipetting from primary/secondary tubes, nucleic acid extraction, reaction setup and
amplification, and result reporting. Here, we demonstrate the transition of a manual Trichomonas
vaginalis (TV) LDT onto the cobas® 6800 System. Methods: Using the cobas omni Optimization kit (Opt
kit), initial assay optimization on a general purpose real-time PCR instrument was performed to scale-up
the established LDT formulation and to mimic UC conditions. Aliquots of de-identified, TV-positive
clinical specimens collected in PreservCyt® Solution were used to assess assay performance. Results: The
transition workflow, utilizing the Opt kit, demonstrated the impact of reaction volume scaling and
facilitated the optimization of an established TaqMan®-based T. vaginalis LDT prior to on-boarding onto
cobas® 6800 System using the UC. Evaluation of the amplification curves and Ct values across three runs
demonstrates that: (1) the enzyme included in the Opt kit is compatible with the unmodified primers
and probes; and (2) higher sample volume led to more comparable Ct values to the expected. Transition
of the LDT onto the cobas® 6800 System demonstrated acceptable amplification curve morphology and
expected Ct values for both T. vaginalis and the Internal Control. Conclusions: The two-phase transition
of LDT assays to the cobas® 6800/8800 Systems is facilitated by the use of the cobas omni Opt kit.
Ultimately, this approach allows for a primer design compatibility assessment and reagent volume/ratio
adjustment with the cobas omni Master Mix reagents, utilizing instrumentation currently on-hand
within clinical laboratories.
Session Number: 225
Session Number: 225
Abstract Title:
Comparison of Four Clia Waved Molecular Point of Care Testing Sys. for Influenza A, B, Rsv, and Group-A
Streptococci Detection
M. Noorbakhsh, B. Gsmalsed; Sutter Hlth.Shared Lab., Livermore, CA
Abstract Body:
Quick and reliable detection of major respiratory pathogens is emerging as a valuable infection control
and point of care (POC) tool. We compared four CLIA-waved POC molecular testing systems for their
performance and other characteristics that make them suitable for use in small clinics or urgent care
facilities. Patient specimens with known Influenza (Flu) A/B, RSV, and Group-A Streptococci (GAS) status
were used for this study. We also compared other factors important for POC testing such as level of
detection (LoD), testing turn-around-time (TAT), ease-of-use, cost of reagents, as well as instrument foot
print, price, and throughput. Methods: The molecular testing instruments are Alere-i (Abbott), Cobas
Liat (Roche), GeneXpert IV (Cepheid), and Solana (Quidel). Capability of detecting each respiratory
pathogens (Flu-A, Flu-B, RSV and GAS) were tested on 30 patient specimens with known results.
FilmArray was used as reference method for Flu A/B and RSV, culture method was used for GAS. All
discrepant results were verified by reviewing patients’ charts. Serial dilutions of commercial Flu A/B
positive control samples were used to determine the LoD of each testing systems. Instrument
dimensions, list price, were provided by the manufacturers or through their websites. The ease of use
and TAT were determined by testing personnel. Results: Percent agreements with the reference
methods and LoD is summarized in the following table. Conclusion: GeneXpert appeared to be the most
user friendly platform, which also provides random access modules for different tests. Liat has the
smallest footprint, and Alere-i showed the shortest TAT, and offered the lowest cost of supplies and
instrument. Although, Liat and GeneXpert IV demonstrated the highest result accuracy for testing Flu
A/B, RSV, GAS and lowest level of detection for Flu A/B, the major findings indicate all four platforms
perform acceptable accuracy required for point of care testing in detecting Flu A/B, RSV, and GAS.<br
/><p><a
href="http://files.abstractsonline.com/CTRL/b1/4/e12/d8b/aef/4d4/6b6/25a/c55/3ab/9c4/cc/g7286_2.
src="http://files.abstractsonline.com/CTRL/b1/4/e12/d8b/aef/4d4/6b6/25a/c55/3ab/9c4/cc/g7286_2.J
PG" alt="" border="0" /></a></p>
Session Number: 225
Session Number: 225
Abstract Title:
Comparison of Simplexa Flua_B & Rsv Direct, Filmarray Respiratory Panel V1.7, Prodesse Proflu+ Assay
and Laboratory-Developed Real-Time Pcr Assays for Detection of Influenza A, Influenza B and Rsv in
Pediatric Respiratory Specimens
H. Wang, R. Eltringham, D. Salamon, A. L. Leber; Nationwide Children's Hosp., Columbus, OH
Abstract Body:
Background: Rapid molecular detection of Influenza A, B and respiratory syncytial virus (RSV) is of great
clinical importance to guide therapy and cohort patients. We evaluated the clinical performance and
workflow of the Simplexa Flu A/B & RSV direct (Simplexa), FilmArray Respiratory panel v1.7 (FA),
Prodesse Proflu+ Assay (Proflu) and individual Laboratory developed real-time PCR assays (LDTs) for
detection of Influenza A, B and RSV. Methods: In total, 171 respirators samples (99% Nasopharyngeal
swabs) with standard of care respiratory testing ordered (FA) were selected based on the FA results in
addition to availability, integrity and specimen volume. Both known positive or negative specimens were
included. The residual specimens were tested on Simplexa, Proflu and LDTs. A true positive result was
defined as positive by at least 2 of 4 tests. The workflow for each test was analyzed based on availability
of two 3M Integrated Cyclers, 16 FilmArray instruments, two Cepheid SmartCycler II instruments and
three ABI thermocyclers. Results: The majority of the specimens were collected from patients < 18 yrs
(n=167, 97.7%) with an average age of 4.7yr. More male than female subjects were included (60.8% vs
39.2%). The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and
total agreement of each test for detection of each analyte are presented in Table 1 and workflow
parameters are presented in Table 2 below: <p><a
href="http://files.abstractsonline.com/CTRL/0c/4/bfe/86c/dd1/44a/a86/217/7a1/cb3/7cc/85/g4385_2.j
src="http://files.abstractsonline.com/CTRL/0c/4/bfe/86c/dd1/44a/a86/217/7a1/cb3/7cc/85/g4385_2.jp
g" alt="" border="0" width="600" height="713" /></a></p> Conclusions: There was variation in
performance characteristics depending on the analyte and the testing platform. FA, Simplexa and LDT
performed similarly for detection of Influenza A in pediatric specimens; however, FA and LDTs
demonstrated increased sensitivity for Influenza B and RSV compared to Simplexa and ProFlu. Simplexa
and FA require minimal hands-on-time and a shorter run time. The choice of testing platforms may
differ based on a laboratory’s patient population and resources.
Session Number: 282
Session Number: 282
Session Title: The Proof is in the Pattern: Epidemiology of Microbes
Abstract Title:
Molecular Epidemiological Investigation Revealed Serotype Switching Observed During Major Dengue
Outbreaks (2007-2015) in Nepal
S. Dumre1, R. Bhandari Dumre2, P. Chinnawirotpisan3, C. Klungthong3, G. Shakya4, B. Marasini5, S.
Shrestha6, P. Ghimire7, J. Karbwang8, K. Na-Bangchang9, S. Fernandez10, K. Hirayama8; 1Kantipur Coll.
of Med. Sci., Tribhuvan Univ., Kathmandu, Nepal, 2Sch.
Abstract Body:
Background: Global expansion of dengue virus (DENV) is a serious public health concern. Although DENV
was introduced in Nepal in 2006, its molecular epidemiology remains largely unclear. We uncovered it
through serotype/genotype analysis of circulating DENV strains. Methods: We employed the archived
serum samples (n = 972) collected during 2007-2015 from DENV infected individuals with linked clinical
and demographic data. Serotyping was performed by reverse transcription PCR. Complete envelope (E)
gene was amplified and sequenced for all four DENV serotypes from Nepal. Maximum-likelihood (M-L)
trees were constructed along with relevant global sequences from GenBank to understand the potential
origin of the Nepal viral strains. Results: During the study period, dengue affected at least 25 districts in
Nepal with an estimated case fatality rate of 1.5%. Majority were secondary infections (67%) and adults
(81%) with 13% severe manifestations. DENV was also detected in the hill districts suggesting their
expanding nature and possible adaptations towards higher altitudes (>1400 meters) in Nepal. All four
serotypes were confirmed; however, a clear serotype switching was observed during the major
outbreaks in the country. While DENV-3 was the major one in 2007-8, DENV-1 and -2 caused the large
outbreak in 2010-11 followed by a predominance of DENV-2 in 2013-14, and a re-emergence of DENV-1
in 2015. Nepal DENV 1 strains belonged to genotype-V and formed two distinct clades, which showed
spacio-temporal variation during these outbreaks. DENV 2, 3, and 4 strains were clustered into
cosmopolitan genotype, genotype-III, and genotypes-I/ IIB, respectively. M-L trees revealed that the vast
majority of Nepal DENV strains were closely related to India and contemporaneous Singapore strains.
India is the most plausible origin of Nepal DENV due to its physical proximity, extensive cross-border
activities (open border) and similar geo-climatic features supporting the phylogenetic relations.
Conclusions: DENV serotype switching occurred in Nepal during the major outbreaks. We have also
outlined genetic diversity of DENV serotypes in Nepal. This information is not only useful for Nepal in
epidemic preparedness but also aids in the understanding of evolutionary dynamics of DENV at regional
and global level. These findings also underscore the need of cross-border collaboration in dengue
control.
Session Number: 282
Session Number: 282
Abstract Title:
Molecular Epidemiology of Legionella pneumophila Isolates in Michigan
S. Altamimi1, M. Perri1, H. Misikir1, D. Vager1, S. McElmurry2, P. Kilgore2, M. Zervos1; 1Henry Ford
Hlth.System, Detroit, MI, 2Wayne State Univ., Detroit, MI
Abstract Body:
Background: Legionella pneumophila is a water borne pathogen that mainly affects the lungs among
other organs, causing a severe form of pneumonia. Little is known about molecular epidemiology.
Methods: Environmental Legionella samples were collected from home water throughout Flint and
Genesee County, Michigan (year 2016) and human samples were obtained from Michigan Department
of Health and Human Services (MDHHS) from patients in Oakland, Wayne and Genesee County,
Michigan. Water samples were originally cultured for Legionella using buffered charcoal yeast extract
agar (BCYE) and BCYE with polymyxin B, anisomycin and vancomycin (PAV), and all of the resulting
Legionella isolates were sub-cultured onto BCYE, and incubated as the water samples, for 48 hours in
humidified ambient conditions at 35 degrees °C. Technical assistance was obtained from the CDC for
processing water cultures. Pulsed-Field Gel Electrophoresis (PFGE) was performed to determine the
relatedness of the Legionella isolates. Genomic DNA was prepared and digested with SfiI (New England
BioLabs, Beverly, MA). Isolates were placed in the same PFGE strain group if their SfiI restriction patterns
were ≥ 80% similar by Dice co-efficient Results: There were 6 strain types that had more than one
isolate from different patients or the environment and patients. Among 18 environmental strains there
were 6 strain types and among 33 human isolates there were 14 strain types. Common strains were
seen in 8 strain types, the strains groups had up to 40 strains including 25 from humans and 15 from
environmental isolates. Three isolates were common to water and humans. Table 1 shows a summary of
findings. <table class="AbstractTable" id="{33FD0BAF-F065-4DE8-AE94-7BEA66005A8F}"><caption
colspan="1"></td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1">Strain
Type</td><td rowspan="1" colspan="1">Total no strains</td><td rowspan="1" colspan="1">Sample
source</td></tr><tr><td rowspan="1" colspan="1">1</td><td rowspan="1" colspan="1">12</td><td
rowspan="1" colspan="1">Environmental</td></tr><tr><td rowspan="1" colspan="1">7</td><td
rowspan="1" colspan="1">14</td><td rowspan="1" colspan="1">Human</td></tr><tr><td
colspan="1">Human, Environmental</td></tr><tr><td rowspan="1" colspan="1">18</td><td
rowspan="1" colspan="1">3</td><td rowspan="1" colspan="1">Human</td></tr><tr><td rowspan="1"
colspan="1">Environmental</td></tr><tr><td rowspan="1" colspan="1">8</td><td rowspan="1"
colspan="1">Human</td></tr><tr><td rowspan="1" colspan="1">19</td><td rowspan="1"
colspan="1">2,3,4</td><td rowspan="1" colspan="1">3</td><td rowspan="1"
colspan="1">Environmental</td></tr><tr><td rowspan="1"
colspan="1">10,11,12,13,14,15,16,17</td><td rowspan="1" colspan="1">8</td><td rowspan="1"
colspan="1">Human</td></tr></table> Conclusion: Common banding patterns between human and
the environmental isolates was rare, however did occur among the human strains and could be of use to
assist in epidemiologic investigation. Common strains among humans suggest a common source
including potentially from the environment. This study supports the need for obtaining culture for
Legionella from water sources and patients with clinical illness to better understand epidemiology of
this infection.
Session Number: 282
Session Number: 282
Abstract Title:
Molecular Epidemiology of Emergent Pathogens Associated with Canine Infectious Respiratory Diseases
in the United States
G. Maboni, A. Lorton, R. Berghaus, P. Bartlett, I. Fernandez, S. Sanchez; Univ. of Georgia, Athens, GA
Abstract Body:
Canine infectious respiratory disease (CIRD) is an endemic syndrome with multiple viral or bacterial
pathogens being involved sequentially or synergistically to cause disease1. The clinical signs caused by
the different agents are similar, which makes differential diagnosis challenging2. There is limited
information about the prevalence of pathogens associated with CIRD in the United States. To attain new
insights into the disease epidemiology, we conducted surveillance using molecular methods to target
the main viral and bacterial agents associated with CIRD in respiratory samples. Additionally, we aimed
to develop a novel probe-based multiplex qPCR to simultaneously detect and differentiate two species
of Mycoplasma (M. canis and M. cynos). Nasal, oropharyngeal, tracheal swabs, and lung tissues from
clinically ill dogs (n=562) were processed at Athens Veterinary Diagnostic Laboratory (University of
Georgia, USA) between 2011 and 2017. Canine Adenovirus 2 (CAdV-2), Canine Distemper Virus (CDV),
Canine Parainfluenza Virus (CPIV), Bordetella bronchiseptica, Coronavirus, Influenza, Streptococcus equi
subsp. zooepidemicus, M. canis and M. cynos were detected by standard or Real-Time PCR. For the
development of the multiplex qPCR for M. cynos and M. canis, primers and probes were designed
targeting species-specific gene regions identified in the 16S/23S rRNA intergenic spacer region. Results
revealed that CPIV (29%), M. canis (23.6%) and M. cynos (24.5%) were the most commonly detected
pathogens followed by Influenza (H3N2) (11.2%), B. bronchiseptica (9%), Coronavirus (4.6%), CAV
(2.5%), CDV (2%) and S. equi subsp. zooepidemicus (0%). Nasal-pharyngeal and oropharyngeal swabs
had the highest percentage of positive results. Co-infections occurred in 46 specimens, which were
positive for 2 to 5 different CIRD agents. The probe-based multiplex qPCR was successfully developed
and specifically detected M. cynos or/and M. canis. In summary, while confirming that CPIV is one of the
main pathogens associated with CIRD, this study highlights the role of newly emerging bacteria, such as
M. canis and M. cynos, as well as the importance of co-infections. These results may aid veterinarians to
adopt strategies to improve empiric antibiotic selection to treat CIRD. Further analysis will elucidate the
role of co-infections in clinically ill and asymptomatic dogs. Our novel multiplex qPCR for M. canis and
M. cynos provides an efficient diagnosis alternative that will allow for accurate disease therapy and
control.
Session Number: 282
Session Number: 282
Abstract Title:
Implementing A Multi-Pathogen Model for Forecasting New York Flu Season Using A Cloud Based
Epidemiological Network
J. Nawrocki, B. Galvin, J. Jones, A. Hoffee, L. Meyers; BioFire Diagnostics, LLC, Salt Lake City, UT
Abstract Body:
Background: Predicting onset and severity of influenza is of interest to healthcare professionals. Two
major factors in implementing effective flu season forecasting models include sourcing reliable data and
selecting the appropriated forecasting method. We seek to solve these problems by sourcing data from
a real-time epidemiological web tool, BioFire FilmArray® Trend, and use machine learning techniques to
build robust, multivariate time series models. FilmArray Trend is an epidemiologic surveillance system
that receives FilmArray® Respiratory Panel (RP) results from more than 20 clinical sites across the US,
with over 350,000 tests dating back to 2012. Our model, built to predict influenza A positivity is a novel
approach in that it uses multiple respiratory pathogens to make the predictions. We seek to apply our
model to forecast the 2017-2018 flu season in New York, and potentially improve the forecasts by
including environmental factors as model inputs. Methods: We used a long short term memory (LSTM)
neural network for predicting influenza A seasonality, 28 days in advance, in the United States using
FilmArray Trend data. The model utilized percent positivity of multiple pathogens since 2012 as inputs.
The 2016-2017 flu season was held out for validation. The most predicative combination of pathogens
were influenza A and human rhinovirus (HRV), while coronavirus contributed the least to predicting flu
seasonality. We will include regional environmental factors, such as weather and humidity, in order to
potentially increase the predictive power of the model. Results: We will apply our model to the 2017-
2018 flu season in New York as a regional validation, making predictions throughout the season. A
comparison of the predicted and actual flu season will be reported in spring, 2018 and compared to the
Center for Disease Control (CDC) influenza like illness (ILI). Conclusion: Our demonstration that influenza
A and HRV positivity rates can be used to forecast the 2016-2017 influenza A seasonality is promising. By
applying this model to an active flu season, we can better understand the accuracy and utility of the
model as well as potentially increase the effectiveness by including environmental factors.
Session Number: 282
Session Number: 282
Abstract Title:
Molecular Epidemiology of Pneumococcal Infections in Aged Patients At the Far East of Russia
A. V. Martynova; Far Eastern Federal Univ., Vladivostok, Russian Federation
Abstract Body:
Background: Pneumococcal infections remain major public health problem worldwide for aged
population.Aims: was to perform multilocus sequence typing (MLST) in strains of S.pneumoniae gained
in patients elder of 60 years at the territory of Primorsky region as the most southern part of the Russian
Far East. The strains were taken since Jan 2014 till Dec 2015. Methods: MLST was conducted with
housekeeping genes on standard method on recommendations of MC Enright (1998) et al. Results:there
were taken 50 isolates ( group 1, from patients with community-acquired pneumonia), 30 isolates (
group 2, from patients with invasive pneumococcal infections such as bacteriemia and menigitidis).
Isolates form 1st group included 24 serotypes ( 26 of non-typable) and 22 sequence types ( ST) according
to MLST of which 7 were novel, 8 were of Taiwanese clones (TW-28,19,26) of 19F serotype, 3 were of
R6, EU38, SP95. In group 2 of 20 strains there was revealed 8 serotypes ( 12 of non-typable) and 6 ST of
Taiwanese clones.The pattern of antimicrobial agents resistance from the strains of 2nd group was 35%
to erythromycin, 15% to tetracycline, 5% to fluoroquinolones. Conclusions:multilocus sequence typing
allows us to suggest the epidemiological significance of Taiwanese isolates of S.pneumoniae in our
region
Session Number: 282
Session Number: 282
Abstract Title:
Moderator
Lixia Liu; New Mexico Dept. of Health, Albuquerque, NM
Abstract Body:
Session Number: 290
Session Number: 290
Session Title: It's No Stretch of the Imagination: Reliable Colistin Antimicrobial Susceptibility Testing is
within Reach
Abstract Title:
Moderator
Eileen M. Burd; Emory Univ. Sch. of Med., Atlanta, GA
Abstract Body:
Session Number: 290
Session Number: 290
within Reach
Abstract Title:
Evaluation of Rapid Polymyxin NP Test for Early Detection of Colistin Nonsusceptibility in Resource
limited setting
P. Gupta, S. Sengupta, A. Kaur; Medanta, The Medicity, Gurugram, India
Abstract Body:
Background<br />India has 2nd highest prevalence (57%) of carbapenem resistance among Klebsiella
pneumoniae, leading to increased use of colistin. This has led to raised colistin resistance and pan drug
resistant strains in healthcare. Challenges in polymyxin susceptibility reporting persist as CLSI guidelines
recommend broth microdilution method (BMD) as standard. This is laborious and time consuming for
routine clinical practice. In clinical laboratory, the methods usually adopted are agar diffusion (E test,
disc diffusion, and automated systems) which are unreliable. In this study, we aimed to evaluate rapid
polymyxin Nordman Poriel test (RPNPT) as a reliable, rapid method, comparing it with the routine
modalities of colistin susceptibility testing.<br />Method<br />160 non-duplicate clinical isolates of K.
pneumoniae with known colistin susceptibility (colistin susceptible = 100, colistin resistant =60)
recovered from blood cultures were tested by BMD, rapid polymyxin test (RPNPT), gradient agar
diffusion method (E strip), and automated method (Vitek2, bioMeriuex France). RPNPT method was
followed as described by Nordman et al. The isolates were incubated aerobically in test reagent
containing cation adjusted Muller Hinton broth, red phenol indicator, 10% (D+) - glucose and 5μg/ml
colistin sulfate for 4 hours. Colistin non-susceptibility was identified by color change from orange to
yellow.<br />Results <br />Considering BMD as the standard reference method, E test and Vitek2 failed
to demonstrate colistin non-susceptibility in 15 and 7 isolates respectively, whereas RPNPT failed to
demonstrate non-susceptibility in 3 isolates. The non-susceptibility was detected by RPNPT in as early as
2 hours whereas in other methods results were available in 12 to 24 hours.<br />Conclusion <br
/>Resource limited nations like India where colistin resistant organisms are acquiring the status of
endemicity in healthcare; implementation of RPNPT routinely can be a simple and practical solution. The
rapidity of the test result can contribute in preserving polymyxin as the “last resort” antibiotic. Clinicians
can be informed early, for institution of appropriate antimicrobial therapy contributing to stewardship
measures. The results also help in implementation of contact isolation measures by infection control
team, preventing the dissemination of these multi drug resistant bugs within the hospital settings.
Session Number: 290
Session Number: 290
within Reach
Abstract Title:
Performance of Microscan Colistin Well in Detecting Colistin Resistance in A Clin. Microbiology
Laboratory
A. Kim1, J. D. Lutgring2, M. Karlsson2, D. Campbell2, A. C. Brown2, E. M. Burd3; 1CFD Res. Corp.,
Huntsville, AL, 2CDC, Atlanta, GA, 3Emory Univ. Dept. of Pathology and Lab. Med., Atlanta, GA
Abstract Body:
Background: The prevalence of antimicrobial resistance is increasing, which has led to a renewed
interest in using colistin as a therapeutic option. The emergence of the plasmid-mediated mcr gene
threatens the utility of colistin. Clinical microbiology laboratories need to be able to detect colistin
resistance but options are limited. This study evaluated whether growth in the MicroScan colistin well
was an accurate indicator of colistin resistance compared with either reference broth microdilution
(BMD) or gradient diffusion strips (ETEST®). Methods: At a single institution, from May 1, 2017 to
October 25, 2017, all Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii with
growth in the MicroScan colistin well were included in the study. Each isolate was retested at the CDC
using BMD, ETEST®, and examination of the colistin well (4 µg/ml) on a MicroScan Neg Breakpoint
Combo 44 Panel. All isolates displaying a colistin minimum inhibitory concentration (MIC) of ≥4 µg/ml by
BMD were considered resistant and evaluated for the presence of mcr-1 and -2 by real-time PCR.
Results: Of the 63 isolates that grew in the colistin well, we excluded 16 samples (25.4%) that were
polymicrobial (including an organism of interest and an organism intrinsically resistant to colistin) and 3
isolates (4.8%) that were misidentified (organism was intrinsically resistant to colistin), leaving 44
isolates for further evaluation. Reference BMD confirmed 41 of the 44 isolates (93.1%) to be colistin-
resistant, indicating that MicroScan overestimated colistin resistance in 3 instances (6.8%). Of the 41
isolates confirmed to be colistin-resistant by BMD, ETEST® detected resistance in 27 isolates (65.9%). All
41 isolates tested negative for the mcr gene by PCR. Conclusions: The MicroScan colistin well showed a
higher rate of categorical agreement than gradient diffusion strips compared to reference BMD.
Laboratories lacking capacity to perform colistin susceptibility testing by reference BMD may consider
MicroScan as an alternative tool to detect colistin-resistant bacteria.
Session Number: 290
Session Number: 290
within Reach
Abstract Title:
Evaluation of Polymyxin B and Colistin Antibiotic Susceptibility Testing Methods for Gram-Negative
Bacteria
A. E. Clark1, C. M. Crooks2, R. A. Weingarten1, J. P. Dekker1, K. M. Frank1; 1NIH, Bethesda, MD, 2Univ.
of Wisconsin-Madison, Madison, WI
Abstract Body:
Background: Antimicrobial susceptibility testing (AST) of polymyxin B and colistin is challenging due to
the physical properties of these compounds that interfere with testing. Accurate AST is critical, as
polymyxins are often last-line options for the treatment of patients with multidrug-resistant Gram-
negative infections. We evaluated the performance of the Sensititre® system (ThermoFisher) for routine
colistin and polymyxin B AST of Enterobacteriaceae and non-Enterobacteriaceae isolates. Methods: We
compared the performance of Sensititre® Gram-negative panels against agar dilution performed both in-
house and at a reference laboratory for colistin and polymyxin B AST. A collection of Gram-negative
clinical isolates with polymyxin minimum inhibitory concentrations (MICs) ranging from ≤0.25 to >4
µg/mL was used: A. baumannii (n=7), Achromobacter sp. (n=15), C. freundii complex (n=1), Enterobacter
sp. (n=14), E. coli (n=18), K. pneumoniae (n=27), Pseudomonas sp. (n=22), Pantoea sp. (n=1), R.
ornithinolytica (n=1), and S. maltophilia (n=2). Samples with discrepant MIC results were repeated, and
panels were manually evaluated for bacterial growth. Categorical agreement (CA) was calculated using a
breakpoint of S ≤2 and R >2 µg/mL. Agar dilution was performed in accordance with CLSI guidelines.
Results: A total of 108 Gram-negative isolates were tested. Of these isolates, 20% and 31% were called
resistant to polymyxin B and colistin respectively by the Sensititre® system. The Sensititre® CA was 85%
compared to polymyxin B agar dilution and 90% CA compared with colistin agar dilution results. Repeat
testing and manual evaluation of discrepant Sensititre® panels revealed the presence of skipped wells or
failure of the instrument to accurately detect bacterial growth. Conclusions: Polymyxin AST is
challenging, and although agar dilution appears to be the most reliable method, it remains too laborious
for most routine laboratories. Laboratories choosing to use the Sensititre® system for polymyxin AST
should consider manual evaluation of microdilution results due to the presence of skipped wells or
failure of the instrument to detect growth of some species.
Session Number: 290
Session Number: 290
within Reach
Abstract Title:
A Comparison of Piperacillin-Tazobactam and Colistin Sensitest Mic to Clsi Broth Microdilution Mic for
Gram Negative Challenge Strains
L. Koeth, J. DiFranco-Fisher; Lab. Specialists, Inc., Westlake, OH
Abstract Body:
Background: The Sensitest was recently developed by Liofilchem to provide a manual broth
microdilution option for MIC testing of single antimicrobial agents using a 32-well dried panel. The
piperacillin-tazobactam (P-T) Sensitest includes a wide range of concentrations (0.008-128 ug/mL),
which provides for testing of 2 isolates/panel. The colistin Sensitest is configured to test 4 isolates/panel
(4 rows each containing 0.25-16 ug/mL). There have been challenges in testing piperacillin-tazobactam
by some automated and gradient strip methods. There have also been difficulties in testing colistin by
gradient strip methods and an overall lack of testing methods available for colistin. This study was
performed as an initial evaluation at a single testing site using challenge organisms specific to each of
the two agents. Method: Each strain was tested once with frozen panels (CLSI reference) and by
Sensitest using the same inoculum in cation adjusted Mueller Hinton broth. Incubation conditions
(ambient at 33-37oC for 16-20 hrs) and reading of the MIC were similar for both methods. The strains
tested included A. baumanii, Enterobacteriaceae, P. aeruginosa and quality control strains. The colistin
challenge set consisted of 52 strains, which were from the CDC resistant collections and included
characterized strains with a range of colistin MIC results. The P-T challenge set consisted of 28 strains,
which were from the LSI collection and were chosen because the majority of MIC results were near the
breakpoints (between 16-≥128 µg/mL). Results: Colistin and P-T agreement rates and error rates are
shown in the table.<table class="AbstractTable" id="{A39E8003-160A-4C0F-91FB-
57848AEF1602}"><caption class="AbstractTableCaption"></caption><tr><td rowspan="1"
colspan="1">Antimicrobial Agent</td><td rowspan="1" colspan="1">n</td><td rowspan="1"
colspan="1">EA</td><td rowspan="1" colspan="1">CA</td><td rowspan="1" colspan="1">Minor Error
Rate</td></tr><tr><td rowspan="1" colspan="1">Colistin</td><td rowspan="1"
colspan="1">52</td><td rowspan="1" colspan="1">98.1%</td><td rowspan="1"
colspan="1">100%</td><td rowspan="1" colspan="1">NA</td></tr><tr><td rowspan="1"
colspan="1">Piperacillin/tazobactam</td><td rowspan="1" colspan="1">28</td><td rowspan="1"
colspan="1">100%</td><td rowspan="1" colspan="1">75%</td><td rowspan="1"
colspan="1">25%</td></tr><tr><td rowspan="1" colspan="5">EA – essential agreement (within ± 1
dilution); CA – category agreement; NA – not applicable</td></tr></table> Although essential
agreement rate was 100% for P-T, the category agreement was 75% as a result of 7 strains with MICs
that differed by one dilution and were near the breakpoints. Conclusions: There was excellent
correlation of Sensitest and reference broth microdilution MIC results for both piperacillin-tazobactam
and colistin. Additional testing, with a larger number of isolates and testing sites, is warranted. The
Sensitest method is a simple MIC method that would provide an option, other than gradient diffusion,
for testing of a single agent as a supplement to a clinical laboratory’s automated system.
Session Number: 290
Session Number: 290
within Reach
Abstract Title:
Colistin Susceptibility Testing for Enterobacteriaceae by Broth Microdilution and Agar Dilution Vs. Rapid
Polymyxin Np
A. Bryson, P. Ramanan, P. Kohner, N. Cole, J. Uhl, R. Patel, A. Schuetz; Mayo Clinic, Rochester, MN
Abstract Body:
Background: Polymyxins, like colistin, serve as last resort antibiotics against multidrug resistant Gram-
negative bacteria; however, resistance to polymyxins among Enterobacteriaceae has been increasing
worldwide. Colistin resistance can arise though chromosomal mutations or plasmid-mediated
acquisition of mcr-1, mcr-2, or mcr-3. The Clinical and Laboratory Standards Institute (CLSI) established
epidemiological cutoff values (ECVs) for Enterobacteriaceae and colistin, but additional and faster
methods would be helpful to identify polymyxin resistance. Here we use 117 clinical Enterobacteriaceae
isolates to evaluate three methods of colistin testing: 1) broth microdilution (BMD), 2) agar dilution
(AD), and 3) rapid polymyxin NP (RPNP). We also used a laboratory developed test (LDT) PCR to detect
mcr-1 and mcr-2. Methods: We compared colistin MICs by AD and BMD for 117 Enterobacteriaceae
isolates from a variety of clinical sources. We included 24 different species with the most abundant
being Enterobacter cloacae complex (29.1% of all isolates), Escherichia coli (19.7%) and Klebsiella
pneumoniae complex (11.1%). Organisms with intrinsic resistance (e.g., Proteus mirabilis) made up 12%
of all isolates. BMD served as the reference method. MIC ≤2 µg/mL was wildtype (WT); MIC ≥4 µg/mL
was non-wild-type (NWT) as per CLSI ECVs. AD and BMD results were compared to RPNP with results
read at 2 hours. Molecular detection of mcr-1 and mcr-2 was performed by a LDT PCR. Results:
Categorical and essential agreement between AD and BMD was 98.0% and 67.3%, respectively. There
were no very major errors and major errors were 3.8% (2/53). Sensitivity and specificity of RPNP were
90.7% and 96.2%, respectively, as compared to BMD. One E. coli isolate, which had an MIC of 4 µg/mL
(NWT) for both AD and BMD was positive for mcr-1. No isolates were positive for mcr-2. Excluding
organisms with intrinsic resistance, BMD categorized 39.8% of isolates as WT, 51.5% as NWT, and 8.7%
as indeterminate (due to skipped wells). E. cloacae complex isolates accounted for 89% of skipped wells
for BMD and 100% for AD. Conclusions: AD demonstrated an acceptable categorical agreement with
BMD of 98.0%, with no very major errors. The essential agreement was low (67.3%), and the major error
rate was 3.8% (2/53). The RPNP assay demonstrated adequate sensitivity and specificity, with same-day
results. Consistent with previously published literature, the majority of skipped wells occurred with E.
cloacae complex isolates. Only one isolate was mcr-1 positive.
Session Number: 290
Session Number: 290
within Reach
Abstract Title:
In Pursuit of the Holy Grail: the Colistin Np Test As A Rapid, Cost Effective and Reliable Screen for Colistin
Resistance
C. Kingsburgh, M. M. Kock, B. Mitton, N. M. Mbelle, K-A. Strydom; Natl. Hlth.Lab. Services, Univ. of
Pretoria, Pretoria, South Africa
Abstract Body:
Background: Colistin minimum inhibitory concentration (MIC) determination is associated with several
issues. Broth microdilution (BMD) is regarded as the gold standard, but requires expertise, is labour
intensive and cannot routinely be implemented in all settings. A need for accurate, rapid and user-
friendly methods of detecting colistin resistance exists, esp. in light of the emergence of plasmid
mediated colistin resistance mechanisms. In this study an in-house prepared colistin NP test was
compared against colistin BMD and Etest as a possible rapid screen for colistin resistance. Methods: A
total of 32 stored Enterobacteriaceae isolates were evaluated using colistin BMD, Etest (bioMèrieux)
and an in-house prepared colistin NP test. These isolates comprised of 25 E. coli (of which 17 confirmed
mcr-1 positive) isolates and 7 K. pneumoniae. The BMD was performed according to CLSI M07-A10 and
regarded as the gold standard. The in-house colistin NP test was prepared as previously described with
the modification of only adding colistin sulphate powder (Abtek Biological Ltd, UK). The Etest and BMD
results were interpreted according to EUCAST clinical breakpoints v.7.1. Results: Twenty-five (78%) of
the isolates tested resistant using BMD, with MICs ranging from 4 µg/mL to 64 µg/mL (average MIC 11
µg/mL). Seven of the isolates tested sensitive using BMD, with an average MIC of 0.25 µg/mL. The
essential and categorical agreement between Etest and BMD were both 94%. A 6% very major error rate
was detected with the Etest. Thirty-one (97%) of the isolates showed a categorical agreement between
BMD and the colistin NP. The colistin NP had one false negative result (3%), which was obtained from a
K. pneumoniae isolate with an unknown mechanism of resistance and a MIC of 8 µg/mL. The sensitivity,
specificity, positive, and negative predictive values of the Etest was 92%, 100%, 100%, and 78%, whilst it
was 96%, 100%, 100% and 88% for the colistin NP test. Conclusions: The colistin NP test is a rapid, user-
friendly, inexpensive test that shows potential as a screening assay for colistin resistance, esp. in
resource limited settings where access to BMD is often limited.
Session Number: 290
Session Number: 290
within Reach
Abstract Title:
Novel Media for Identification of Colistin Heteroresistant Enterobacter
D. Hufnagel, D. Weiss; Emory Univ., Atlanta, GA
Abstract Body:
Current trends in bacterial antibiotic resistance threaten multitudes of modern medical procedures from
transplants, to in-dwelling medical devices, to control of bacterial infections. The increasing frequency of
multi-drug resistant bacterial isolates has caused physicians and researchers to revisit the use of the
antibiotic colistin. Colistin is a cationic antimicrobial that binds and disrupts the negatively charged
membrane of Gram-negative bacteria. An issue with colistin is the inaccuracy and numerous problems
involved with susceptibility testing of isolates. Etest strips contain a gradient of antibiotic and are placed
on top of bacteria growing on an agar plate creating a halo if bacterial growth is inhibited. Etests are
currently not recommended for colistin susceptibility testing due to inaccurate results. A common
colistin resistance mechanism is to decorate the bacterial lipid A with positively charged residues
through the PhoPQ and PmrAB two component systems to repel the positively charged drug. The Weiss
lab has found that certain bacteria have a sub-population of resistant cells that can actively grow in the
presence of antibiotic, instead of the entire culture being recalcitrant to treatment, as occurs in classic
antibiotic resistance. This phenomenon, termed heteroresistance, is quite common in carbapenem-
resistant Enterobacteriaceae. I have designed and detailed a novel media termed DIAG that can not only
detect colistin resistant and susceptible strains, but also low frequency colistin heteroresistant
Enterobacter cloacae. Heteroresistance can cause antibiotic treatment failure in a mouse model of
sepsis, and certain heteroresistant isolates are undetected by susceptibility testing, showing the
importance for accurate identification of colistin heteroresistant isolates. DIAG contains decreased
cations and doesn’t inhibit the resistant sub-population of heteroresistant isolates. Detection of
heteroresistant isolates on DIAG is dependent on PhoPQ. Heteroresistant isolates that normally appear
as susceptible by Etest are able to grow alongside the Etest strip to high concentrations of antibiotic on
DIAG plates, accurately showing the underlying highly drug resistant subpopulation. DIAG holds great
promise for unearthing antibiotic resistant subpopulations and properly advising physicians on the
resistance profiles of bacteria.
Session Number: 290
Session Number: 290
within Reach
Abstract Title:
Performance of Colistin Mic Determination of the Accelerate Phenotest Bc Kit for Res. Use Only
J. Towne, R. Humphries; Accelerate Diagnostics, Tucson, AZ
Abstract Body:
Background: Susceptibility testing of colistin (CST) has been a challenge for laboratories due to several
technical issues, including variability of stock powders and solutions, the propensity for CST to adsorb to
plastics, and the absence of U.S. Food and Drug Administration-endorsed clinical breakpoints by which
to interpret CST minimum inhibitory concentrations (MIC). This study compared MIC agreement of the
Accelerate PhenoTest™ BC kit (AXDX) research-use-only (RUO) CST results to those of reference broth
microdilution (rBMD). CLSI CST breakpoints and epidemiological cut-off values (ECVs) were utilized to
interpret both rBMD and AXDX MICs. Methods: A challenge set (n=33) of Gram-negative bacteria used
previously by the CLSI to evaluate CST tests was assessed. This included 5 Acinetobacter baumannii
(ABA; 2 resistant), 10 Pseudomonas aeruginosa (PAE; 2 resistant) and 18 Enterobacteriaceae (8 non
wild-type by CLSI ECV, 4 with mcr-1 resistance gene). Isolates were seeded into BD BACTEC™ Aerobic
Plus bottles containing 10 mL of diagnostic human blood and bacteria at a concentration of ~50 clones
and incubated on the BD BACTEC™ FX until they flagged positive. In parallel, rBMD was performed as
defined by the CLSI M07-A4 standard. Essential agreement (EA; i.e., MIC +/- 1 log2 dilution) and
categorical agreement (CA) with CLSI breakpoints/ECVs was assessed. Results: Upon sub-culture from -
80oC frozen, 1 ABA and 1 PAE were found to no longer be CST resistant. Using results from parallel
testing, CST EA of AXDX with rBMD was 97.0% (32/33) and evaluable EA was 96.6% (28/29). One isolate,
an ABA with a rBMD MIC of 1 µg/mL and AXDX MIC of 4 µg/mL, was out of EA. This isolate had MICs
ranging from 1 to 2 µg/mL when tested previously. CA was 97.0% (32/33), with a sole major error (false
resistance) for the ABA which was out of EA. All 4 isolates (Escherichia coli) with mcr-1 yielded an MIC of
≥8 µg/mL by AXDX (non wild-type). Conclusions: The AXDX RUO CST test performed well when
compared to rBMD.
Session Number: 326
Session Number: 326
Session Title: Global Clinical Microbiology: It's A Small World After All
Abstract Title:
Novel Point-Of-Care Electricity-Free Centrifugation of Blood for Resource Challenged Diagnostic
Microbiology Lab. Using Ultra-Low Cost Hand-Powered Plastic/Paper Centrifuge
A. Gautam1, B. Subedi2, A. K. Gupta1, N. Koirala1; 1Dr.Koirala Res. Inst. for Biotechnology, Kathmandu,
Nepal, 2Pokhara Univ., Pokhara, Nepal
Abstract Body:
Background We are witnessing a great paradigm shift from central laboratory-based diagnosis to point-
of -care based diagnosis. The currently used centrifuges are heavy, large, expensive and impractical for
field clinics, with no electricity access. The design and fabrication of hand-powered centrifuge is based
on principle of an ancient whirligig string toy operated by spinning. We evaluated the electricity-free
centrifugation potential of the hand-powered centrifuge using human blood at point-of-care level.
Methods We designed and fabricated plastic/paper centrifuge of 12-cm diameter size. It was composed
of two circular 1-mm thick plastic/paper discs used for stationery purpose which was bound by an
adhesive tapes with two small holes in the center. Two sample capillary holders were glued horizontally
to the discs. An extra-strong 62-cm long fishing line was used for spinning. Phlebotomy was performed
aseptically during remote field clinics and 2-3 ml blood was transferred to small tube. Then it was placed
inside the sample holder and rotated by hand for 3-4 minutes. This resulted in a good separation of
plasma from the cellular blood components. Results We found that hand-powered centrifuge can
adequately separate the plasma from anti-coagulated blood within 2-3 minutes. The overall cost and
weight of centrifuge is $ 0.5 USD and about 25-30g respectively, which is more practical and cost-
effective than modern electric centrifuges. We were able to show good qualitative agreement in terms
of centrifugation and separation of pure plasma by the hand-powered centrifuge Conclusion Re-
purposing of a simple toy has resulted in a novel point-of-care electricity-free blood centrifuging device
for remote diagnostic laboratory. Additionally the device and methodology provides a practical
alternative when the serum or plasma is required for point-of-care field testing or analysis by diagnostic
kits and device (e.g. testing for a number of infectious diseases. <p><a
href="http://files.abstractsonline.com/CTRL/d1/c/6ae/f32/ddf/4bd/9ab/7b3/1fc/e48/3f8/0a/g2188_1.j
src="http://files.abstractsonline.com/CTRL/d1/c/6ae/f32/ddf/4bd/9ab/7b3/1fc/e48/3f8/0a/g2188_1.jp
Session Number: 326
Session Number: 326
Abstract Title:
Clinical Evaluation of A Rapid Blood Culture Identification Panel for the Diagnosis of Bacteremia among
Critically Ill Patients in Uganda
M. Workneh1, H. Kajumbula2, O. Abrahim1, F. Nalubega2, O. Mbabazi3, K. C. Carroll1, Y. C. Manabe1, S.
J. Reynolds4; 1Johns Hopkins, Baltimore, MD, 2Makerere Univ., Kampala, Uganda, 3Infectious Diseases
Inst., Kampala, Uganda, 4NIH, Bethesda, MD
Abstract Body:
Background In low-income settings like Uganda, limited availability of blood cultures and long turn-
around time limits ability to appropriately diagnose bacteremia. The Biofire™ FilmArray BCID panel is a
multiplex PCR system designed to identify 24 pathogens commonly causing bacteremia and 3 antibiotic
resistance genes. The goal of this study was to compare the FilmArray BCID to culture-based biochemical
methods for identification of organisms responsible for bacteremia. Methods Septic patients 18 years
and older were recruited from the Intensive Care Unit at Mulago Hospital and the Uganda Cancer
Institute in Kampala, Uganda. Two sets of blood cultures collected from independent venipuncture sites
were inoculated into BD Bactec bottles and loaded into the Bactec 9120 blood culture system
immediately upon delivery to the laboratory. Positive blood cultures were identified using Gram-stain
and routine biochemical tests. In addition, an aliquot was tested on the FilmArray BCID according to
manufacturer’s instructions. Results 73 patients were enrolled in the study between July and October
2017. 16 had positive blood cultures (22%). 13 were tested by the FilmArray BCID and all 16 samples
were tested by routine microbiologic methods. The majority of organisms were Gram-negative, 69% by
the FilmArray BCID and 64% by routine biochemical testing. Organisms identified included Acinetobacter
baumannii (n=4), Klebsiella pneumoniae (K.pneumoniae) (n=2), Escherichia coli (n=1), Pseudomonas
aeruginosa (n=1), Salmonella spp. (n=1), methicillin-resistant Staphylococcus aureus (n=1), Candida
tropicalis (n=1), Enterococcus spp. (n=1). The Salmonella isolate was identified as Enterobacteriaceae by
the FilmArray BCID. 1 of the 2 K.pneumoniae isolates was a metallo-beta-lactamase producer by
meropenem-EDTA combined disc test. All identified Enterobacteriaceae were KPC negative by the
FilmArray BCID. Polymicrobial growth was identified in 3 specimens by FilmArray BCID compared to in 1
specimen by routine methods. Conclusion These early results suggest increased prevalence of Gram-
negative organisms in this setting in contrast to multi-center evaluations in the United States where
majority of isolates are Gram-positive organisms. Relatively common organisms in this setting, such as
Salmonella spp. are only identified to the family level of Enterobacteriaceae by the FilmArray BCID.
FilmArray BCID may be able to identify polymicrobial growth more readily but misses non-KPC
carbapenemases that are more common in this setting.
Session Number: 326
Session Number: 326
Abstract Title:
Performance of Lab. Professionals Working on Malaria Microscopy in Tigray North Ethiopia
M. Alemu1, D. Tadesse2, T. Hailu2; 1Bahir Dar Univ., Bahir Dar, Ethiopia, 2Mekelle Univ., Mekelle,
Ethiopia
Abstract Body:
Background: Microscopic analysis of stained blood smear is the most suitable method of malaria
diagnosis. However, gaps were observed among clinical laboratory professionals in microscopic
diagnosis of malaria. Objective: To assess the performance of laboratory professionals on malaria
microscopy in Tigray, North Ethiopia. Methods: A cross sectional study was conducted in December
2015 among 46 clinical laboratory professionals. Data was collected via on-site assessment and panel
testing. The slide panel testing was composed of positive and negative slides. The kappa score was used
to estimate the agreement between participants and reference reader. Results: A total of 46 laboratory
professionals have taken part in this study. Most of the study participants were male gender (78%), in
the age of 20-30 years (65.2%) and served for less than five years in the health institutions (52.2%). The
overall sensitivity and specificity of laboratory professionals in detection of malaria parasites was 63%
and 95.7%, respectively. Out of the total 46 laboratory personnel involved in the study, only 1 (2.2%), 2
(4.3%) and 7(15.2%) of the participants correctly reported all the six, five and four slides, respectively.
The overall agreement between the study participants and the reference reader in malaria detection
was 79 % (kappa = 0.62). Participating in refresher training on malaria microscopy (AOR=7, CI= 1.5-36.3)
and malaria epidemic investigation (AOR= 4.1 CI= 1.1-14.5) had statistical significant association with
detection rate of malaria parasites. Conclusion: Clinical laboratory professionals showed low
performance in malaria microscopy. Most of the study participants were graded ‘in-training’ in
laboratory diagnosis of malaria. Table 1. Performance of laboratory personnel in identification of malaria
parasites, North Ethiopia 2016.<table class="AbstractTable" id="{9DA5CD8E-C75C-43A8-A2F6-
EC22A803B674}"><caption class="AbstractTableCaption"></caption><tr><td rowspan="1"
rowspan="1" colspan="2">Participant reader</td><td rowspan="1" colspan="2">Expert reader</td><td
rowspan="1" colspan="1">+ve</td><td rowspan="1" colspan="1">-ve</td><td rowspan="1"
colspan="1">Sensitivity</td><td rowspan="1" colspan="1">Specificity</td><td rowspan="1"
colspan="1">Agreement</td><td rowspan="1" colspan="1">Kappa</td></tr><tr><td rowspan="1"
colspan="2">Species identification</td><td rowspan="1" colspan="1"></td><td rowspan="1"
colspan="1">P. falciparum (n=92)</td><td rowspan="1" colspan="1">+ve</td><td rowspan="1"
rowspan="1" colspan="1">P. vivax (n=92)</td><td rowspan="1" colspan="1">+ve</td><td rowspan="1"
rowspan="1" colspan="1">Mixed infection<br />(n=46)</td><td rowspan="1" colspan="1">+ve</td><td
colspan="1"></td><td rowspan="1" colspan="1"></td><td rowspan="1"
colspan="1"></td></tr></table>
Session Number: 326
Session Number: 326
Abstract Title:
Poor Performance of Serology in Detection of Hepatitis C Virus in Blood Donors in Nigeria
N. F. Isong1, E. A. Ochang2, R. A. Bolarinwa3, A. O. Aboderin3; 1Univ. of Uyo Teaching Hosp., Uyo,
Nigeria, 2Univ. of Calabar Teaching Hosp., Calabar, Nigeria, 3Obafemi Awolowo Univ.Teaching Hosp.
Complex, Ile-Ife, Nigeria
Abstract Body:
Background: Sub-Saharan Africa has the highest estimated prevalence of hepatitis C virus infections.
Detection and donor blood screening continues to depend on serology which may miss several cases
and allow further transmission. We evaluated the serological and molecular prevalence and HCV
genotypes among blood donors in a tertiary hospital in South-West Nigeria. Methods: Study was cross
sectional and approved by the Obafemi Awolowo University Teaching Hospital (OAUTH) Ethics and
Research Committee (IRB/IEC/0004553). After initial screening for fitness to donate blood, blood
specimens were collected from all consenting donors. Demographic data and risk evaluation was done
using interviewer-administered questionnaire. Serum specimens were screened for anti-HCV antibody
using rapid test (Micro point Diagnostics,Nantong, China) and HCV antibody ELISA (Diagnostic Bioprobes,
Milano, Italy). Plasma specimens were screened for HCVRNA using Nested Reverse Transcriptase
Polymerase Chain Reaction (RT-PCR). RNA was extraction was done with ZR viral RNA kit while 5′
untranslated region of viral isolates were amplified using One Taq One Step RT-PCR and Quick load
Master Mix (New England Bio Labs Inc. Massachusetts). HCV positive samples were genotyped by direct
gene sequencing. Results: A total of 205 donors were recruited over a three month period. Twenty four
(11.7%) had detectable HCVRNA while 13 (6.3%) and 15 (7.3%) donors were positive for anti HCV by
rapid test and ELISA respectively. There was a significant difference between HCV detection by
molecular test and anti-HCV ELISA (F=3.213, p =0.031) as well as between molecular test and rapid test
(F-4.222, p <0.01). All HCVRNA were genotype 1 with 1b in 20 (83.3%) and 1a in four (16.3%) donors.
Significant risk factors for HCV infection were previous surgical procedures (x2=14.913, p<0.001) and
sexually transmitted infection (x2=4.930, p =0.026). Conclusion: Every ninth intending donor in this
environment is HCV infected and the current practice of serologic screening alone is allowing continued
transmission of HCV to recipients of blood and blood products. A review of National Guideline is
urgently required.
Session Number: 326
Session Number: 326
Abstract Title:
Magnetic Bead Assay A Rapid Cost Effective Approach for Diagnosis of Tuberculous Meningitis (Tbm) in
Low Resource Settings
K. Sharma, M. Modi, M. Sharma, A. Sharma, P. Ray; PGIMER, Chandigarh, India
Abstract Body:
Background: Rapid and specific diagnosis of tubercular meningitis (TBM) is of paramount importance to
decrease associated morbidity and mortality. Although there have been many recent advances in MTB
detection technologies, there still remains a major need to develop simpler point-of-care techniques. In
an effort towards such a diagnostic test for resource-poor settings, we have designed a test based on
detecting amplified DNA via magnetic beads bridging flocculation technique. The assay is inexpensive
and rapid (60 minutes), with a sensitivity approaching a single cell of Mycobacterium tuberculosis.
Methods: In the present study we evaluated magnetic beads bridging flocculation technique on 110
culture confirmed CSF samples and 200 clinically suspected TBM patients, who were culture and smear
negative but responded to ATT, in these patients composite reference standard as described in previous
studies were taken as reference for evaluation of test. We also evaluated this test from 100 non TB
control CSF samples. DNA extracted from H37RV was used as positive control. Test was also evaluated
with DNA extracted from various NTM and other bacteria for specificity. Results: Analytical sensitivity of
test is: single copy of M.tuberculosis . Analytical specificity: it is highly specific as there is no cross
reactivity with DNA extracted from various NTMs and other bacteria. In culture positive samples 104 out
of 110 (94.54%) were positive by this new test, in clinically suspected cases 179 out of 200 (89.5%) were
positive by this test. Specificity of the test was100% as it was negative in all the control patients and
DNA extracted from NTM and other bacteria. Conclusion: The assay is an affordable test (less than 1
dollar), with a sensitivity approaching a single cell of Mycobacterium tuberculosis. Magnetic bead assay
(MBA) is robust and cost effective method for diagnosis of TBM in low resource and high endemic
settings..
Session Number: 326
Session Number: 326
Abstract Title:
The Implementation of Diagnostic Microbiology Lab. in A Low Resource Setting, Experience from
Cambodia
J. Letchford, R. Martin, E. J. Baron, J. C. McLaughlin; Diagnostic Microbiol. Dev. Program, Los Altos, CA
Abstract Body:
Despite International Health Regulations 2005 and TB, Malaria and HIV programs, few diagnostic
microbiology laboratories exist in low resource settings (LRS) with the capacity for accurate results. We
describe the implementation of diagnostic bacteriology in Cambodia, using a balance of strengthening
and supportive activities. From 2008 to 2017, the Diagnostic Microbiology Development Program
(DMDP), in collaboration with Ministry of Health (MoH) and laboratory strengthening partners (LSP),
implemented diagnostic microbiology in five provincial hospital laboratories and strengthened three
national laboratories where a microbiology service was in place. We placed eight short term (3-6
months) and eight long-term (6-12 months) experienced expat mentors in laboratories to train more
than 40 government staff and eight Cambodian mentors. Cambodian mentors were recruited by DMDP
after graduating from the Technical School for Medical Care or University of Health Sciences. These
DMDP Cambodian mentors became the backbone of long-term onsite diagnostic strengthening. From
2013, we assisted Bureau of Medical Laboratory Services to strengthen the National Medical
Microbiology Laboratory Network by conducting quarterly meetings, encouraging sharing of experiences
and promoting continuous education. Other activities included implementation of a central media
production laboratory, review and feedback of monthly activity reports (MAR) and mentoring of hospital
doctors by DMDP expat and Cambodian doctors to improve awareness and understanding of a
diagnostic microbiology service. Government microbiology laboratories have increased from six in 2008
to 13 in 2017. Proficiency testing scores remain consistently above 85% and in 2016, laboratory
assessment scores of five laboratories ranged from 64% to 84%. Review of MARs led to development of
monthly cumulative pathogen reports and annual cumulative antibiograms allowing reporting of
pathogens (e.g., Burkholderia pseudomallei, Streptococcus suis) and resistance patterns (e.g., CRE, ESBL
and MRSA) to MoH. Laboratory strengthening requires expert mentoring for at least two years, group
meetings every quarter, close collaboration with international partners and fulltime in-country
leadership. The results of our collective efforts contribute to improved patient care and to national,
regional and global awareness of pathogen prevalence, emerging infectious disease and antimicrobial
resistance.
Session Number: 326
Session Number: 326
Abstract Title:
Malaria Diagnostic Practices in U.S. Laboratories, 2017
C. Prestel1, K. R. Tan2, F. Abanyie2, R. Jerris3, J. Gutman2; 1Emory Univ. Sch. of Med., Atlanta, GA,
2CDC, Atlanta, GA, 3Children's Hlth.care of Atlanta, Atlanta, GA
Abstract Body:
Background: In the United States (US), gold standard for malaria diagnosis is blood smear examination
by light microscopy; rapid diagnostic tests (RDTs) for malaria antigens must be confirmed with
microscopy. Given that malaria is not endemic in the US, the availability and quality of these diagnostic
tests may be limited, possibly leading to delays in diagnosis and treatment, and increased morbidity and
mortality. Methods: A nationwide convenience survey of United States laboratories was conducted from
June 26th to July 24th, 2017 to document challenges with malaria diagnosis. The survey, sent out via the
American Society for Microbiology listservs, explored malaria diagnostic test availability, techniques, and
reporting practices. Results were assessed using the Clinical and Laboratory Standards Institute (CLSI)
guidelines for malaria testing and reporting and compared to results from a similar 2010 survey (1).
Basic statistical analysis was performed using RStudio; Fisher exact test and chi-square test were used to
assess the significance of comparisons.Results: After excluding incomplete and duplicate responses,
results from 175 laboratories were included. Only 1% reported receiving no specimens, 31% reported
receiving 1-10 specimens for malaria per year, while 15% receive 100 or more. The majority (74%)
diagnosed five or fewer cases of malaria per year. Most (91%) had at least one type of malaria diagnostic
test available on-site; 90% of all surveyed laboratories performed blood smears on-site and 40%
performed RDTs on-site. Over half (63%) provided initial blood smear results within 4 hours, and 80%
provided complete results, including speciation, within 24 hours. Of those with RDTs on-site, 94% had
follow up testing with microscopy. Although diagnostic testing for malaria was available 24 hours a day,
seven days a week at 74% of responding laboratories (141), only 12% (17) met CLSI guidelines for
analysis and reporting of malaria testing. This rate of compliance with CLSI guidelines (12%) was higher
than reported in a similar survey in 2010 (3%; p < 0.05). Conclusion: The majority of laboratories
responding to this survey had the capability for timely diagnosis of malaria, however, few were in
compliance with CLSI guidelines. Inexperience might be a factor with this non-compliance; many
laboratories see few to no cases of malaria per year. Although reported adherence to CLSI guidelines
increased since the 2010 survey, updates or a review of standard operating procedures are needed to
improve laboratory compliance to CLSI guidance.
Session Number: 326
Session Number: 326
Abstract Title:
Endemic Malaria in West French Guiana: A 7 Years Retrospective Study
M-F. Manca1, A. Jollivet1, C. Kezza1, G. Carles1, C. Misslin-Tritsch1, A. Barrelet1, J. Clouzeau1, J. Miller2,
R. Boukhari1, P. Boex1, L. Musset3, J-F. Carod1; 1West French Guiana Hosp. Ctr., Saint-Laurent-du-
Maroni, French Guiana, 2Microbiol. Technical
Abstract Body:
Background: French Guiana is a French territory located in South America where malaria is endemic. The
laboratory of Western French Guiana Hospital also serves the general practitioners of the area and the
health centers of Western French Guiana. Until 2005, the majority of malaria cases in this area was due
to Plasmodium falciparum. Most cases are now due to P. vivax. The purpose of this study was to
evaluate and analyse the results of malaria diagnosis made by the laboratory of Western French Guiana
hospital between 2011 and 2017. Methods: Malaria was diagnosed using conventional microscopic
analysis by Giemsa staining thin and thick peripheral blood smears plus rapid diagnostic testing
(Immunochromatographic OptiMAL assay) (Biosynex SA, Illkirch,F rance). Since March 2017, LAMP
technology (Illumipro, Meridian Bioscience, Cincinnati, USA) replaced the thick smear. This retrospective
descriptive study was conducted using the laboratory database. The analyses were performed using
Stata®13.1. Results: Between the 1st of January, 2011 and the 18th of December, 2017, 14,476 samples
were examined. Among these, 217 were positive for malaria (1.5%, CI95% 1.3-1.7). The prevalence
remained stable between 2001 and 2016. In 2017, a significant increase was observed: 3% (p<0.05). The
majority of the positive cases were diagnosed in patients consulting at the emergency unit of the
Hospital (80.2%). The median age of malaria patients was 28 years old (IQR: 12) with no difference in
prevalence observed between female and male. No seasonal pattern was found. 47.5% of cases were
due to P. vivax, 41.9% to P. falciparum, and the prevalence of mixed P. falciparum and P. vivax infection
was 9.2%. P. malariae was found in 1.4% of the cases. The place of residence for all cases was along the
Maroni River, in French Guiana (n=54,27%) or Suriname 27% (n=58/217), respectively. of the positives
cases were located in Surinam (border city) and are all (100%) of Brazilian origin. 57% of the positives
cases from Saint-Laurent-du-Maroni were also Brazilian nationals. Conclusion: The analysis of the
laboratory database showed a stable occurence of infections mostly related to P. vivax but also
associated with P. falciparum. Most of the cases were related to Brazilian nationals. This may be
correlated to illegal gold mining activity which is mainly handled by Brazilians “garimpeiros”. The
implementation of Molecular biology, enhancing the sensitivity of the diagnosis, may play a role in
increasing the number of diagnosed cases.
Session Number: 326
Session Number: 326
Abstract Title:
Moderator
Jean-Francois Carod; West French Guiana Hosp. Ctr., Saint-Laurent-du-Maroni, French Guiana
Abstract Body:
Session Number: 346
Session Number: 346
Session Title: Here YEast, Here YEast!: Overcoming Challenges of Yeast Identification
Abstract Title:
Identification of Candida Glabrata Complex Species: Use of Vitek Ms Ruo and Clinprotools to Overcome
Limitations of the Vitek Ms Ivd and Bruker Biotyper Databases
X. Hou1, M. Xiao1, S. Chen2, F. Kong2, H. Wang1, X. Fan1, Y-C. Xu1; 1Peking Union Med. Coll. Hosp.,
Beijing, China, 2Univ. of Sydney, Sydney, Australia
Abstract Body:
Background: Candida glabrata species complex includes Candida glabrata sensu stricto and two major
cryptic species, Candida nivariensis and Candida bracarensis. Distinction of these species is relevant for
epidemiological purposes and for antifungal management. Materials/methods: In this study, two
commercial MALDI-TOF MS systems (Vitek MS system [bioMérieux] and the Bruker MS system [Bruker
Daltoniks]) employing the Vitek MS RUO and Bruker ClinProTools programs, respectively were evaluated
for the identification of 33 isolates of C. glabrata complex (17 C. glabrata, 14 C. nivarensis, 2 C.
bracarensis). Results: C. glabrata sensu stricto was identified correctly by both systems with distinct
principle components compared with the two cryptic species. All C. nivariensis and C. bracarensis could
not be identified to species level by the Vitek MS v2.0 IVD and Bruker Biotyper MS v3.1, but were all
correctly identified by the Vitek MS RUO. The generic algorithm model from ClinProTools software
showed 100% recognition capability and cross validation for the discrimination of C. nivariensis and C.
bracarensis. Spectra peak statistics revealed that five markers (3292.47Da, 4152.64Da, 6245.75Da,
6585.15Da and 7304.68Da) can reliably distinguish between C. nivariensis and C. bracarensis, with area
under the curve (AUC) values of 0.928, 0.9998, 1, 1 and 1, respectively. A small “in-house” Bruker
spectral database was established incorporating spectra of two clinical isolates representing C.
nivariensis and C. bracarensis identified in this study. After complementation with the “in-house”
database, all the remaining 14 C. nivariensis and C. bracarensis isolates were correctly identified to
species level (score >2.00). Conclusions: MALDI-TOF MS enabled rapid and reliable identification of C.
glabrata complex isolates. The use of the Vitek MS RUO system, Bruker ClinProTools software and the
addition of MSP to Bruker MS databases representing the local diversity of isolates assisted with
achieving differentiation of cryptic species within C. glabrata complex.
Session Number: 346
Session Number: 346
Abstract Title:
Growth of Candida Auris and Three Related Species in Aerobic Blood Culture Media and on Selective and
Differential Plated Media
A. Akinlade, R. Scott, L. King, P. Warns, L. Crowther, B. Morrow, V. White, R. Pfeltz; BD Life Sci., Sparks,
MD
Abstract Body:
Background: This seeded study provides specific growth media recovery data for the emerging,
antifungal-tolerant pathogen Candida auris (CA) and three phylogenetically related but less frequently
isolated Candida with which it may be misidentified. Methods: BACTEC™ plastic blood culture bottles of
Plus Aerobic/F (0, 3, 10 mL bagged blood), Peds Plus™/F (0, 1, 5 mL), and Standard/10 Aerobic/F (0, 3, 10
mL) media were inoculated with 10-100 CFU expected per bottle of 23 organisms: 11 CA, eight C.
haemulonii (CH), two C. duobushaemulonii (CD) and two C. pseudohaemulonii (CP). The 621 bottles
(N=3) were incubated in a BACTEC FX™ automated culture instrument and time to detection (TTD) data
was analyzed by ANOVA with Tukey Pairwise Comparisons. The same inocula were plated on nine BBL™
and Difco™ media (N=4; 828 plates): Sabouraud Dextrose Agar Emmons (SDA-E), CHROMagar™ Candida,
and seven selective media containing antifungals (five with cycloheximide, CXD). Plates were incubated
at 35oC and 42oC (N=2), and colonies counted daily for five days; < 10 CFU on either plate per pair was
deemed sporadic recovery. Results: All culture bottles detected within 120-hrs except one Peds Plus
with CH and no blood (121.9 hrs). Differences in overall mean TTDs between media or blood levels were
relatively small, within 3.2 and 5.5 hrs, respectively. Organism was the factor accounting for the most
data variability, with TTD means (range) all differing significantly at 24.7 (15.6-49.6) hrs for CA, 37.0
(31.2-43.6) for CD, 51.9 (29.1-114.8) for CP, and 60.7 (41.7-121.9) for CH (P < 0.05). All strains had
expected CFU at 35oC on CHROMagar, WL Differential Medium (0.0004% CXD), and except for sporadic
recovery of one CH, on SDA-E; six CA had expected CFU on Campy-CSM Agar (0.01% CXD) as did six CA
on BCYE-PAC Agar (0.008% anisomycin). Some CD, CH and CP grew on Dermatophyte Test Medium
(0.05% CXD), Mycosel™ Agar (0.04% CXD), SABHI with CC Agar (0.05% CXD), and Selective 7H11 Agar
(0.001% amphotericin B) at 35oC. Only CA grew at 42oC, with expected CFU from all strains on
CHROMagar and SDA-E, and on WL Differential Medium except for one strain with sporadic recovery;
four and three CA had expected CFU on Campy-CSM and BCYE-PAC, respectively. Conclusions: CA and
related strains reliably detected in BACTEC aerobic blood culture media irrespective of blood volume.
Recovery was optimal on CHROMagar for all conditions, on WL Differential Medium at 35oC and for CA
on SDA-E at 35oC or 42oC. CA thermotolerance and 0.04% CXD sensitivity was confirmed1, but growth
on 0.01% CXD was strain-dependent.
Session Number: 346
Session Number: 346
Abstract Title:
Moderator
Robert Tibbetts; Henry Ford Hlth. Sys.
Abstract Body:
Session Number: 346
Session Number: 346
Abstract Title:
Abstract Body:
scores.
Session Number: 346
Session Number: 346
Abstract Title:
Spectroscopy
Abstract Body:
Session Number: 378
Session Number: 378
Session Title: The Animals among Us
Abstract Title:
Detection of the Rat Lungworm, Angiostrongylus Cantonensis, in Giant African Snails in Trinidad
K. Kines1, Y. Qvarnstrom1, J. Seetahal2, L. Gyan2, V. Lashley2, C. Wharwood2, A. Balfour2, S. Rahaman3;
1CDC, Atlanta, GA, 2Ministry of Agriculture, Land and Fisheries, Trinidad, Trinidad and Tobago, 3Ministry
of Hlth., Trinidad, Trinidad and Tobago
Abstract Body:
Background: Angiostrongylus cantonensis, the rat lungworm, is the most common infectious cause of
eosinophilic meningitis in humans. A. cantonensis is endemic in Southeast Asia and the Pacific Basin, as
well as in areas of Africa, South America and the Caribbean. The known geographic range of A.
cantonensis has expanded in recent decades, partially due to habitat expansion of the rat and snail
hosts. Lissachatina fulica, the giant African snail, is an invasive and highly destructive agricultural pest
species recently introduced in Trinidad. Giant African snails are known intermediate hosts for A.
cantonensis and there is growing concern about public health implications of this introduction. Although
human angiostrongyliasis cases have been reported from several other Caribbean islands, no human
cases have been detected yet in Trinidad. An environmental investigation was initiated to determine the
prevalence of A. cantonensis infection in giant African snails in Trinidad. Methods: Giant African snails
were collected from multiple locations on the island of Trinidad in 2013, 2014 and 2017. DNA was
extracted using the QIAGEN PowerSoil kit and real-time PCR was used to detect the presence of A.
cantonensis larvae in the mantle tissue of the snails. Results: A total of 211 giant African snails were
tested for A. cantonensis. Infected snails were detected for all three sampling periods: 12% (7/58) in
2013, 2% (2/85) in 2014 and 16% (11/68) in 2017. Three of the 9 locations sampled in 2017 had A.
cantonensis-positive snails. Conclusions: These results revealed that A. cantonensis is present in giant
African snails in Trinidad. Giant African snails are classified as a notifiable pest in Trinidad and
eradication efforts are ongoing, with a special focus on locations where A. cantonensis was detected.
Session Number: 378
Session Number: 378
Abstract Title:
Role of Stray Dogs of Slum Area in Visceral Leishmaniasis in Eastern Nepal
K. Bantawa, D. Subba Limbu, P. Subba; Central Campus of Technology, Tribhuvan Univ., Nepal, Dharan,
Nepal
Abstract Body:
Background: Visceral leishmaniasis (VL) is a fatal parasitic vector-borne disease, also known as kala-azar.
Worldwide incidence of VL is 500,000 cases per year among them about 90% of cases occur in India,
Nepal, Bangladesh, Sudan and Brazil. During the past 5-6 years, the number of reported cases has
increased on the Indian subcontinent. With the goal to reduce annual incidence of VL in endemic areas
to <1 case per 10,000 people by 2015, Kala-azar elimination program was launched by governments of
Bangladesh, India and Nepal supported by WHO. Leishmania donovani, the etiologic agent of VL, is still
considered as anthroponotic but no clear conclusions have been made regarding animals as risk factors
or reservoir hosts. Identification of reservoir host is essential for controlling the kala-azar. In 2008, L.
donvani was found in person (6.1%), cows (5%), buffaloes (4%) and goats (16%) in endemic areas of
Dharan. To determine the possible role of homeless stray dogs of slum area, this study as performed in
active VL transmission area of Dharan city of eastern Nepal. Methods: All dog surveys were done by
veterinarians in March 2017. Blood samples were collected from 64 (42 female and 22 male) stray dogs
found in slum areas of Dharan-11, 15, 16 and 17. The samples (3ml) were collected by venipuncture into
tubes containing Na2EDTA. All tubes were immediately stored in ice box and transferred to the
laboratory of Central Campus of Technology. DNA extraction was done by phenol- chloroform method.
All DNA samples were allowed to come at ambient temperature and stored in TS1 buffer. All DNA were
analyzed by PCR (Sigma Aldrich PCR kit catalog number P0476) specific for small ribosomal (115 bp)
genes of Leishmania spp. using sense primer 18s-L-F 5'CGTAGTTGAACTGTGGGCTGTGC-3' and antisense
primer 18s-L-R 5' ACTCCCGTGTTTCTTGTTTCTTTGAA-3'. To confirm that amplified DNA corresponded to
Leishmania spp., set of positive samples from BPKIHS were sequenced. Result: The rate of Leishmania
positivity in homeless dogs was 7.82% (5/64), female dogs accounted for 9.53% (4/42) and male dogs for
4.54% (1/22). Statistical analysis showed that there is no significant relationship between gender of dogs
and L. donovani. Conclusion: The findings indicate that both domestic and stray dogs may act as
reservoir host for L. donovani and can play role in the transmission cycle of VL. Regular screening of dogs
and other domestic animals for VL should be implemented in control programs of VL.
Session Number: 378
Session Number: 378
Abstract Title:
Reemergence of Novel Reassortant Avian Influenza H14n3 in Asia
N. Siddique; Natl. Agricultural Res. Ctr., Islamabad, Pakistan
Abstract Body:
Background: Since the isolation of original four isolates of H14 Avian Influenza Virus in central Asia
during 1982, these rare viruses had never been reported in any Asian country. Exceptionally, the four
H14 isolates recovered from Wisconsin and California during 2010-11. Pakistan homes a wide variety of
migratory birds flocking up from Siberia, Russia and Europe during winters through Indus flyway. In 2014
during Avian Influenza virus surveillance three H14N3 isolates recovered from Duck, Geese and Pigeon
from Live bird market in Pakistan. Methods: The clinical specimens (cloacal and tracheal swabs) were
collected and subjected to virological evaluation through embryonated SPF chicken egg inoculation.
Subtype identification was determined by HA, HI techniques along with RT-PCR and QRT-PCR procedures
using sequence specific primers and probes. The isolated H14N3 AIVs were subjected to whole genome
sequencing. The purified PCR products were directly used for cycle sequencing reactions in a genetic
analyzer. Phylogenetic analysis was conducted using MEGA -4. The gene sequences were submitted to
GenBank. Results: The whole genome sequencing revealed introduction of a unique reassortant
Eurasian- American avian strain into Pakistan. Phylogenetically HA gene shares 92% nucleotide sequence
identity with H14 from Wisconsin and California. The NA genes showed 97.7% homology with European
H11N3. The M, PB1, PB2 and PA genes were Asian in origin whereas NS and NP genes showed maximum
sequence identity with the Pakistani H3N1 and H4N6. Sequence analysis revealed a LP amino acid motif
(PDKQAK ) at HA cleavage site, avian-like receptor specificity (Q243, G241) and 7 N- linked glycosylation
sites while 2 glycosylation sites were lost due to S23I and N278G mutations.The amino acids known to
be associated with sensitivities to antiviral drugs were found conserved except the unique mutation
Ile27Val on matrix protein. Seroprevalence was negligible in various birds species Conclusions: These
observations suggested that these AIVs may persist in environmental stasis for long periods of time,
undetected by surveillance, until a time when agent, host, or environmental factors allow for
reemergence in susceptible hosts within a region. Some IAV strains are preferentially maintained within
individual migratory flyways and may be endemic to certain regions of the world The various point
mutations in these novel reassortant H14N3 and close relationship with American, European and Asian
AIVs also reflect the genetic diversity and intercontinental spread.
Session Number: 378
Session Number: 378
Abstract Title:
Moderator
Abstract Body:
Session Number: 394
Session Number: 394
Session Title: SUNDAY - CPHM Late-breakers
Poster Board Number: SUNDAY - CPHM LB1
Abstract Title:
Combatting Antibiotic Resistance through rapid phenotype testing with IDAST
J. Holder, S. Gruszka, G. Kourepenos, P. Dow, C. McBrine, P. Cavanagh, B. Hoefler, J. Fiering; Draper,
Cambridge, MA
Abstract Body:
The current standard of care for diagnosing bacterial infections requires days of lab testing to determine
what the appropriate treatment should be. Today, the slow time to antibiotics susceptibility results
forces clinicians to treat empirically; the negative impact of this are severe and many-fold. Draper is
developing a platform technology called IDAST for rapid diagnosis of bacterial infections leading to
better antimicrobial stewardship. IDAST identifies (ID) the causative agent and tests the antibiotic
susceptibility (AST) in less than one hour. Draper’s proprietary IDAST technology utilizes engineered
bacteriophage that encode luciferase expression (lumiphage) for phenotypic determination of the
identity and physiological state of bacterial cells for evaluation whether an antibiotic is having a lethal
effect and at what drug concentration. A distinguishing feature of IDAST technology is that it does not
require growth of bacteria prior to testing thus vastly reducing the time to results and enabling direct
from sample testing with proven efficacy in blood, urine, and blood culture. Draper is developing devices
that can be deployed in a microbiology lab using automated liquid handling and at the Point of Care
(PoC) utilizing plastic based microfluidic chips capable of removing blood components from bacteria and
resulting in >33 fold increase in sensitivity for bacteria. We have developed lumiphage cocktails and
training sets of 96 blood derived strains for each species: S. aureus, P. aeruginosa, and E. coli with 100,
81, and 46 % strain recognition respectively. We demonstrate clinical validation in our alpha program
with hospital derived positive blood cultures by detecting 100, 98, and 117% respectively relative to our
training sets. Our alpha-AST tests were performed for 1 hour and indicated by SIR classification to be
100% correlated with results from Etest strips for Linezolid, Gentamycin, Amikacin, and 97% correlated
for vancomycin. Cocktails are being developed for greater species and strain detection while ASTs
workflows are being developed to accommodate drug susceptibility phenotypes requiring induction and
phenotypic lag.
Session Number: 394
Session Number: 394
Abstract Title:
Optimization of Multiplex Quantitative Polymerase Chain Reaction Based on Response Surface
Methodology and an Artificial Neural Network-Genetic Algorithm Approach
P. Pan1, Y. Chen2, D. Yu1; 1Hangzhou First People’s Hosp., Zhejiang Chinese Med. Univ., Hangzhou,
China, Hangzhou, China, 2Dept. of Clinical Lab., Zhejiang Hosp., Hangzhou, China, Hangzhou, China
Abstract Body:
Background: The construction of a reliable and dynamic mathematical model for multiplex qPCR that
analyzes the effects of interactions between variables and results is especially important. Response
surface methodology (RSM) and back-propagation neural network-genetic algorithm (BPNN-GA) were
employed to analyze interactions between various parameters and the results of multiplex qPCR.
Methods: An integrative analysis of multiplex qPCR was performed using three viruses (RSV, INF, and
HMPV). RSM-central composite designs (CCDs) were used to construct a 5-factor 5-level uni- and
multiplex qPCR experimental plan. The data were analyzed using models constructed from RSM and the
BPNNs-GA. Mathematical models built using these two methods were evaluated for their capacity and
goodness of fit to modle the results of multiplex qPCR. Results: The mean absolute error (MAE) and the
mean square error (MSE) of the predictive models based on RSM were less than those of the BPNN-GA
for both uni- and multiplex PCR for all three viruses tested. The Coefficient of Determination(R2) of the
RSM for multiplex qPCR of the three viruses used were 0.847, 0.746, and 0.864, whereas those for the
BPNNs-GA were 0.451, 0.709, and 0.528. Ultimately, optimal parameters of multiplex qPCR were
determined by RSM. Conclusion: Dynamic models of uni- and multiplex qPCR can be constructed using
both RSM and BPNNs-GA methods. However, RSM was better able to predict multiplex qPCR results
than BPNNs-GA and was more applicable for the optimization of the conditions in our study.<br /><p><a
href="http://files.abstractsonline.com/CTRL/cc/9/f38/475/4ee/4af/7ba/61b/66b/c5e/605/8a/g8040_1.j
src="http://files.abstractsonline.com/CTRL/cc/9/f38/475/4ee/4af/7ba/61b/66b/c5e/605/8a/g8040_1.jp
Session Number: 394
Session Number: 394
Abstract Title:
Identification of Bloodstream Pathogens from an Enhanced Blood Culture Instrument using a Multiplex
Molecular Diagnostic System
J. Green1, A. Clark1, C. Carter1, C. Chandler1, U. Spaulding1, M. Rogatcheva1, E. Amiott1, S. Thatcher1,
P. Deol2, B. Katzin2, E. Miller3; 1BioFire Diagnostics, LLC, Salt Lake City, UT, 2bioMérieux Inc., Durham,
NC, 3bioMérieux Inc., Durham, UT
Abstract Body:
Life threatening bloodstream infections (BSI) require fast and reliable identification of causative
pathogens to positively impact patient outcomes. Advancements in blood culture instrumentation are
leading to faster times to positivity (TTP) and when paired with rapid molecular diagnostics can
significantly reduce turnaround time for identification. Faster TTP may result in lower organism titers
that may impact molecular detection. However organism titers in positive blood culture (PBC) are rarely
characterized. The bioMérieux BacT/ALERT® VIRTUO™ system is a high-throughput automated blood
culture instrument providing faster TTP for many BSI organisms. PBCs generated by the VIRTUO system
were tested on the BioFire® FilmArray® Blood Culture Identification Panel (BCID) to evaluate if faster
TTP, and potentially lower titers, affect pathogen detection and identification by a molecular system. BSI
organisms, 5 fungi and 12 bacteria, were independently added to blood samples, seeded into
bioMérieux BacT/ALERT SA, SN, FA Plus, and FN Plus blood culture bottles, and incubated in the VIRTUO
system; fungal samples were also incubated in the bioMérieux BacT/ALERT® 3D system for direct
comparison. TTP was recorded for all PBCs and testing performed on the BioFire BCID Panel for PBCs
collected ≤1 hour of positivity. Titers were determined for 4/5 fungal and 5/12 bacterial PBCs; fungal
titers from both systems were directly compared and all titers were compared to historical 3D system
values. On average fungal TTP on the VIRTUO system was 3.1h faster (range of 1.3-6h) compared to the
3D system. Bacterial TTPs were 6.9h faster on average (range of -29-9.6h) compared to historical 3D
data. Fungal titers from VIRTUO PBCs were on average 4.5-fold lower compared to the 3D system;
bacterial titers from VIRTUO PBCs were similar to historical 3D titers. The BioFire BCID Panel detected
100% (69/69) of seeded organisms in PBC for both VIRTUO and 3D; all titers were above thresholds for
sensitivity. The VIRTUO system results in faster TTP than the 3D system in this study. Faster TTP resulted
in reduced fungal titers; no significant difference in bacterial titers. Faster TTP and lower organism titers
did not impact molecular identification of PBCs (69/69 PBCs correctly identified) by the BioFire BCID
Panel as titers remain above the limits of detection of a molecular system. Advancements in blood
culture instrumentation coupled with a robust molecular test, like the BioFire BCID Panel, can provide
actionable results faster for critically ill patients.
Session Number: 394
Session Number: 394
Abstract Title:
A Diverse Panel of Clinical Pseudomonas aeruginosa for Research Use
k. dommaraju, E. C. Snesrud, M. R. Galac, M. D. Julius, A. E. Hector-Mason, A. Ong, Y. I. Kwak, A. R. Jones,
M. K. Hinkle, P. T. Mc Gann; WRAIR, Silverspring, MD
Abstract Body:
Background: Pseudomonas aeruginosa is a leading cause of infections in humans and is associated with
significant morbidity and mortality. Treatment of P. aeruginosa is complicated by the intrinsic resistance
of this pathogen to multiple antibiotics and its tendency to develop resistance to other antibiotics via
mutation. As a result, there is a constant search for novel treatments and therapeutics to combat this
organism. Herein, we describe a panel of 100 P. aeruginosa isolates that represent a diverse cross-
section of P. aeruginosa clinical isolates. Methods: 2,409 clinical P. aeruginosa isolates, collected globally
between 2003 and 2017, were sequenced using an Illumina Next Seq. A core-genome multi-locus
sequence typing (cgMLST) scheme was developed and used to select. 300 distantly related strains. This
set was refined further by pan-genome analysis; SNPs in the core genome and accessory gene content
were taken into account, maximizing the phylogenetic distance and overall gene content, respectively,
of a final 100 isolate panel. Results: All 100 isolates have extensive attendant data, including date of
isolation, clinical source (e.g. blood, urine etc.), antibiotic susceptibilities to 11 antibiotics, traditional
MLST, and a complete list of all antibiotic resistance genes (AbR) genes carried. Furthermore, WGS data
for all 100 isolates will be available through NCBI in the near future. The 100 selected isolates span 95
different traditional MLST categories and include the most common sequence types (STs) identified in
clinical isolates. Antibiotic resistance varies considerably among the strains, with 3 isolates non-
susceptible and 18 isolates susceptible to all 11 antibiotics tested. This is also reflected in the antibiotic
resistance gene content. All isolates carry the five P. aeruginosa intrinsic AbR genes, aph(3')-IIb, blaOXA-
50, blaPAO, catB7, and fosA, but up to 11 additional antibiotic resistance genes are also present in some
strains. Notably, two isolates carry the carbapenemase genes blaVIM- 6 and blaVIM-11, respectively,
and one isolate carries the carbapenemase blaKPC-2. Conclusions: We describe a panel of 100 highly
diverse P. aeruginosa selected from a large repository of over 2,400 clinical isolates collected worldwide.
The panel has been meticulously selected based on cgMLST to represent the most diverse collection of
P. aeruginosa of clinical relevance. Comprehensive attendant meta-data is also provided for every strain
making this panel a valuable resource P. aeruginosa-focused research.
Session Number: 394
Session Number: 394
Abstract Title:
Delafloxacin: Activity against Fastidious Organisms Tested by EUCAST vs CLSI Methodology
G. Rossolini1, D. Zinzi2, A. Nuti2, A. Capriati2, D. Shortridge3, S. McCurdy4, S. Cammarata4, M.
Sanguinetti5; 1Univ. of Florence, Florence, Italy, 2Menarini Ricerche, Florence, Italy, 3JMI Lab.,, North
Liberty, IA, 4Melinta Therapeutics, New Haven, CT,
Abstract Body:
Delafloxacin (DLX) is a new fluoroquinolone with broader activity against Gram-negative and Gram-
positive, including MRSA, atypical and anaerobe pathogens. DLX is FDA approved and under evaluation
by EMA for the treatment of ABSSSI. For fastidious organisms, CLSI and EUCAST MIC methods use
different media. The study assessed DLX susceptibility of 898 isolates of nine fastidious species from DLX
Surveillance within the SENTRY Antimicrobial Surveillance Program using EUCAST method and compared
the data with those previously obtained with CLSI. MICs were determined at 3 different laboratories
using MH-F broth panels manufactured at each laboratory according to the EUCAST Media Preparation
v5.0, 2017. As no quality control (QC) ranges are available from EUCAST for DLX, levofloxacin was tested
for QC. For quality assurance purposes, S. pneumoniae ATCC 49619, H. influenzae ATCC 49247, and H.
influenzae ATCC 49766 were tested across the laboratories. DLX MIC results were overall similar by
EUCAST and CLSI methods, with differences usually within 1 doubling dilution. With most species, DLX
MIC results by the CLSI method were slightly higher than those by the EUCAST method (Tab. 1). All
tested species were highly susceptible to DLX. In particular, S. pyogenes DLX MICs using EUCAST and
CLSI methods ranged from 0.002-0.03 mg/L. S. pneumoniae DLX MICs using EUCAST and CLSI methods
ranged from 0.002-0.25 mg/L. H. influenzae DLX MICs ranged from 0.000125-0.002 and 0.000125-0.004
mg/L, using EUCAST and CLSI methods respectively. Fig.1 reports the comparison between the two
methods for S. pyogenes, S. pneumoniae and H. influenzae. No significant differences were observed
between the results obtained by different laboratories with QC strains. This study reports for the first
time DLX MIC by EUCAST versus CLSI methods for 898 fastidious isolates. The results showed an overall
good correlation between the two methods for the nine species studied.<br /><p><a
href="http://files.abstractsonline.com/CTRL/26/7/7a0/405/c0f/40f/a9d/9a4/1b9/bf0/44e/34/g8034_2.
PNG" target='_blank' address=no ><img
src="http://files.abstractsonline.com/CTRL/26/7/7a0/405/c0f/40f/a9d/9a4/1b9/bf0/44e/34/g8034_2.P
NG" alt="" border="0" width="600" height="527" /></a></p>
Session Number: 394
Session Number: 394
Abstract Title:
Rapid Susceptibility Evaluation of Ceftalozane/Tazobactam by Flow Cytometry Directly from Positive
Blood Cultures
S. Quintas1, R. Teixeira-Santos2, S. Costa-de-Oliveira2, A. Silva-Dias2, I. Martins-Oliveira2, A. Rodrigues1,
C. Pina-Vaz2; 1Faculty of Med., Univ. of Porto, Porto, Portugal, 2FASTinov, SA, Porto, Portugal
Abstract Body:
Background: Ceftolozane/tazobactam is a novel antibacterial and β-lactamase-inhibitor combination
that has shown appreciable activity against wild-type Enterobacteriaceae and potent activity against P.
aeruginosa. Moreover, it shows activity against extended-spectrum β-lactamases, AmpC β-lactamase, a
loss in porin channels, or the overexpression of efflux pumps in P. aeruginosa. Classic susceptibility
methods are based on culture so take time to give results. FASTinov has been developing antimicrobial
susceptibility tests (AST) directly from blood cultures using a disruptive technology based on flow
cytometry (FC). Methods: Fifty-four blood cultures (BC) were spiked with 24 Enterobacteriaceae and 30
Pseudomonas spp. and inoculated with human blood (Cambridge Bioscience) according to Puttaswamy
et al, 2011. After flag positive, the bacteria were extracted from BC and incubated for 1 h, at 37ºC, 180
rpm in the FASTinov® gramneg kit; it includes four different concentrations of cetalozane/tazobactam
(1/4, 2/4, 4/4 and 8/4 µg/mL), kindly provided by Merck Sharp & Dohme, Lda, and a fluorescent probe.
The cells were then analyzed in an Accuri C6 plus (BD) flow cytometer (FC) and the intensity of
fluorescence of treated cells compared with control (non-treated). Cut-off values were previously
determined in order to classify the bacteria phenotype: susceptible-S, intermediate-I or resistant-R. In
parallel, BC were subcultured and E-test to ceftalozane/tazobactam performed the day after. The results
obtained from both FC and E-test (reference method) were compared. Results: Forty-four bacteria were
susceptible (S), 8 resistant (R) and 2 intermediate (I) to ceftalozane/tazobactan according to Etest. The
categorical agreement between the two methods was 88% for Enterobacteriaceae and 97% for
Pseudomonas spp. The overall CA was 93%, which shows great accuracy of the method. For
Enterobacteriaceae, there was no minor error (mE); 1/17 major error (ME) and 2/7 very major error
(VME). For Pseudomonas spp., the minor error was 1/30 and there were no major or very major errors.
The results obtained using EUCAST or CLSI protocols were similar. Conclusions: A fast and accurate test
is now available for determination of antimicrobial susceptibility (AST) directly on BC in a time-to-result
of 2 hours versus 48h of the routine method. This makes flow cytometry a preferential method for AST
evaluation. The results obtained also showed great activity of ceftalozane/tazobactam against
Enterobacteriaceae and Pseudomonas spp.
Session Number: 394
Session Number: 394
Abstract Title:
Compatibility of High- and Maximum-Containment Virus Inactivation Protocols with Identification of
Bacterial Co-Infections by MALDI-TOF Mass Spectrometry
M. Matson1, F. Stock2, W. Shupert1, T. Bushmaker1, F. Feldmann1, W. Bishop2, K. Frank2, J. Dekker2, D.
Chertow2, V. Munster1; 1NIH/NIAID Rocky Mountain Lab., Hamilton, MT, 2NIH Clinical Ctr., Bethesda,
MD
Abstract Body:
Background: Diagnostics in patients infected with high- and maximum-containment viruses present
unique challenges, and viral inactivation protocols compatible with downstream analyses are essential.
The West Africa Ebola virus disease (EVD) epidemic highlighted the shortcomings of current practice. An
EVD patient treated in Germany developed severe gram-negative sepsis, but the etiological species
could not be identified due to the inaccessibility of advanced diagnostic tools outside containment1.
Furthermore, a recent study utilizing an unbiased sequencing approach of EVD patient blood suggests
bacterial sepsis may frequently complicate EVD2. Our unpreparedness to efficiently and
comprehensively manage patients such as these in advanced healthcare facilities must be addressed.
The aim of this study was to identify a validated viral inactivation protocol that is compatible with
downstream analysis for bacterial co-infection with MALDI-TOF MS. Methods: Using a panel of bacteria,
we assessed six validated virus inactivation protocols3,4,5 for compatibility with bacterial identification
on a Bruker MALDI Biotyper system. A routine protocol was utilized for each tested bacteria as a
standard. Results: Inactivation with TRIzol or gamma irradiation is compatible with MALDI-TOF MS and
produced confident and accurate results adherent to the '10% rule'6,7,8. Conclusions: The TRIzol
inactivation protocol together with MALDI-TOF MS bacterial speciation is simple, economical, and rapid
and is widely applicable for samples containing viruses such as Ebola, Nipah, and Lassa. MALDI-TOF MS
is becoming the microbiological diagnostic platform of choice and is a robust alternative to identification
by biochemical analysis or 16S sequencing. The burgeoning threat of emerging viruses necessitates
development of viral inactivation protocols that are compatible with a full range of downstream
analyses to maximize preparedness for inevitable outbreaks.<br /><p><a
href="http://files.abstractsonline.com/CTRL/f7/4/669/1eb/c80/433/498/a93/a77/313/340/3c/g7456_1.
src="http://files.abstractsonline.com/CTRL/f7/4/669/1eb/c80/433/498/a93/a77/313/340/3c/g7456_1.j
Session Number: 394
Session Number: 394
Abstract Title:
Validation of a Rapid Diagnostic Test Directly on Whole Blood for Detection of Blood Stream Infection
C. Pickens, C. Qi, M. Malcyznski, H. Donnelly, M. Breganio, N. Borkowski, R. Wunderink; Northwestern
Univ., Chicago, IL
Abstract Body:
Background: Several rapid diagnostic tests have been introduced to address the problem of prolonged
turnaround time for blood culture results; however, no clinically available test identifies bloodstream
infection (BSI) directly from whole blood. The T2Bacteria Panel RUO (T2B Panel) is a novel diagnostic test
that can detect as few as 1cfu/mL of six different organisms directly in whole blood with results in 3-5
hours. The primary aim of this study was to understand the operating characteristics of the T2B Panel
using blood culture as the gold standard. The second aim was to determine the sensitivity and specificity
of the T2B Panel for detection of a pathogen when blood culture was negative, but urine or respiratory
culture was positive.<br />Methods: Whole blood samples were collected from patients in the medical
intensive care unit with suspected sepsis or septic shock.<br />Results: 119 samples were eligible for
testing, 95 were retrospectively collected and 24 were prospectively collected. The sensitivity and
specificity of the test was calculated for each organism using blood culture as the gold standard.<table
class="AbstractTable" id="{78FF9371-3816-42AC-80F9-F8F78F5A0204}"><caption
rowspan="1" colspan="1">Sensitivity (N positive/N total)</td><td rowspan="1" colspan="1">Specificity
(N negative/N total)</td></tr><tr><td rowspan="1" colspan="1">A. baumannii</td><td rowspan="1"
colspan="1">NA (0/0)</td><td rowspan="1" colspan="1">96% (114/119)</td></tr><tr><td rowspan="1"
colspan="1">E. coli</td><td rowspan="1" colspan="1">67% (4/6)</td><td rowspan="1"
colspan="1">93% (105/113)</td></tr><tr><td rowspan="1" colspan="1">E. faecium</td><td
rowspan="1" colspan="1">67% (2/3)</td><td rowspan="1" colspan="1">96%
(111/116)</td></tr><tr><td rowspan="1" colspan="1">K. pneumoniae</td><td rowspan="1"
colspan="1">100% (1/1)</td><td rowspan="1" colspan="1">90% (106/118)</td></tr><tr><td
rowspan="1" colspan="1">P. aeruginosa</td><td rowspan="1" colspan="1">100% (1/1)</td><td
rowspan="1" colspan="1">92% (109/118)</td></tr><tr><td rowspan="1" colspan="1">S.
aureus</td><td rowspan="1" colspan="1">100% (10/10)</td><td rowspan="1" colspan="1">94%
(103/109)</td></tr><tr><td rowspan="1" colspan="1">Overall</td><td rowspan="1" colspan="1">90%
(18/21)</td><td rowspan="1" colspan="1">94% (648/693)</td></tr></table><br />Table 2 shows the
T2B Panel ability to detect a primary urinary (n = 19) or respiratory infection (n = 20) when blood culture
was negative. Note some samples had multiple microbes present.<br /><table class="AbstractTable"
id="{B73C26B4-EAFE-4A75-9B13-615E3F5B7066}"><caption
rowspan="1" colspan="1">Sensitivity</td><td rowspan="1" colspan="1">Specificity</td></tr><tr><td
rowspan="1" colspan="1">A. baumannii</td><td rowspan="1" colspan="1">75% (3/4)</td><td
rowspan="1" colspan="1">97% (113/116)</td></tr><tr><td rowspan="1" colspan="1">E. coli</td><td
rowspan="1" colspan="1">60% (3/5)</td><td rowspan="1" colspan="1">95%
(106/111)</td></tr><tr><td rowspan="1" colspan="1">E. faecium</td><td rowspan="1"
rowspan="1" colspan="1">K. pneumoniae</td><td rowspan="1" colspan="1">56% (5/9)</td><td
rowspan="1" colspan="1">94% (106/113)</td></tr><tr><td rowspan="1" colspan="1">P.
aeruginosa</td><td rowspan="1" colspan="1">53% (8/15)</td><td rowspan="1" colspan="1">92%
(107/116)</td></tr><tr><td rowspan="1" colspan="1">S. aureus</td><td rowspan="1"
rowspan="1" colspan="1">Overall</td><td rowspan="1" colspan="1">61% (28/46)</td><td
rowspan="1" colspan="1">96% (643/671)</td></tr></table><br />Conclusions: The T2B Panel was 90%
sensitive and 94% specific for the detection of six different organisms compared to blood culture. The
calculated specificity is limited by comparison to blood culture because many studies demonstrate that
blood culture itself is poorly sensitive. Thus, the reported false positive results of the T2B Panel may be
true positives. As further support, the T2B Panel detected organisms in the blood when blood culture
was negative but evidence of infection in the urine or lung was available. A positive T2B Panel result in
this setting may indicate poor source control, inappropriate antibiotics or poor host defenses.
Session Number: 394
Session Number: 394
Abstract Title:
Seroprevalence of Entamoeba histolytica at a Voluntary Counseling and Testing Center in Tokyo: Data
from 2017
Y. Yanagawa1, M. Nagashima2, T. Shinkai2, Y. Kikuchi1, H. Gatanaga1, S. Oka1, K. Sadamasu2, K.
Watanabe1; 1Natl. Ctr. for Global Hlth.and Med., Tokyo, Japan, 2Tokyo Metropolitan Inst. of Publ. Hlth.,
Tokyo, Japan
Abstract Body:
Background: Amebiasis, a disease caused by Entamoeba histolytica, is a re-emerging public health issue
linked with sexually transmitted infections (STIs) in Japan, and control measures against its spread will
be required for the upcoming 2020 Tokyo Olympics. We have previously shown that HIV-infected
individuals have a high risk of contracting STI-amebiasis, but few data exist on HIV-negative individuals.
We also have shown that asymptomatic, seropositive individuals often harbor colonic ulcers and shed
cysts in their stools, a potential transmission source for STI-amebiasis. Methods: The stocked serum
samples from 718 anonymous individuals who underwent STI testing at a voluntary counseling and
testing center in Tokyo were subjected to enzyme-linked immunosorbent assaying to determine the
seroprevalence anti-E.histolytica IgG antibodies. Results: The overall seroprevalence of E. histolytica was
2.7% (19/718) (3.3% among males, 1.0% among females) with seropositivity values for HIV-1, Rapid
Plasma Reagin test (RPR) and Treponema pallidum hemagglutination assay (TPHA) of 0.42%, 2.9% and
9.8%, respectively. All the E. histolytica seropositive individuals were HIV negative. The E. histolytica
seropositivity rate was highest in the age group 40-49 years (5.9%) (Figure). The regression analyses
showed that TPHA seropositivity was an independent risk factor for E. histolytica seropositivity (Table).
Conclusions: E. histolytica seroprevalence was 6.3 times higher than that of HIV-1 and was at the same
level as a recent syphilis infection (RPR positive). Our results indicate that STI-amebiasis is now
spreading among HIV-negative, TPHA-positive individuals.<br /><table border="1" cellpadding="1"
class="DisplayTable" id="{C608DFDA-0EA4-4BFD-915C-81931871ABC0}"><caption>Risk analyses for
anti-E. histolytica antibody positivity (Cox proportional hazard regression model)</caption><tr><td
colspan="1">Univariate analysis</td><td rowspan="1" colspan="1">Univariate analysis</td><td
rowspan="1" colspan="1">Multivariate analysis</td><td rowspan="1" colspan="1">Multivariate
analysis</td></tr><tr><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td
rowspan="1" colspan="1">Odds ratio [95% CI]</td><td rowspan="1" colspan="1">p value</td><td
rowspan="1" colspan="1">Odds ratio [95% CI]</td><td rowspan="1" colspan="1">p
value</td></tr><tr><td rowspan="1" colspan="1">Male</td><td rowspan="1" colspan="1"></td><td
rowspan="1" colspan="1">3.34 [0.76 - 14.6]</td><td rowspan="1" colspan="1">0.11</td><td
rowspan="1" colspan="1">-</td><td rowspan="1" colspan="1">-</td></tr><tr><td rowspan="1"
colspan="1">Age (by 10 years)</td><td rowspan="1" colspan="1"></td><td rowspan="1"
colspan="1">1.56 [1.08 - 2.25]</td><td rowspan="1" colspan="1">0.02</td><td rowspan="1"
colspan="1">1.47 [0.99 - 2.18]</td><td rowspan="1" colspan="1">0.06</td></tr><tr><td rowspan="1"
colspan="1">HIV positive</td><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1">-
</td><td rowspan="1" colspan="1">-</td><td rowspan="1" colspan="1">-</td><td rowspan="1"
colspan="1">-</td></tr><tr><td rowspan="1" colspan="1">Syphilis</td><td rowspan="1"
colspan="1">RPR positive</td><td rowspan="1" colspan="1">1.89 [0.24 - 14.8]</td><td rowspan="1"
colspan="1">0.55</td><td rowspan="1" colspan="1">-</td><td rowspan="1" colspan="1">-
</td></tr><tr><td rowspan="1" colspan="1">Syphilis</td><td rowspan="1" colspan="1">TPHA
positive</td><td rowspan="1" colspan="1">5.89 [2.24 - 15.5]</td><td rowspan="1"
colspan="1"><0.01</td><td rowspan="1" colspan="1">5.02 [1.89 - 13.4]</td><td rowspan="1"
colspan="1"><0.01</td></tr><tr><td rowspan="1" colspan="1">Syphilis</td><td rowspan="1"
colspan="1">RPR and TPHA positive</td><td rowspan="1" colspan="1">1.99 [0.25 - 15.7]</td><td
rowspan="1" colspan="1">0.51</td><td rowspan="1" colspan="1">-</td><td rowspan="1"
colspan="1">-</td></tr><tr><td rowspan="1" colspan="1">Chlamydia positive</td><td rowspan="1"
colspan="1"></td><td rowspan="1" colspan="1">1.34 [0.17 - 10.5]</td><td rowspan="1"
colspan="1">0.78</td><td rowspan="1" colspan="1">-</td><td rowspan="1" colspan="1">-
</td></tr></table><br /><p><a
href="http://files.abstractsonline.com/CTRL/2d/0/c65/4ef/e91/452/abb/9bb/5d7/fe0/195/ce/g7906_2.
src="http://files.abstractsonline.com/CTRL/2d/0/c65/4ef/e91/452/abb/9bb/5d7/fe0/195/ce/g7906_2.J
Session Number: 394
Session Number: 394
Abstract Title:
Autoantibody Levels Correlate with Severe Malaria in Plasmodium falciparum Patients
J. L. Rivera-Correa1, A. L. Conroy2, R. O. Opoka3, C. C. John2, A. Rodriguez1; 1New York Univ. Sch. of
Med., NY, NY, 2Indiana Univ. Sch. of Med., Indianapolis, IN, 3Makerere Univ., Kampala, Uganda
Abstract Body:
Background: Malaria is a parasitic disease caused by Plasmodium parasites and it’s still one of the
leading infectious diseases in the world, affecting mostly children under the age of 5 in Sub-Saharan
Africa1. Severe anemia (SMA), cerebral malaria (CM) and acute kidney injury (AKI) are some of the most
common malaria-associated complications, which account to both great morbidity and mortality of the
infection. Our lab has recently identified autoantibodies that target the membrane lipid
phosphatidylserine (PS) on uninfected erythrocytes as a major promoter of malarial anemia both in
human patients and in a rodent model2. Other autoimmune antibodies, such as anti-DNA antibodies
that have a major role in promoting autoimmune pathologies in different autoimmune disorders, are
also present in malaria patients3. The aim of this work was to identify the presence of autoantibodies
against both PS and DNA in severe malaria. We hypothesized that these two types of autoantibodies
could correlate distinctly between these different malaria-associated complications. Methods: We
tested this hypothesis in a cohort of 382 Ugandan children (n=66 community controls, n=160 SMA,
n=156 CM) by using ELISA techniques for both anti-PS and anti-DNA IgG antibodies. AKI was a common
complication occurring in 56 (36.6%) of children with CM and 37 (23.9%) of children with SMA. Results:
Both types of autoantibodies were elevated in children with severe malaria compared to community
controls and were further elevated in children who died within 24 months of hospital discharge. Our
results showed differential correlation between these anti-PS and anti-DNA antibodies and malaria
associated complications. In multivariable linear regression models, anti-PS IgG antibodies were
positively associated with lactate dehydrogenase as a marker of red blood cell hemolysis and negative
associated with hemoglobin, while anti-DNA IgG antibodies were independently associated with
creatinine and AKI. Conclusion: Collectively, these results attribute a possible role of autoantibodies in
severe malaria and suggest autoantibodies as distinctive clinical parameters of these malaria-associated
complications.
Session Number: 394
Session Number: 394
Abstract Title:
Gastric Microbiota Associate with histological features
G. Yu1, T. Shimazu2, R. Meier1, D. C. Koestler1, M. S. Humphrys3, J. Ravel4, S. Tsugane2, A. M.
Goldstein5; 1Univ. of Kansas Med. Ctr., kansas City, KS, 2Natl. Cancer Ctr., Tokyo, Japan, 3Inst. for
Genome Sci., Baltimore, MD, 4Univ. of Maryland Sch. of M
Abstract Body:
Helicobacter pylori (Hp) is the major causative risk factor of gastric cancer. However, only <3% of Hp-
infected individuals develop gastric cancer, suggesting that other factors contribute to gastric
carcinogenesis. In this study, we examined the association between gastric microbiota, gastric cancer
risk factors and clinical features in a Japanese gastric cancer screening population aged 40-69 years with
no history of H. pylori eradication therapy who responded to a validated food frequency questionnaire.
Gastric microbial composition was profiled in the antral biopsies from 110 Hp+ and 150 Hp- subjects by
sequencing the V3-V4 region of the 16S rRNA gene using the Illumina HiSeq platform. Histological review
of antral biopsies was performed. Multivariate linear regression models stratified by Hp status were
used to examine the associations of risk factors/clinical features with microbiota alpha diversity/taxa
relative abundance. Permutation multivariate analysis of variance was used to test for an association
between risk factors/clinical features and beta diversity. Among Hp- individuals, we found no statistically
significant associations between any microbial features and age, sex, pack-years of smoking and intake
of green/yellow vegetables, fruit and salt. A suggestive association between total energy of dietary
intake and alpha diversity (P(Shannon index)=0.06) was observed, along with significant associations
between total energy of dietary intake and several taxa (P(Actinobacteria)=0.008), P(Firmicutes=0.05)
and P(Streptococcus)=0.007). Among Hp+ individuals, we found that microbial features (including alpha,
beta diversity and taxa relative abundance) significantly differed by clinical features (including Hp
density, chronical inflammation, polymorphonuclear neutrophil activity, intestinal metaplasia and
glandular atrophy), but not by the epidemiologic risk factors. In addition, we found that microbial
composition differed by Hp status. Our results revealed a possible association of microbial features with
gastric cancer risk factors in Hp- subjects and significant associations of microbial features with clinical
features in Hp+ subjects. Our results suggest a link between gastric microbiota and gastric
carcinogenesis and therefore warrant further studies.
Session Number: 394
Session Number: 394
Abstract Title:
VirMAP, a Fast and Comprehensive Tool for Viral Detection
N. J. Ajami, M. C. Wong, M. C. Ross, R. E. Lloyd, J. F. Petrosino; Baylor Coll. of Med., Houston, TX
Abstract Body:
Background: Despite the increased use of targeted assays to detect common viral etiologies, untargeted
methods using next-generation sequencing are gaining traction especially for undiagnosed diseases and
biosurveillance. In addition to the development of practical and inexpensive methods to process
specimens, bioinformatic approaches resulting in actionable data are urgently needed. Presently, viral
characterization from metagenomic data suffers from high background noise and signal crosstalk that
confounds current methods. Methods: To overcome these issues, we created VirMAP, a method that
considers both nucleotide and protein sequence information irrespective of its source to achieve high
resolution taxonomic classification. VirMAP employs both de-novo and mapping assembly strategies to
maximize recovery of viral sequence information independent of genome coverage or read overlap.
Results: We have applied VirMAP to >20,000 simulated, lab-made, environmental, and clinical datasets,
resulting in the identification of previously unreported viral strains and classification of identified strains
to higher taxonomic levels marked by a significant reduction in noise. Conclusions: This innovative
method offers new opportunities to support viral detection and surveillance through a reliable and
unbiased approach yielding optimal precision and recall values.
Session Number: 395
Session Number: 395
Session Title: CPHM02 - Antimicrobial Susceptibility Testing: Novel Methods
Poster Board Number: SUNDAY - 205
Abstract Title:
Rapid Antimicrobial Susceptibility Testing (Ast) Via Laser Light Scattering Technology for Gram Negative
Organisms from Screen Positive Urine
K. M. Riederer, R. Witherell, J. T. Fishbain; St. John Hosp. and Med. Ctr., Grosse Pointe Woods, MI
Abstract Body:
Background: Rapid screening for urinary tract infection and organism identification is currently available
using narrow angle forward laser scattering technology followed by matrix assisted laser desorption
ionization-time of flight mass spectrometry (MALDI-TOF MS). Rapid AST (RAST) could add critical
information for treatment. We evaluated a new RAST method using 4 antibiotics for Gram negative
pathogens. Methods: Adult inpatient urine submitted for culture was screened for presence of >104
CFU/ml by monitoring growth kinetics (216 Dx, BacterioScan). Organisms were identified by MALDI-TOF
MS (Biotyper, Bruker) via direct smear from positive turbid cuvettes. Gram negatives were diluted in
Mueller Hinton broth and inoculated to antibiotic cuvettes for RAST (216 Dx). CLSI breakpoint
concentrations of Ciprofloxacin (CIP), Ceftriaxone (CRO), Cefazolin (KZ) and Meropenem (MEM) for
sensitive (S) and intermediate (I) interpretation were used. RAST growth kinetics along the no drug
control curve were considered resistant (R). RAST and parallel disk diffusion were interpreted in blinded
fashion then compared to MIC reports (Vitek 2, bioMerieux) from urine culture isolates (gold standard).
Categorical agreement was calculated and discordance classified as minor (I versus S or R), major (false
R) or very major (false S) errors. QC for E. coli (pan S), E. cloacae (pan R) and K. pneumoniae CIP, KZ (R)
CRO, MEM (S) was included. Time required for RAST results was assessed. Results: 1015 urines were
screened and 112 AST performed on Gram negative organisms. 90 AST were evaluated; 22 were
polymicrobic and excluded (15 with ≥ 3 organisms, 7 with 2 organisms on culture). RAST concordance
with MIC interpretation for all 4 drugs was 94.4%. Five errors from separate samples were noted: 1 CIP
minor (K. pneumoniae), 2 CRO minor (K. pneumoniae, E. coli), 1 MEM major (A. baumannii) and 1 KZ
very major (E. coli). RAST categorical agreement with MIC interpretation for individual drugs (CIP, CRO,
MEM, KZ) was 98.9, 97.8, 97.7 and 98.8% respectively. RAST results were available within 4 hours.
Conclusions: Rapid AST via laser light scattering technology provides early phenotypic evidence of
antimicrobial susceptibility and resistance. RAST at CLSI breakpoint concentrations correlates well with
standard MIC interpretation. Urine screening, Gram negative organism identification and susceptibility
information can be available within 8 hours to guide treatment. Further testing is needed to determine
RAST reliability with additional organisms and antimicrobial drugs.
Session Number: 395
Session Number: 395
Abstract Title:
Maldi-Tof Ms Identification and Phenotypic Ast Delivering True Mics in Hours, Directly from Clin. Positive
Blood Cultures
M. Klintstedt1, Y. Molin1, M. Sandow1, S. Vincentsson1, C. Goransson1, J. Goransson1, J. Grawé1, A-K.
Smekal2, H. M. Riedel3, M. Gullberg1; 1Q-linea, Uppsala, Sweden, 2Uppsala Univ. Hosp., Uppsala,
Sweden, 3Uppsala Univ., Uppsala, Sweden
Abstract Body:
Background: Rapid pathogen identification (ID) and antimicrobial susceptibility testing (AST) are
important factors in the battle against antimicrobial resistance to improve antimicrobial stewardship
and provide optimal treatment to the septic patient. Here we present data from a combination of
MALDI-TOF mass spectrometry (MS) identification of pathogens and a novel prototype AST system,
ASTar™ (Q-linea), using clinical blood cultures from Uppsala University Hospital (UUH).
Materials/Methods: Clinical positive blood cultures were collected during a two-week period and
directly plated on agar plates. Both aerobic and anaerobic BACT/ALERT® VIRTUO® (bioMérieux) blood
culture flasks, cultured in a BACT/ALERT® VIRTUO® system (bioMérieux), were included. After a short-
time incubation (4-6 hours) colonies were run on a Microflex™ LT MALDI-TOF System (Bruker), according
to UUH routine procedures. Simultaneously, separate aliquots were taken and transported to Q-linea
(400 meters, ~5 minutes transport) for automated AST analysis using a prototype ASTar™ system. The
500 µl aliquot was subjected to automated sample preparation followed by inoculation into discrete
concentrations of different antimicrobials in dilution ranges. Time-lapse images were taken, and
proprietary algorithms translated growth results into minimum inhibitory concentration (MIC) values
within 6 hours. Reference MICs were obtained from the EUCAST Development Laboratory. Results:
Rapid culturing and MS identification gave results with scores above 1.95 for all samples and above 2.0
for 23 out of 26 samples. One sample flagged as polymicrobial by the MS was excluded for AST analysis
as well as two others, reported as mono-microbials, but showed multiple pathogens after overnight
incubation on plates. Of the 25 samples run for AST analysis, one gave rise to a fail in the AST analysis
yielding a total of 24 samples in the study with an overall Essential Agreement (EA) over 90% and
Categorical Agreement (CA) over 95%. Conclusions: This study evaluated a combination of MALDI ID
following short-time incubation on plates, and automated AST using a prototype AST system, directly
from positive blood cultures. The rapid ID gave correct result in 92% of the samples, failing on two
polymicrobial samples whereas the AST were in good concordance with 18-hour MIC. Results indicate a
future workflow using MALDI pathogen identification and ASTar™ AST analysis delivering complete
results within 4-6 hours from a positive blood culture.
Session Number: 395
Session Number: 395
Abstract Title:
Maldi-Tof Ms As An Alternative Tool for Rapid Detection of Vancomycin-Resistant “Enterococcus
faecium”
R. L. Ribeiro, T. C. A. Pinto, J. M. Morais, F. S. P. Rocha, L. M. Teixeira; Univ.e Federal do Rio de Janeiro,
Rio de Janeiro, Brazil
Abstract Body:
Vancomycin-resistant Enterococcus faecium (VREfm) has been appointed by the World Health
Organization as one of the current twelve most important antimicrobial resistance threats for public
health worldwide. VREfm is a leading cause of opportunistic infections, especially in hospital
environments, being associated with therapeutic challenges and a high ability to disseminate. Thus,
rapid detection of this microorganism, in both colonized and diseased patients, is essential to prevent,
control and properly treat enterococcal infections. Nevertheless, identification of VREfm can be slow,
expensive or even inaccurate by the existing phenotypic (conventional and automated) and genetic
methods. In the present study, we evaluated the ability of MALDI-TOF MS to discriminate between
VREfm (91 strains) and vancomycin-susceptible E. faecium (VSEfm; 31 strains) isolates recovered from
human sources (intestinal colonization and enterococcal infections) in Brazil. All 122 strains were
previously identified and evaluated regarding vancomycin susceptibility by phenotypic and/or genotypic
methods. For MALDI-TOF MS analysis, fresh colonies were directly applied to the target plate and
covered with CHCA matrix solution. Measurements were performed with a Microflex LT mass
spectrometer using default parameters and generating spectra in the range of 2,000-20,000 m/z.
Spectra were then analyzed with the BioNumerics software v7.6. All 122 strains were correctly identified
to the species level, with scores >2.0. By using BioNumerics a comparative analysis of the spectra was
performed. Overall, 30 peaks (biomarkers) ranging from 2,212 to 10,225 m/z were detected. Twelve of
these biomarkers were exclusively associated with VREfm: four of them were present in 100% and eight
were present in more than 90% of the 91 VREfm strains and absent in all 31 VSEfm isolates. Our results
indicate that MALDI-TOF MS is a promising alternative procedure to discriminate between VREfm and
VSEfm isolates from human sources, highlighting its usefulness for rapid and cost-effectiveness routine
application in clinical laboratories.
Session Number: 395
Session Number: 395
Abstract Title:
Detection of Kanamycin Modifications in Bacteria-Free Supernatants Using Ultra-High Performance
Liquid Chromatography Tandem Mass Spectrometry
C-Y. Chen, J. J. Perez; USDA, Wyndmoor, PA
Abstract Body:
Background: Early detection of antibiotic resistant bacteria is important in establishing proper treatment
and minimizing unnecessary use of broad-spectrum antibiotics. Conventional antibiotic susceptibility
tests (AST) are time-consuming and labor-intensive, and thus, there is a need for development of
accelerated tests since few alternatives are available, particularly for the family of aminoglycosides.
Methods: Ultra-High Performance Liquid Chromatography Tandem Mass Spectrometry (UHPLC-MS/MS)
was used to develop assays for kanamycin modifications, specifically acetylation and phosphorylation.
Escherichia coli strains carrying plasmid-borne aminoglycoside acetyltransferase (AAC) or
phosphotransferase (APH) were collected either from agar plates or from broth cultures, washed in
water and incubated with kanamycin solutions for various durations at 37°C. Cell-free supernatants
were minimally processed for MS by adding ion pairing reagent and standardization control before
injection into the instrument and performing qualitative and quantitative analyses. Results: Full scan and
tandem MS/MS analysis was performed using QTrap® 6500+ (AB Sciex) and Q-Exactive hybrid
quadruple-Orbitrap MS (ThermoFisher) for unit- and high-resolution analysis, respectively. Detection of
modified kanamycin (acetylation and phosphorylation) was achieved after as little as 30 min incubation
time (tested for up to 24 hr) and over a range of kanamycin concentrations (0.5 – 50 mg per liter). High
resolution MS confirmed the structural predictions of the modified kanamycin and detected the product
ions with high mass accuracy (~ 7 ppm). Conclusion: The two newly developed UHPLC-MS/MS analytical
techniques for kanamycin modifications resulting from AAC and APH enzymes are rapid and sensitive,
capable of detecting modified products at concentrations 50-100-fold lower than the MIC. The MS assay
could be a viable faster alternative to the AST or molecular detection methods, although it will not be
able to evaluate resistance not resulting from kanamycin modifications. The analysis can be directly
integrated into routine procedures and does not require lengthy preparation. These can be further
developed into assays for other members of the aminoglycoside family and for different modes of
enzymatic modification such as aminoglycoside nucleotidyltransferase (ANT).
Session Number: 395
Session Number: 395
Abstract Title:
A Rapid and Novel Detection Method for Confirmation of Urinary Tract Infections and Antimicrobial
Susceptibility
K. Falconer, S. Gillespie, R. Hammond; Univ. of St Andrews, St Andrews, United Kingdom
Abstract Body:
Background: Urinary Tract Infections affect 150 million people worldwide annually. Up to a third of
patients receive inappropriate antibiotic treatment due to the time consuming nature of current urine
culture and antimicrobial susceptibility testing methodology. The Scattered Light Integrating Chamber
(SLIC) provides a rapid and simple to use method for the high-resolution monitoring of bacterial growth
and antimicrobial response in real time straight from the patient’s urine. Methods: The two most
common uro-pathogens; E.coli and E. faecalis were used as model organisms for mock urine studies,
which allowed the limit of detection of SLIC and time to positivity to be calculated. A proof-of-principle
study was used to confirm the clinical utility of SLIC with 36 patient urine samples tested against a panel
of 5 different antibiotics to determine antimicrobial susceptibility. Results: Our study has demonstrated
that a UTI can be confirmed in less than 10 minutes. E. coli infection with a bacterial load of 103
eCFU/ml or greater can be detectable in 33 seconds and E. faecalis can be detectable in under 6
minutes. A proof of concept study demonstrated that it is possible to determine the presence or
absence of infection for a range of uro-pathogens and determine their antimicrobial susceptibility
directly from the sample in under 30 minutes with a sensitivity of 100% and specificity of 82%.
Conclusion: Rapid confirmation of UTI and subsequent antimicrobial susceptibility is desperately
needed. SLIC can answer two important questions in minutes; - Does this patient have a bacterial
infection? - If yes, then what is the most effective antibiotic for this patient? SLIC’s use and value in
assisting with the diagnosis and antimicrobial therapy of UTIs is particularly promising as it offers AST
results in minutes rather than hours.
Session Number: 395
Session Number: 395
Abstract Title:
Novel Platform for Rapid Ast with Expanded Antibiotic Menus
E. Stern1, A. Vacic1, K. Flentie1, B. R. Spears1, N. Purmort1, F. Giok1, K. DaPonte1, S. Scott1, D. Puff1, F.
Floyd1, Z. Zhang1, P. Reilly1, E. Viveiros1, N. Phelan1, C. Krebill1, T. Cerier1, A. N. Flyer1, D. C. Hooper2,
D. L. Smalley3, M. Ferraro4; 1SeL
Abstract Body:
Background: Prompt delivery of proper antibiotic therapies to infectious disease patients is essential for
improving patient outcomes, decreasing hospital lengths-of-stay, and combating the spread of
antibiotic-resistant pathogens. Antibiotic stewardship programs address these goals by coordinating
hospital efforts to rapidly deliver the most effective antibiotics for each patient, which requires accurate
antimicrobial susceptibility testing (AST). There is thus a strong clinical need for an AST platform that
quickly provides minimum inhibitory concentration (MIC) results for extended antibiotic test menus (30+
drugs). SeLux has developed an automated, rapid, phenotypic AST platform that enables simultaneous,
same-shift susceptibility testing full dilution series of a wide array of antibiotics, including newly
approved drugs. The core of this high-throughput, low-cost technology is an endpoint assay for bacterial
surface area, which enables delineation of truly resistant bacteria from filamented or swelled
(spheroplast or protoplast) organisms as can occur prior to lysis. Methods: AST was performed with the
SeLux platform and compared against the CLSI broth microdilution reference method on a total of 800
bacterial isolates obtained from clinical laboratories and 300 FDA-CDC and BEI “challenge” isolates.
Isolates comprised 20 species and multiple genera of non-fastidious bacteria. Each isolate was tested
against 20 antibiotics representative of the major antibiotic classes. Results: SeLux’s platform returned
definitive susceptibility results within 5 hours from >90% of the isolates tested to date. Essential
Agreement (EA) with the reference method was ≥90% for all combinations tested. Categorical
Agreement (CA) was also ≥90%. Overall averages are below. <table class="AbstractTable"
id="{CFA5BDC3-8EF4-4339-88C5-8B8CDDD22A47}"><caption
colspan="1"></td><td rowspan="1" colspan="1">Enterobacteriaceae</td><td rowspan="1"
colspan="1">P. aeruginosa</td><td rowspan="1" colspan="1">A. baumanii</td><td rowspan="1"
colspan="1">Staphylococcus</td><td rowspan="1" colspan="1">Enterococcus</td></tr><tr><td
rowspan="1" colspan="1">EA</td><td rowspan="1" colspan="1">95.0%</td><td rowspan="1"
colspan="1">96.1%</td><td rowspan="1" colspan="1">97.6%</td></tr><tr><td rowspan="1"
colspan="1">CA</td><td rowspan="1" colspan="1">93.7%</td><td rowspan="1"
colspan="1">94.2%</td><td rowspan="1" colspan="1">96.5%</td></tr></table> Conclusion: By
speeding the reporting of AST results, SeLux’s platform should enable hospitals to simultaneously
improve patient care, decrease lengths-of-stay, and meet antibiotic stewardship goals. Furthermore, the
platform’s compatibility with 384- and 1536-well microplates enables complete dilution-range testing
for a broad array of drugs, should transform the rate with which newly approved antibiotics gain use in
the clinic, and may speed antibiotic development.
Session Number: 395
Session Number: 395
Abstract Title:
Combined Infection Confirmation and Antibiotic Susceptibility in Under Five Hours
K. Babcock, S. Markakis, S. Strenn, C. Schneider, P. Harris; Affinity Biosensors, Santa Barbara, CA
Abstract Body:
Current diagnostics for microbial infection require a two-step process: incubation using growth media,
followed by identification and/or phenotypical AST. For blood stream infections, an average of 15 hours
is required to confirm infection, followed by a minimum of 7 hours to an AST result, with much longer
times found in most labs. We have developed a unified platform that greatly reduces both the time to
confirm infection and the time to a true phenotypical AST result. The system is easily automated,
accommodates standard antibiotic panels, is extensible to most sources of infection, and interfaces
naturally with most identification platforms. The system employs a mechanically resonant microchannel
optimized to measure microbe concentration in broth microdilution cultures in 15 seconds, and is
automated to measure growth in a 96 well plate format. Samples were spiked with Gram negative
microbes of various species at concentrations from 1/ml to 106/ml. The cultures were sampled every
few minutes until growth by replication was confirmed. These cultures were then used to inoculate 96
well plate antibiotic panels (LifeScale GN3) to run rapid ASTs, again by monitoring growth. The
fundamental performance, and best-case time-to-results, were measured first by spiking growth media.
Performance under clinical conditions was then tested by spiking urine and blood samples at clinically
realistic levels. Large content (RBCs, protein rafts, etc.) was removed by centrifuging 3 minutes @ 1000g,
and the supernate was diluted at least 10:1 with MH broth and processed on the system. For urine
samples spiked with E. coli at 5x104/ml, growth was confirmed after 120 minutes of incubation. The
culture was used to inoculate the well plate containing 14 antibiotics, which was measured after an
additional hour of incubation, and MIC’s were reported 4 ½ hours after the urine sample was spiked. For
blood samples, additional time was required to confirm infection due to platelets which the system
cannot distinguish from microbes. For whole blood spiked with Klebsiella pneumonia at 10-100/ml,
approximately 7 hours is required for microbes to outnumber platelets and allow their presence to be
confirmed, with a total time to AST result of 9 hours. The culture at confirmation provides an excellent
source for identification (e.g., MALDI or molecular methods). Ongoing work aims to demonstrate that
comprehensive clinical results, including confirmation, identification, and AST, can be produced in 4-10
hours from sample draw for UTI’s, BSI’s, and CSF infections.
Session Number: 395
Session Number: 395
Abstract Title:
Scattered Light Integrating - Collector Point-Of-Care (Near Patient) Clin. Breakpoint Analysis
R. J. H. Hammond, K. J. Falconer, S. H. Gillespie; Univ. of St Andrews, St Andrews, United Kingdom
Abstract Body:
Background: The burden of Anti-Microbial Resistance (AMR) is a growing problem globally. In the O’Neill
report (May 2016) it was stated that one of the key milestones in stopping AMR as a global problem was
effective rapid antibiotic susceptibility tests (AST). Here, we present a device that determines
susceptibility rapidly and could help turn the tide of AMR. SLIC (Scattered Light Integrating Collector) is a
sensitive device for the detection of both bacterial and fungal based on the scattering of laser light.
Methods: Proof a concept studies were carried out initially to establish the lower limit of detection. This
was found to be 10-50 CFU/mL. This exquisite sensitivity allowed us to commence work establishing
rapid MICs. Starting with an inoculum of 105 mL bacteria and using a relevant range of antibiotic
concentrations the MIC can be established in less than one microbial doubling period. Results: The rapid
and sensitive detection SLIC affords allows for fast growing organisms such as E. coli and S. aureus to
have their MICs established in less than 30 minutes, for any antibiotic. For slow growing organisms such
as M. bovis we are able to establish an MIC in <2 hours. The technology can also be used for fastidious
and difficult to grow organisms such as H. influenzae and Mycoplasma spp. Figure 1. Real-time lytic
effect of meropenem on a culture of ATCC 35218 E. coli observed over a 30 minute time period
Conclusion: As bacterial quantification is continuously monitored we are able to see the action of
antibiotics in real time. Using this facility, we can readily distinguish between lytic antibiotic such as
meropenem from bactericidal but non-lytic antibiotics such as gentamicin. This provides the opportunity
to gain new insights into the mechanisms of action and the effect antibiotics have or microbes in a new
way. We believe that SLIC has the potential to change the way and AMR research progresses in the
coming years.<br /><p><a
href="http://files.abstractsonline.com/CTRL/3f/5/065/4cf/6d7/4ad/f8e/05f/9d7/af9/d5b/d0/g5319_1.j
src="http://files.abstractsonline.com/CTRL/3f/5/065/4cf/6d7/4ad/f8e/05f/9d7/af9/d5b/d0/g5319_1.jpg
" alt="" border="0" width="600" height="483" /></a></p>
Session Number: 395
Session Number: 395
Abstract Title:
Comparison of the Accelerate Pheno®, Vitek®2, Verigene®, and Maldi Biotyper® Systems for Microbial
Identification and Antimicrobial Susceptibility Testing
J. G. Schneider, C. Emery, T. Davis, R. Relich, B. Bocian, J. Wood, J. Manaloor, B. Schmitt; Indiana Univ.
Sch. of Med., Indianapolis, IN
Abstract Body:
Background: Historically, microbial identification and antimicrobial susceptibility testing methods have
been limited by the need to test pure cultures of bacterial isolates. Because of the rapidly increasing
prevalence of multi-drug resistant pathogens, quick and reliable methods that obviate the need for
isolate procurement are essential for the timely implementation of appropriate antimicrobial therapy,
especially for bloodstream infections. We present results comparing bloodstream pathogen
identification and antimicrobial susceptibility testing (AST) using the Accelerate Pheno™ (AXDX),
VERIGENE®, MALDI-TOF, and VITEK®2 systems. Methods: One hundred forty-eight patients, including 32
pediatric patients, with monomicrobial Gram-negative bacteremia were enrolled; blood culture broth
aliquots were tested on the VERIGENE® and Accelerate Pheno® systems in tandem after bottles flagged
positive by the BACTEC® FX instrument. Bacterial isolates were routinely identified by mass
spectrometry (Bruker MALDI Biotyper) and AST was performed by the VITEK®2 (GN73 card). Results: For
bacterial identification, the Accelerate Pheno® (AXDX) had a positive percent agreement (PPA) of 94.6%
and an accuracy of 99.7% compared to VERIGENE®, along with a PPA of 95.8% and an accuracy of 99.8%
compared to the MALDI Biotyper®. VERIGENE® required a mean time of 2.1 h for identification from
time of set-up while AXDX averaged 1.3 h. Mean time for MALDI Biotyper® confirmatory testing was
36.3 h from time of blood culture positivity. For AST, the AXDX had an overall categorical agreement of
93.1%, including 91.2% for AST in all ESBL isolates, compared to the VITEK®2. Routine isolate AST by
VITEK®2 required a mean of 36.2 h compared to a mean of 6.6 h by AXDX. Discussion: Overall, the
Accelerate Pheno® provides reliable results that are comparable to other methods, including molecular
and proteomics-based methods for organism identification and phenotypic and molecular methods for
AST. The rapidity of the result TAT has the potential to significantly reduce TAT for positive blood culture
identification and AST resulting, and thus can likely further aid in effective antimicrobial stewardship.
Session Number: 395
Session Number: 395
Abstract Title:
Direct Antimicrobial Susceptibility Testing of Positive Blood Cultures Comparing the Accelerate Pheno™
and Vitek®2 Systems
J. G. Schneider, C. Emery, T. Davis, R. Relich, J. Wood, B. Bocian, B. Schmitt; Indiana Univ. Sch. of Med.,
Indianapolis, IN
Abstract Body:
Background: Current diagnostics for identification and antimicrobial susceptibility testing (AST) are
based primarily on testing bacterial culture isolates. As rates of multi-drug resistant pathogens rise,
reliable and rapid diagnostic tests are necessary in the selection of effective antimicrobial therapy,
which may have significant implications for patient outcomes and antibiotic stewardship. We present
results comparing antimicrobial susceptibility testing (AST) of positive blood cultures using the
Accelerate Pheno™ (AXDX) and VITEK®2 systems, along with direct-inoculation AST using the VITEK®2.
Methods: Blood cultures from 148 patients, including 32 pediatric patients, with monomicrobial Gram-
negative bacteremia were included in our study. All blood culture broths were tested on the Accelerate
Pheno™ and by direct inoculation on the VITEK®2 in tandem. For the latter, bacterial cell suspensions
were made by pelleting bacteria from broth in serum separator tubes and resuspending the pellets in
saline. VITEK®2 GN73 cards were inoculated with cell suspensions adjusted to the turbidity
approximating a 0.5 McFarland standard. All AST outcomes were compared to VITEK®2 results obtained
from testing isolated colonies of bacteria. Results: Compared to direct inoculation on the VITEK®2, the
AXDX had an overall categorical agreement of 91.5%. Measured against isolated colonies by VITEK®2,
the AXDX had a categorical agreement of 93.1%. When comparing direct-inoculation AST to VITEK®2
isolated colonies using VITEK®2, there was 97.9% categorical agreement. Direct-inoculation of VITEK®2
testing required a mean of 13.0 h for AST, compared to a mean of 6.6 h by AXDX and 36.2 h for isolated-
colony AST by VITEK®2 Conclusions: Overall, antimicrobial susceptibility testing by Accelerate Pheno™
and direct VITEK®2 inoculation from positive blood cultures can aid in expediting appropriate antibiotic
treatment, which may have significant implications for patient outcomes and antimicrobial stewardship.
Session Number: 395
Session Number: 395
Abstract Title:
Direct Phenotypic Antimicrobial Susceptibility Testing is An Accurate Method to Guide De-Escalation and
Improve Patient Care
B. E. Whitacre, M. V. Powers-Fletcher; Univ. of Cincinnati, Cincinnati, OH
Abstract Body:
Background: Rapid initiation of broad-spectrum antibiotic therapy is standard of care for patients
presenting with sepsis. De-escalation of empiric treatment to targeted therapy is essential to combat
increasing rates of resistance, decrease the risk of adverse effects, and reduce healthcare costs. Despite
the use of molecular resistance detection assays, de-escalation is often delayed for days until the results
of phenotypic antimicrobial susceptibility testing (AST) return. A more rapid approach to phenotypic AST
may improve this process. The purpose of this quality improvement project was to determine if direct
phenotypic AST (dAST) from positive blood culture bottles could be used as an accurate method to guide
de-escalation. Methods: This study combined results from positive blood cultures (BacT/ALERT®)
inoculated with either clinical specimens as part of routine patient care or quality control (QC)
organisms. dAST was performed using a modified Kirby-Bauer method. A swab saturated with broth
from a positive culture was used to inoculate a Mueller-Hinton plate, streaking for a lawn of growth.
Gram stain morphology or rapid molecular identification (Verigene®) determined which panel of agents
was tested. Plates were incubated for 12-16 h before results were recorded and interpreted using the
clinical interpretive breakpoints published by CLSI. Either the expected QC values or the results derived
from clinical work-up of isolated colonies (Vitek® 2) were used as the comparator. Results: A total of 73
tests were performed during the study period; nine samples were excluded because they were mixed or
failed to grow. The remaining tests included 48 Gram positive (GP) and 16 Gram negative (GN)
organisms. For the GP organisms finalized, the dAST results correlated with the comparator with 92%
agreement; for GN, agreement between dAST and comparator was 96%. For all organisms tested, there
were six major errors and five very major errors. Results were available 12-16h following Gram stain;
current time to phenotypic AST results at our institution is 72h, on average. Conclusions: dAST from
positive blood culture broth is an accurate means of predicting definitive phenotypic susceptibility
results. Because these results can be made available up to 60h faster than current methods, this
approach could have a significant impact on antimicrobial stewardship efforts and improve patient care.
Research efforts should be made to standardize this approach so that it may be widely applied by clinical
laboratories.
Session Number: 395
Session Number: 395
Abstract Title:
Performance of Colistin Mic Determination of the Accelerate Phenotest Bc Kit for Res. Use Only
J. Towne, R. Humphries; Accelerate Diagnostics, Tucson, AZ
Abstract Body:
Background: Susceptibility testing of colistin (CST) has been a challenge for laboratories due to several
technical issues, including variability of stock powders and solutions, the propensity for CST to adsorb to
plastics, and the absence of U.S. Food and Drug Administration-endorsed clinical breakpoints by which
to interpret CST minimum inhibitory concentrations (MIC). This study compared MIC agreement of the
Accelerate PhenoTest™ BC kit (AXDX) research-use-only (RUO) CST results to those of reference broth
microdilution (rBMD). CLSI CST breakpoints and epidemiological cut-off values (ECVs) were utilized to
interpret both rBMD and AXDX MICs. Methods: A challenge set (n=33) of Gram-negative bacteria used
previously by the CLSI to evaluate CST tests was assessed. This included 5 Acinetobacter baumannii
(ABA; 2 resistant), 10 Pseudomonas aeruginosa (PAE; 2 resistant) and 18 Enterobacteriaceae (8 non
wild-type by CLSI ECV, 4 with mcr-1 resistance gene). Isolates were seeded into BD BACTEC™ Aerobic
Plus bottles containing 10 mL of diagnostic human blood and bacteria at a concentration of ~50 clones
and incubated on the BD BACTEC™ FX until they flagged positive. In parallel, rBMD was performed as
defined by the CLSI M07-A4 standard. Essential agreement (EA; i.e., MIC +/- 1 log2 dilution) and
categorical agreement (CA) with CLSI breakpoints/ECVs was assessed. Results: Upon sub-culture from -
80oC frozen, 1 ABA and 1 PAE were found to no longer be CST resistant. Using results from parallel
testing, CST EA of AXDX with rBMD was 97.0% (32/33) and evaluable EA was 96.6% (28/29). One isolate,
an ABA with a rBMD MIC of 1 µg/mL and AXDX MIC of 4 µg/mL, was out of EA. This isolate had MICs
ranging from 1 to 2 µg/mL when tested previously. CA was 97.0% (32/33), with a sole major error (false
resistance) for the ABA which was out of EA. All 4 isolates (Escherichia coli) with mcr-1 yielded an MIC of
≥8 µg/mL by AXDX (non wild-type). Conclusions: The AXDX RUO CST test performed well when
compared to rBMD.
Session Number: 395
Session Number: 395
Abstract Title:
Evaluation of Lifescale Instrument for Rapid Antimicrobial Susceptibility Testing of Gram Negative
Organisms from Positive Blood Culture Bottles
M. L. Faron, D. Gerstbrein, B. W. Buchan, N. A. Ledeboer; The Med. Coll. of Wisconsin, Milwaukee, WI
Abstract Body:
Background: Bloodstream infections are a serious health risk to patients, and the delay in appropriate
antibiotic administration can cause significant increase in mortality. Current antimicrobial susceptibility
testing requires 48-72 hours from bottle positivity, often leading to broad-spectrum empirical
treatment. In this study, we evaluated the preliminary version of the LifeScale (Affinity Biosensors, Santa
Barbara, CA) instrument that detects microbe mass and can report minimal inhibitory concentration for
gram negative rods (GNR) in 6 hours from positive blood culture and compared the results to BD
Phoenix (BD Diagnostics, Sparks MD) as the gold standard. Materials/Methods: Positive blood culture
bottles that were observed to have GNR within a Gram stain were de-identified and enrolled into the
study. An aliquot of 1.5mL of positive blood culture media was centrifuged to remove blood cells and
1mL of supernatant was added to 12mL of filtered Mueller-Hinton broth. Bacterial cells were counted by
the instrument to determine the dilution required to produce an inoculum load of 5x105 cells/ml.
Dilution and dispensing into a Sensititre GN2F plate was performed by an automated pipette station.
Dilutions were plated for purity and the inoculated plate added to the LifeScale instrument. An initial
bacterial count is performed to confirm the starting concentration. After 2.5 hours of incubation, the
instrument measures again looking for growth in the no-antibiotic control wells. If growth meets pre-set
thresholds, minimal inhibitory concentrations are determined for each antibiotic by comparing the
growth difference of each well from the initial reading. If thresholds are not met, the instrument re-
incubates the plate and performs measurements a second set of measurements. FDA breakpoints were
used for interpretation. Results: In total 31 monomicrobial aerobic GNR were tested including 12 E. coli,
9 Klebsiella, 3 P. aeruginosa, 3 Enterobacter cloacae complex, 2 S. marcescens and 2 Acinetobacter.
Three specimens failed to obtain growth in control wells and did not report a MIC. Overall categorical
agreement and essential agreement was >90% for 10/13 and 11/13 antibiotics. Average time to result
was 5.6 hours with approximately 15 minutes of hands on time. Discussion: The LifeScale instrument
was highly accurate in determining the MIC of pathogens directly and can reduce turnaround time by 42
hours or more. Future studies will compare LifeScale specific antibiotic plates to microbroth dilutions.
Session Number: 395
Session Number: 395
Abstract Title:
Fully Automated Antimicrobial Susceptibility Test Sys. Delivering Phenotypic Mics in Hours from Positive
Blood Cultures for Fastidious- and Non-Fastidious Pathogens
M. Klintstedt, Y. Molin, M. Sandow, L. Levén, J. Grawé, C. Goransson, J. Goransson, M. Gullberg; Q-linea,
Uppsala, Sweden
Abstract Body:
Background: Rapid identification (ID) and antimicrobial susceptibility testing (AST) are important factors
in the battle against antimicrobial resistance to improve antimicrobial stewardship and provide the
septic patient with the right antimicrobial treatment. Fastidious bacteria are a virulent group of
pathogens isolated from around 10% of positive blood cultures. Currently, there is no rapid AST system
for fastidious bacteria available, and clinicians are forced to rely on empiric therapy until traditional AST
results are available. Here we present data from a new system for automated susceptibility testing
directly from positive blood cultures including fastidious pathogens, ASTar™ (Q-linea). The AST results
presented here were obtained on a prototype system using blood cultures with spiked clinical isolates.
The tested panel contained 21 different antimicrobials in multiple concentrations per antimicrobial,
covering clinical breakpoints set by EUCAST. In total 12 different pathogens were tested in this study
including the fastidious pathogens Streptococcus spp. and Haemophilus spp., as well as non-fastidious
pathogens including Pseudomonas spp., yielding over 57 unique bug-drug combinations.
Materials/methods: Blood culture flasks spiked with blood from healthy individuals and clinical isolates
were cultured until signaled positive. From each blood culture flask, a 500 µl aliquot was subjected to
automated sample preparation, followed by inoculation into discrete concentrations of different
antimicrobials in dilution ranges. Time-lapse images were taken, and proprietary algorithms translated
growth results into minimum inhibitory concentration (MIC) values. Results: Within 6 hours an overall
essential agreement and categorical agreement of more than 95% compared to 18-hour MIC were
achieved, in all bacteria-antimicrobial agent combinations tested. Around 30% of all isolates tested had
a resistant phenotype, and many had a MIC equal to or close to the breakpoint. Conclusions: This study
showed that the ASTar™ system can deliver rapid phenotypic AST results for pathogens, including
fastidious pathogens. The results were in concordance with 18-hour MIC, with excellent reproducibility.
In combination with established rapid pathogen ID technologies, ASTar™ enables healthcare providers
to provide correct antimicrobial treatment within 6 hours directly from positive blood cultures. This
means fewer hospital days, less overuse of broad spectrum antibiotics, and reduced costs for society.
Session Number: 395
Session Number: 395
Abstract Title:
Rapid Standard Inoculum Generator Directly from Positive Blood Culture Using An Electrical Biosensor
Technology
M. Herget1, O. Knopfmacher1, A. Estabrook1, N. Rajan1, J. Hurst1, T. Abbey1, K. Vo1, C. Hogan2, N.
Banaei3; 1Avails Med., Inc., Menlo Park, CA, 2Stanford Hlth.care Clinical Microbiol. Lab., Palo Alto, CA,
3Stanford Hlth.care Clinical Microbiol. Lab., Men
Abstract Body:
Introduction: Blood stream infections are life threatening and every hour a patient is not treated with an
effective antibiotic the survival chance decreases by 10%. This is aggravated by the emergence of
multidrug resistant bacteria where more and more antibiotics fail to be effective. Accelerating antibiotic
susceptibility testing (AST) from positive blood cultures to provide targeted antibiotic therapy early on is
key to improve patient survival. In this work, we aim to develop a new method based on an electrical
biosensor technology to generate a standardized inoculum (eMcFarland) directly from positive blood
cultures within 2 hours, referred to as eQuant method. The eMcFarland is used for subsequent AST.
Minimal inhibitory concentrations (MICs) generated with the eMcFarland in broth microdilutions are
compared to MICs by gold standard AST from colonies. Methods: Patient derived positive blood cultures
were de-identified and analyzed by MALDI-TOF for the identification of the bacterial pathogen. eQuant
was performed on blood cultures that were positive for gram-negative rods or Staphylococcus and
Enterococcus spp. and an eMcFarland was generated and used in subsequent broth microdilution AST.
In parallel, the same cultures were subcultured by the clinical laboratory for AST on the following day
using the automated Beckman Coulter MicroScan system. Broth microdilution MICs using the Avails’
eMcFarland inoculum from positive blood culture were compared to MICs obtained by the gold
standard AST for category and essential agreements. Results: With 11 clinical blood cultures that were
positive for E. coli (3x), K. pneumoniae (2x), S. marcescens (1x), P. vulgaris (1x), P. mirabilis (1x), S.
aureus (1x), P. aeruginosa (1x), we performed 130 ASTs (15-20 antibiotics per sample). Comparing our
eMcFarland/broth microdilution MICs with MicroScan AST results, 3.8% (5 ASTs) were associated with a
minor error, 0.8% (1 AST) were associated with a major error and none of the ASTs were associated with
a very major error. The overall essential agreement was 92.3 %. Conclusions: Avails eQuant technology
provides a standardized inoculum (eMcFarland) directly from positive blood culture within 1 to 2 hours
eliminating the need for time-consuming subcultures. We validated the Avails’ eMcFarland inoculum for
broth microdilution AST from 11 clinical positive blood cultures. We hypothesize that eMcFarland can be
used in conjunction with all AST methods, providing physicians with AST results at least 18 hours earlier
compared to current methods.
Session Number: 395
Session Number: 395
Abstract Title:
Development and Evaluation of A Laboratory-Developed Taqman Array Card (Tac) for Antimicrobial
Resistance (Amr) Detection
S. Pholwat1, J. Liu1, M. Taniuchi1, I. Thaipisutikul2, P. Ratanakorn3, S. Foongladda2, E. Houpt1; 1Dept.
of Med., Univ. of Virginia, Charlottesville, VA, 2Faculty of Med. Siriraj Hosp., Mahidol Univ., Bangkok,
Thailand, 3Faculty of Vet. Sci., Mahidol Uni
Abstract Body:
Background: Antimicrobial resistance (AMR) is a significant public health issue worldwide. Monitoring
and surveillance of resistance is one action for addressing and preventing AMR. Phenotypic
antimicrobial susceptibility testing is laborious and unable to test multiple antimicrobial agents
simultaneously. A rapid high throughput AMR detection tool will be useful to monitor AMR prevalence.
Methods: We designed and developed an easy-to-perform genotypic TaqMan array card with 90
sequence specific PCR reactions to detect 85 antimicrobial resistance associated genes or mutations for
10 highly important antimicrobial classes used in human and veterinary medicine. This included
cephalosporins, quinolones, macrolides, penicillins, aminoglycosides, polymyxins, folate pathway
inhibitors, tetracyclines, phenicols and carbapenems. The proposed AMR-TAC layout was shown in
figure 1. Results: The 90 qPCR assays were tested for performance against 85 well-characterized
Enterobacteraciae isolates from the FDA-CDC AR bank (CDC, Atlanta, GA, USA) on 384 well PCR plates.
Sanger sequencing was performed for confirmation when discordances were observed. Sixty two of the
90 assays revealed near-perfect concordance on the 85 isolates (i.e., 686 true positives, 4582 true
negatives, 2 discrepant). The remaining 28 assays are still undergoing optimization. Comparing the
association between genotypic (sequencing) and phenotypic DST of 31 E. coli isolates for 23
antimicrobial agents, we observed an overall sensitivity of 81-100% across the 23 drugs, specificity 62-
100%, and accuracy 81-100%. Kappa agreement was moderate to perfect between the two methods (κ =
0.58 - 1). Conclusion: This TaqMan array card yields an accurate susceptibility result compared to the
standard phenotypic DST.<br /><p><a
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Session Number: 395
Session Number: 395
Abstract Title:
Machine Learning Approach for Predicting Antimicrobial Resistance Using Genome Sequencing Data
B. Han, B. Metcalf, S. Chochua, L. McGee, B. Beall, Y. Li; CDC, Atlanta, GA
Abstract Body:
Background: The emergence and spread of antimicrobial resistance (AMR) is a major concern in public
health. Conventional detection of AMR involves a phenotypic test to determine antibiotic minimum
inhibitory concentration (MIC), which relies on obtaining a viable isolate and takes long processing time.
Whole genome sequencing (WGS) technology showed great potential to improve AMR detection. Here
we developed a simple software pipeline that used raw sequencing reads to predict MIC values for six β-
lactam antibiotics for Streptococcus pneumoniae, an important human pathogen. MIC prediction
performance was optimized by comparing different machine learning algorithms. Methods: Phenotypic
MIC values for penicillin, amoxicillin, meropenem, cefotaxime, ceftriaxone, and cefuroxime were
measured for 2713 pneumococcal isolates, which were identified through Emerging Infections
Program/Active Bacterial Core surveillance, using the micro-broth dilution method. All isolates were
sequenced on the Illumina platform. Single nucleotide variations (SNVs) at three pneumococcal genes
critical to conferring β-lactam resistance (pbp1a, pbp2b, and pbp2x) were identified using the kestrel
software. The SNVs were used as features in three different machine learning algorithms (LDA, SVM, and
random forest) to predict log2 transformed MIC values. Model training and five-fold cross-validation
were performed using the Python “SKlearn” package. Prediction performance was evaluated based on
goodness of fit (R2). Results: We obtained 16,278 phenotypic MIC values for the 2713 isolates and
identified 1247 SNV sites (features) in the three pbp genes. Cross-validation results showed prediction
performance (R2) for the six antibiotics ranging from 0.78 to 0.92, 0.83 to 0.93, and 0.89 to 0.97, for
LDA, SVM, and random forest, respectively. For each algorithm, the predictions for Ceftriaxone MIC
were less accurate compared to predictions for the other antibiotics. The average time to generate six
MIC predictions from a raw sequencing file was 145 seconds on a computer with 2 cores and 4G RAM.
The MIC prediction pipeline is available at “github.com/hbqdhr/ML_MIC.git”. Conclusion: We
implemented a machine learning-based MIC prediction pipeline that might serve as an efficient
alternative to phenotypic AMR determination. Prediction performance of different algorithms, although
all above 0.77, differed substantially using the same input features. The random forest algorithm
performed better than the other two algorithms for each of the six antibiotics.
Session Number: 395
Session Number: 395
Abstract Title:
Predicting the Currently Unpredictable: Developing A Tool to Use Whole Genome Sequences to Predict
Antimicrobial Susceptibility for Neisseria Gonorrhoeae
M. W. Schmerer1, H. Pouseele2, J. A. McLean1, J. Cartee1, S. T. Lucking1, P. S. O'Brien3, K. Kneupper4, C.
Wang4, R. Kirkcaldy1, D. L. Trees1; 1CDC, Atlanta, GA, 2Applied Maths, NV, 9830 Sint-Martens-Latem,
Belgium, 3Hawaii State Dept. of Hlth., Pearl Ci
Abstract Body:
Background: Neisseria gonorrhoeae has developed resistance to each antimicrobial recommended for
treatment; dual therapy with azithromycin and ceftriaxone is the only remaining recommended
treatment. Use of culture (necessary for antibiotic susceptibility testing [AST]) for gonorrhea diagnosis
has been largely supplanted in developed countries by nucleic acid amplification tests (NAATs), thus
molecular AST approaches are attractive. We developed a tool that uses raw whole genome sequence
(WGS) data and tested how accurately it predicts gonococcal susceptibility. Methods: The goal is to
predict the susceptibility of five classes of antimicrobials, which all have at least one genetic marker with
high positive and negative predictive values based on previously published work. We used a reference
mapping-based method that identifies mutations directly from raw sequencing reads and then analyzes
them using a decision tree-type algorithm for susceptibility prediction. The tool was developed using
two sets of isolates each of which contained both susceptible and non-susceptible isolates. The first
consisted of 261 location stratified, randomly chosen isolates from the Gonococcal Isolate Sequencing
Project (GISP) isolated during the years 2013 and 2014. The second consisted of 637 isolates from the
years 2016 and 2017, which underwent AST testing and sequencing at the Hawaii and Texas State Public
Health labs. WGS-based results were compared to Minimum Inhibitory Concentration (MIC) values
determined by agar dilution. MIC cutoffs used for reduced susceptibility/resistance were azithromycin
>= 2.0 ug/mL, ciprofloxacin >= 1.0 ug/mL, cefixime >= 0.25 ug/mL, ceftriaxone >= 0.125 ug/mL, penicillin
>= 2.0 ug/mL, and tetracycline >= 2.0 ug/mL. The complete analysis pipeline was carried out in
BioNumerics. A custom python script was used to compare output from BioNumerics to MIC values and
calculate the concordance between the tool and susceptibility category. Results: When tested on the
combined data, the concordance between the tool and the MIC-based categories is 97.1% for
azithromycin, 96.3% for ciprofloxacin, 99.0% cefixime and ceftriaxone, 87.4% for penicillin and 83.4% for
tetracycline. Conclusions: While the loci chosen for the tool are based on previously published data, the
low values for some drugs suggest that these loci alone are insufficient for predicting susceptibility vs
non-susceptibility. Future work will focus on additional loci to improve performance, and validation with
a broad set of isolates untested by the tool.
Session Number: 395
Session Number: 395
Abstract Title:
Detection of Methicillin Resistance in Staphylococcus aureus by Maldi-Tof Mass Spectrometry Using
Direct-On-Target Microdroplet Growth Assay (Dot-Mga) Directly from Positive Blood Cultures
E. Idelevich1, L. Storck1, K. Sparbier2, M. Kostrzewa2, K. Becker1; 1Univ. Hosp. Muenster, Muenster,
Germany, 2Bruker Daltonik GmbH, Bremen, Germany
Abstract Body:
Background: Direct-on-target microdroplet growth assay (DOT-MGA) based on MALDI-TOF MS has
recently been suggested for rapid antimicrobial susceptibility testing. This proof-of-principal study aimed
to investigate this method for detection of methicillin-resistant Staphylococcus aureus (MRSA) directly
from positive blood cultures (BCs). Methods: After pilot tests with agar cultures, six MRSA and six
methicillin-susceptible S. aureus isolates were added to 10 ml human blood in final concentration 10
cfu/ml. The inoculated blood was introduced into BACTEC aerobic bottles and incubated in the BACTEC
system. Each positive BC sample was processed by (i) dilution protocols followed by testing of serial
dilutions in cation-adjusted Mueller-Hinton broth (CA-MHB), and (ii) centrifugation protocols with
differential centrifugation and lysis/centrifugation, followed by dissolving the pellet and standardization
of inoculum to 5x105 cfu/ml in CA-MHB. 6-µl microdroplets containing S. aureus with or without 4 µg/ml
cefoxitin were spotted in triplicate onto MBT Biotarget96 target (Bruker). The targets were incubated at
36°C for 4, 5 and 6 hours, followed by medium removal. After adding matrix, MALDI-TOF MS spectra
were generated (Bruker) and identification scores ≥1.7 were designated as resistance. Broth
microdilution and mecA PCR were used as standard methods. Results: Initial testing of agar cultures
showed principle feasibility of DOT-MGA for S. aureus and reduction of test time by adding 1 µl of formic
acid to each spot, which was adopted for BC experiments. Testing 1:100 dilution from positive BCs
demonstrated best performance: 100% valid results (i.e., growth control detected) already after 5 hours.
At this time point, 100% sensitivity and 100% specificity were reached. Centrifugation protocols revealed
poorer performance achieving with differential centrifugation only 41.7% valid results after 6 hours.
Conclusions: This study demonstrated feasibility of rapid phenotypic detection of methicillin resistance
by MALDI-TOF MS-based direct DOT-MGA from positive BCs. Further optimization and standardization
of sample processing, measurement and evaluation algorithms is warranted.
Session Number: 395
Session Number: 395
Abstract Title:
Very Rapid Phenotypic Susceptibility Testing of Clin. Specimens Using the Novel Astar System
E. Hell, J. Goransson, T. Lindberg, F. Bjornlund, C. Goransson, M. Gullberg, J. Grawé; Q-linea, Uppsala,
Sweden
Abstract Body:
Background: Methods allowing rapid antimicrobial susceptibility testing (AST) are important factors for
timely and accurate antimicrobial treatment, as well as for reducing the global threat of antimicrobial
resistance. In this study we show that the innovative AST system, ASTar™ (Q-linea), can be used to not
only deliver on-scale MIC values within 3-6 hours but also to deliver a phenotypic AST with S/I/R
classification in only 30 to 60 minutes. Materials/methods: Urine samples from healthy volunteers were
spiked with bacteria yielding a final titre of 1*10^5 CFU/ml, ~5 ml of the spiked urine samples was used
for analysis. First, pathogens were isolated by an automated sample preparation process that removes
potential mammalian cells and other non-pathogen particles, finally presenting the pathogens in cation-
adjusted Mueller Hinton media. The isolated pathogens were then manually transferred to a prototype
AST consumable, cultured, and imaged using a dedicated prototype AST reader that allows rapid
imaging of large areas yielding a good representation of the sample assayed. While kept at 35°C,
automated time-lapse image series were acquired. Proprietary algorithms evaluated growth and
subsequently translated growth results into S/I/R results. For reference, MIC was obtained by broth
microdilution of the tested strains, incubated and read after 18 hours (+/-2 h). Results: After 30 to 60
minutes incubation with antibiotics we attained phenotypic AST results on gram negative bacteria for
selected antimicrobials directly from a clinical sample matrix, capable of correctly classifying the tested
strain as susceptible or resistant. For selected antibiotics a 30-minutes AST was sufficient, but time-to-
result is affected by the mechanism of action. Up to a total of 90 minutes assay time was needed to
achieve SIR results from all antibiotics in a set of antimicrobials covering; (i) DNA synthesis inhibitors, (ii)
cell wall targeting antibiotics inhibiting cell division, as well as (iii) ribosomes inhibitors. Conclusions: The
results presented here on bacteria isolated from urine samples were in concordance with 18-hour AST-
results and showed that ASTar™ can deliver very rapid phenotypic AST approaching point-of-care
requirements for selected antimicrobials.
Session Number: 395
Session Number: 395
Abstract Title:
In Vitro Test Sys. to Determine Tetracycline Residue Binding to Human Feces
Y. Ahn1, J. Jung1, B. T. Veach2, S. Khare1, K. Gokul1, S. A. Piñeiro3, C. E. Cerniglia1; 1Natl. Ctr. for
Toxicological Res., U.S. Food and Drug Admin., Jefferson, AR, 2Arkansas Regional Lab., Office of
Regulatory Affairs, U.S. Food and Drug Admin., Jeffe
Abstract Body:
The use of antimicrobials, such as tetracycline, in food-producing animals may result in antimicrobial
drug residues (ADR) in edible tissues and meat products from the treated animal and contribute to the
emergence of antibiotic resistant bacteria. Veterinary International Conference on Harmonization (VICH)
document (VICH GL36(R)/ FDA-CVM Guidance for Industry#159) provides guidance on evaluating the
safety of veterinary ADR in the human foods as related to impact on the human intestinal microbiome. A
research gap is the need for additional data and testing requirements to determine the fraction of oral
doses of ADR available to intestinal microorganisms. In the present study, we address this need by
examining the binding of tetracycline to human feces using chemical and microbiological assays. High-
performance liquid chromatography and liquid chromatography with mass spectrometry assay showed
that the 25% (w/v) autoclaved feces dosed with 0.15 and 1.5 µg/ml tetracycline had a binding of
58.2±10.8% and 56.9±9.1%, respectively. Tetracycline binding to unsterilized fecal suspensions gave
similar results. Microbiological assay with two reference bacterial strains validated the results of the
chemical assay. Based on data from chemical and microbiological assays methods, the fraction of dose
available to microorganisms was 0.418 and 0.431 in the 0.15 and 1.5 µg/ml tetracycline, respectively.
This study also provides general factors to be considered when designing and conducting experiments to
determine the percent of antimicrobial agents that is available to microorganisms in the gastrointestinal
tract.
Session Number: 395
Session Number: 395
Abstract Title:
Label-Free Flow Cytometry for Rapid Determination of Antimicrobial Susceptibilities of Low Cfu Samples
from Blood
A. Filbrun1, T-H. Huang1, J. Richardson1, Y-L. Tzeng2, R. Dickson1; 1Georgia Inst. of Technology, Atlanta,
GA, 2Emory Univ., Atlanta, GA
Abstract Body:
Despite the accessibility of appropriate antimicrobial therapies, bacterial infections contribute to global
morbidity and mortality, in large part due to the rapid development of antibiotic resistance.
Confounding the efficacy of conventional bacterial diagnosis is the inability to rapidly diagnose infection-
causing bacteria and determine their antimicrobial susceptibilities, as time to actionable treatment is
the key determinate to ushering positive patient outcomes and reducing antibiotic resistance
proliferation. Particularly problematic, the >60-hour time-to-result of bacterial bloodstream infections
(BSI) diagnosis is at odds with the only known indicator of sepsis survival - time to initiation of
appropriate treatment. Consequently, this significant delay to actionable treatment determinations
negatively influences patient outcomes and favors the misuse and overuse of broad-spectrum
antibiotics leading to increased proliferation of antibiotic resistance. For this reason, our lab developed a
novel antibiotic susceptibility test (AST) platform that enables antimicrobial susceptibilities within as
little as 8 hours of blood collection. Concentrating on three common bloodstream infection-causing
bacteria with resistance threat levels of urgent (Carbapenem-resistant Enterobacteriaceae, CRE) and
serious (extended spectrum β-lactamase producing (ESBL) Enterobacteriaceae, (focusing on Escherichia
coli and Klebsiella pneumoniae) and multi-drug resistant (MDR) Pseudomonas aeruginosa), we combine
our selective bacterial cell recovery and label-free flow cytometry technologies to glean antimicrobial
susceptibilities from low bacterial load samples with minimal incubation. Taken together, our combined
approach portends significant improvement to patient treatment efficacy, while lowering the incidence
of resistance associated with inappropriate antibiotic treatments.
Session Number: 395
Session Number: 395
Abstract Title:
FASTinov® kits forAST and Detection of Main Mechanisms of Resistance on Gram-negative bacilli
Directly from Positive Blood Cultures - Performance Evaluation
R. Teixeira-Santos1, S. Costa-de-Oliveira1, A. Silva-Dias1, I. Oliveira1, R. Gomes2, A. G. Rodrigues2, C.
Pina-Vaz1; 1FASTinov, SA, Porto, Portugal, 2Faculty of Med., Univ. of Porto, Porto, Portugal
Abstract Body:
Background: Antimicrobial resistance is a global problem. According to CDC, more than 50% of
antimicrobial drugs used at hospital settings are inappropriate/unnecessary. Thus, quicker diagnostic
methods are urgently need. FASTinov® kits allow the determination of antimicrobial susceptibility
profile, providing MIC values in case of carbapenems, and detection of enzymatic resistance to beta-
lactamic drugs. In this study, the performance of FASTinov® gramneg and FASTinov® mar kits was
evaluated directly from positive blood cultures (BC) of gram-negative bacilli. Methods: A total of 93 BC
were spiked with well characterized bacteria including recommended AST control strains (54
Enterobacteriaceae and 39 Acinetobacter spp.), inoculated with human blood and incubated until flag
positive. Bacteria was extracted from BC according to IFU, inoculated in the FASTinov®kit and incubated
for 1 h; afterwards, the susceptibility for main antimicrobial drugs and screenings for detection of
resistance mechanisms was analyzed by AccuriTM C6 Flow Cytometer. Regarding Enterobacteriaceae,
whenever the screening was positive for ESBL, AmpC and carbapenemases enzymes, the FASTinov® mar
kit was performed. EUCAST and CLSI protocols were selected on the FASTinov dedicated software. In
order to check the accuracy of FASTinov® gramneg and FASTinov® mar kits, error rates [minor (mE),
major (ME) and very major (VME)], categorical agreement (CA) and accuracy measurements were
calculated. Results: Regarding Acinetobacter spp., the overall CA between FASTinov®kit and broth
microdilution was 97% for EUCAST and 96% for CLSI. The highest ME rate was detected for piperacillin-
tazobactam, and VME was verified for ciprofloxacin. In case of Enterobacteriaceae, the overall CA was
93% for both protocols. The highest ME rate was detected for amoxacillin-clavulanic acid, and VME was
verified for meropenem. Data analysis showed a significant association between meropenem and
imipenem MIC, and cytometric values (p=0.022 and p=0.001, respectively) and a high agreement
(93.06% and 96.70%, respectively). The CA between FASTinov® mar kit and EUCAST protocol was 96%.
ESLB, carbapenemases, and AmpC positive strains, were detected with a sensitivity of 100%. Specificity
of FASTinov® mar kit was 92% for detection of ESBL, 95% for carbapenemases, and 95% for AmpC.
Conclusions: The phenotypic methods developed by FASTinov® provided reliable results regarding AST
and detection of resistance mechanisms (TTR 2 h), that may allow a targeted therapy and the isolation
of the patient in useful time.
Session Number: 395
Session Number: 395
Abstract Title:
Flow Cytometry Antimicrobial Susceptibility Test (Fast) Directly on Positive Blood Cultures for
Pseudomonas Aeruginosa
S. Costa-de-Oliveira1, R. Teixeira-Santos1, A. Silva-Dias1, I. Oliveira1, A. Rodrigues2, C. Pina-Vaz1;
1FASTinov, SA, Porto, Portugal, 2Faculty of Med., Univ. of Porto, Porto, Portugal
Abstract Body:
Background: Current culture-based methods are no longer fit for purpose for the diagnosis of infection,
particularly acute infection like sepsis, owing to the slow turnaround time of results. FASTinov® have
developed a rapid phenotypic test (TTR-2 hours), not growth dependent, in order to evaluate the
susceptibility profile of most common bacteria isolated from blood cultures. Pseudomonas aeruginosa is
one of the most frightening agent posing a great challenge to medical treatment often causing
nosocomial infection on especially debilitated patients. The present work aims to evaluate the
performance of FASTinov® gram negative kit for susceptibility evaluation of Pseudomonas aeruginosa
directly on positive blood culture. Methods: A total of 35 blood cultures (BD) were spiked with well
characterized bacteria including all recommended AST control strains, inoculated with human blood
(Cambridge Bioscience) and incubated until obtain a positive flag. A protocol for extraction of
microorganisms from BD bottles was followed according IFU. The cells were then incubated for 1 hour
accordingly the FASTinov® gram neg kit; afterwards the microplate, with main antimicrobial drugs, was
analyzed by the BD AccuriTMC6 Plus Flow Cytometer. EUCAST and CLSI protocols were selected on the
dedicated software provided. The result was automatically obtained and categorical agreement (CA) and
the minor, major and very major errors were calculated. Results: The overall categorical agreement
between FASTinov® kit and broth microdilution was 0.95 for both protocols. The highest CA was
observed for gentamicin, ceftalozane-tazobactam and colistin (1.00); followed by amikacin and
imipenem (0.97 and 0.96, respectively). The highest major error rate was detected for piperacillin-
tazobactam, and the very major discrepancies were verified for meropenem. <table
class="AbstractTable" id="{D0C5A65B-CEFF-4F97-A137-B5C8A8780C08}"><caption
rowspan="1" colspan="1"></td><td rowspan="1" colspan="4">EUCAST</td><td rowspan="1"
colspan="4">CLSI</td></tr><tr><td rowspan="1" colspan="1"> </td><td rowspan="1"
colspan="1">CA</td><td rowspan="1" colspan="1">mE</td><td rowspan="1" colspan="1">ME</td><td
rowspan="1" colspan="1">VME</td><td rowspan="1" colspan="1">CA</td><td rowspan="1"
colspan="1">mE</td><td rowspan="1" colspan="1">ME</td><td rowspan="1"
colspan="1">VME</td></tr><tr><td rowspan="1" colspan="1">Amikacin</td><td rowspan="1"
colspan="1">0,97</td><td rowspan="1" colspan="1">-</td><td rowspan="1" colspan="1">0,03</td><td
rowspan="1" colspan="1">-</td><td rowspan="1" colspan="1">0,97</td><td rowspan="1"
colspan="1">0,03</td><td rowspan="1" colspan="1">-</td><td rowspan="1" colspan="1">-
</td></tr><tr><td rowspan="1" colspan="1">Piperacillin-tazobactam</td><td rowspan="1"
colspan="1">0,03</td><td rowspan="1" colspan="1">0,08</td><td rowspan="1" colspan="1">-
</td></tr><tr><td rowspan="1" colspan="1">Gentamicin</td><td rowspan="1"
colspan="1">1,00</td><td rowspan="1" colspan="1">-</td><td rowspan="1" colspan="1">-</td><td
colspan="1">-</td><td rowspan="1" colspan="1">-</td><td rowspan="1" colspan="1">-
</td></tr><tr><td rowspan="1" colspan="1">Ciprofloxacin</td><td rowspan="1"
colspan="1">-</td><td rowspan="1" colspan="1">0,10</td><td rowspan="1" colspan="1">-
</td></tr><tr><td rowspan="1" colspan="1">Ceftalozane-tazobactam</td><td rowspan="1"
colspan="1">-</td><td rowspan="1" colspan="1">-</td><td rowspan="1" colspan="1">-
</td></tr><tr><td rowspan="1" colspan="1">Ceftazidime</td><td rowspan="1"
colspan="1">0,94</td><td rowspan="1" colspan="1">0,03</td><td rowspan="1"
rowspan="1" colspan="1">0,04</td><td rowspan="1" colspan="1">-</td><td rowspan="1"
colspan="1">-</td></tr><tr><td rowspan="1" colspan="1">Meropenem</td><td rowspan="1"
colspan="1">0,11</td><td rowspan="1" colspan="1">0,09</td></tr><tr><td rowspan="1"
colspan="1">Imipenem</td><td rowspan="1" colspan="1">0,96</td><td rowspan="1"
rowspan="1" colspan="1">0,96</td><td rowspan="1" colspan="1">0,04</td><td rowspan="1"
colspan="1">-</td><td rowspan="1" colspan="1">-</td></tr><tr><td rowspan="1"
colspan="1">Colistin</td><td rowspan="1" colspan="1">1,00</td><td rowspan="1" colspan="1">-
</td><td rowspan="1" colspan="1">-</td><td rowspan="1" colspan="1">-</td><td rowspan="1"
rowspan="1" colspan="1">-</td></tr><tr><td rowspan="1" colspan="5">CA - categorigal agreement;
mE - minor errors; ME - major errors; VME - very major errors.</td><td rowspan="1"
rowspan="1" colspan="1"></td></tr></table> Conclusions: FASTinov® gram negative kit revealed to be
a fast and accurate tool for Pseudomonas aeruginosa AST achieving high agreement with reference
method.
Session Number: 395
Session Number: 395
Abstract Title:
A Colorimetric Method for Detecting the Choline Kinase Product Phosphocholine
T. Zimmerman, S. Ibrahim; North Carolina A&T State Univ., Greensboro, NC
Abstract Body:
Choline kinase has been recently established as a drug target in Streptococcus pneumoniae . Inhibiting
choline kinase has the effect of slowing cell growth and division. However, the precise role that choline
kinase plays in cell growth and division in Gram positive bacteria is unknown. A low cost, safe, and
accessible method for measuring choline kinase activity needed to developed in order to study choline
kinase function more profoundly. In the context of complex samples like cell extracts, choline kinase
activity until now could only be measured by quantifying the resulting phosphocholine product using
three accepted methods: radioactive methods, massspectrometry, and Nuclear Magnetic Resonance.
The first method is hazardous, and the second and third require significant capital investments and
technical expertise. All three methods are also time consuming. For these reasons a less expensive,
higher throughput, more easily accessible colorimetric assay has been developed. We developed an easy
to implement colorimetric method detect and quantify choline and phosphocholine using wavelengths
in the visible range. We demonstrated that this method can be used to monitor the consumption of
choline in parallel with the production of phosphocholine in enzymatic reactions using cell extracts as
the source of the choline kinase enzyme. In addition, choline kinase enzyme inhibition can be monitored
and characterized. We present here a cost effective, easily accessible method for studying the choline
kinase of Gram-positive pathogens in complex samples. Using this method, the role choline kinase plays
in Gram- positive cells can be further clarified.
Session Number: 395
Session Number: 395
Abstract Title:
Accelerated and Accurate Carbapenemase Detection with the Cpo Complete Test
G. Thomson1, S. AbdelGhani2, K. Thomson1; 1Univ. of Louisville, Louisville, KY, 2Beni-Suef Univ., Cairo,
Egypt
Abstract Body:
Background: The high mortality of infections by carbapenemase-producing organisms (CPOs) stems
from therapeutic and infection control failures. Rapid CPO detection can help prevent the spread of
CPOs. Coupled with carbapenemase classification it can help reduce mortality and improve antibiotic
stewardship by indicating if the new anti-CPO β-lactamase inhibitor combinations are potential
therapeutic candidates or contraindicated. We developed a rapid, manual CPO detection and
carbapenemase classification kit that is capable of adaptation to currently available automated
instruments and can be stored at temperatures up to 38oC. The test, CPO Complete, detects accelerated
carbapenem hydrolysis. A study was designed to evaluate its speed and accuracy of CPO detection and
the potential of phenotypic markers to classify carbapenemases. Methods: The isolates consisted of 306
Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii that were characterized for
type of β-lactamase production by molecular, phenotypic and biochemical tests. Detection tests were
performed on 114 isolates producing KPC, NMC-A or SME class A carbapenemases, 80 isolates producing
NDM, GIM, SPM, IMP or VIM class B carbapenemases, 43 isolates producing OXA class D
carbapenemases, 5 isolates producing 2 carbapenemase classes and 64 non-CPOs. Classification
potential was assessed for a subset of 40 CPOs. The benchtop test format was used to enable immediate
visualization of positive results. Results: The test was 100% sensitive and 98.4% specific (Table) with
76.4% of carbapenemases detected within seconds to 10 minutes and 99.2% detected within 1 hour.
Notably, KPCs in P. aeruginosa were detected within 1 minute, 95.3% of OXA carbapenemases were
detected within 60 minutes, and KPC was detected in A. baumannii. One falsely positive test occurred
with an AmpC-producing Enterobacter aerogenes isolate. All class A, B and D carbapenemases were
correctly classified.<table class="AbstractTable" id="{FB15E32B-429B-41C8-A575-
567FDC962E5B}"><caption class="AbstractTableCaption"></caption><tr><td rowspan="1"
rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1">Mechanisms</td><td
rowspan="1" colspan="1">No. of<br />Isolates</td><td rowspan="1" colspan="2">Isolate Results No
(%)</td></tr><tr><td rowspan="1" colspan="1"></td><td rowspan="1" colspan="1"></td><td
rowspan="1" colspan="1">Positive</td><td rowspan="1" colspan="1">Negative</td></tr><tr><td
rowspan="1" colspan="1">Carbapenemase Producers</td><td rowspan="1" colspan="1"></td><td
colspan="1">Class A</td><td rowspan="1" colspan="1">114</td><td rowspan="1"
colspan="1">Class B</td><td rowspan="1" colspan="1">80</td><td rowspan="1"
colspan="1">80</td><td rowspan="1" colspan="1">0</td></tr><tr><td rowspan="1" colspan="1">Class
D</td><td rowspan="1" colspan="1">43</td><td rowspan="1" colspan="1">43</td><td rowspan="1"
colspan="1">0</td></tr><tr><td rowspan="1" colspan="1">Dual Carbapenemases</td><td
colspan="1">0</td></tr><tr><td rowspan="1" colspan="1">All Carbapenemase Producers</td><td
rowspan="1" colspan="1">242</td><td rowspan="1" colspan="1">242 (100%)</td><td rowspan="1"
colspan="1">0 (0%)</td></tr><tr><td rowspan="1" colspan="1"></td><td rowspan="1"
rowspan="1" colspan="1">Non-carbapenemase Producers</td><td rowspan="1"
colspan="1">64</td><td rowspan="1" colspan="1">1 (1.6%)</td><td rowspan="1" colspan="1">64
(98.4%)</td></tr></table> Conclusion: With 100% sensitivity, 98.4% specificity, and 99.2% of CPOs
detected within 1 hour, plus carbapenemase classifications and room temperature storage, CPO
Complete has the speed, accuracy and convenience to help improve patient management and reduce
the mortality of the CPO pandemic.
Session Number: 395
Session Number: 395
Abstract Title:
Comparison of Three Carbapenemase Detection Methods
D. Ortiz, P. Ren; Univ. of Texas Med. Branch, Galveston, TX
Abstract Body:
Carbapenem antibiotics are often used as a last resort to treat serious gram-negative infections.
However, the rise in carbapenemase-producing carbapenem-resistant enterobacteriaceae (CP-CRE) has
left clinicians with limited treatment options, and subsequently led to hospital outbreaks that have been
associated with high rates of mortality. In an effort to prevent hospital transmission of CP-CREs, the
Centers for Disease Control and Prevention recommends healthcare facilities implement control
measures for patients colonized or infected with CP-CRE. To aid in the identification of CP-CRE, as well as
other carbapenemase-producing organisms, we evaluated the accuracy of three carbapenemase
detection methods: Rapidec Carba NP (bioMérieux), mCIM, and Xpert Carba-R (Cephied). Thirty isolates
previously characterized by whole-genome sequencing from the CDC-FDA Antibiotic Resistance Isolate
Bank were evaluated, including 18 Enterobacteriaceae, 6 Pseudomonas aeruginosa, and 6 Acinetobacter
baumanii. Among all isolates tested, the Rapidec Carba NP, mCIM, and Xpert Carba-R test methods
produced accuracies of 87% (26/30), 83% (25/30), and 80% (24/30), respectively. Among isolates
recommended for testing by CLSI or the package insert, accuracies improved to 100% for all test
methods. The Acinetobacter baumanii isolates used in this study attributed to most of the discrepant
results, which produced Ambler class D carbapenemases OXA-23, OXA-24, OXA-25, OXA-65 and OXA-66.
Compared to the other test methods, the Carba NP is a relatively low cost (~$5) test with a rapid
turnaround time of 2-4 hours, but required the most hands-on time. Alternatively, the mCIM required
minimal hands-on time and was the cheapest (< $1), but test results become available only after an
overnight incubation period (18-24 hours). The easiest test to perform was the Xpert Carba-R, which
displayed the fastest turnaround time (~1.5 hours) with the least amount of hands-on time, but was also
the most expensive. In conclusion, all three test methods are well suited for identifying carbapenemase-
producing organisms inside their respective CLSI or package insert guidelines. However, healthcare
facilities should take into account the advantages and disadvantages of each test method when making
a decision of which test to offer at their institution.
Session Number: 395
Session Number: 395
Abstract Title:
Detection of Ctx-M- Producing Enterobacteriaceae Using Maldi-Tof
R. Figueroa Espinosa, B. Ghiglione, V. Rumi, C. Barberis, C. Vay, J. Di Conza, G. Gutkind; Univ. de Buenos
Aires, Buenoa Aires, Argentina
Abstract Body:
Background: The CTX-M enzymes are the most prevalent extended-spectrum β-lactamases (ESBL), both
in nosocomial and in community settings. Fast, easy to perform sensitive and specific methods for ESBLs
detection are needed to avoid treatment failure and prevent dissemination. MALDI-TOF mass
spectrometry is frequently used as a routine bacterial identification tool to categorize bacterial species.
Direct detection of ß-lactamases production in Enterobacteriaceae by MALDI-TOF is another important
diagnostic challenge for clinical microbiology lab. In this study, an easy, fast and direct MALDI-TOF MS
protocol was designed to detect CTX-M-producing Enterobacteriaceae. Methods: 49 clinical isolates of
Escherichia coli (15), Salmonella enterica (15), Klebsiella pneumoniae (8), Serratia marcescens (8) and
Proteus mirabilis (3) were included in this study. Susceptibility tests were performed according to CLSI.
Isolates were analyzed by PCR and sequencing, and grouped according to the CTX-M variant.
Subsequently, CTX-M- detection was carried out by MALDI-TOF (Bruker) after proteins extraction with
organic solvents. MALDI-TOF spectra were recorded by MALDI Biotyper system and analyzed using
ClinProTool software (Bruker). E. coli transformants producing CTX-M-2, CTX-M-15 and CTX-M-9
enzymes were used as positive controls and isolates lacking any blaCTX-M- as negative controls. Results:
Operating at a mass range of 17,000 to 50,000 Da, three peaks at approximately m/z 28,287, 28,093 and
27,950 Da were detected by MALDI-TOF and they were correlated with the mature protein of CTX-M-2,
CTX-M-15 and CTX-M-14, respectively. After statistical analysis of peaks, two groups of isolates could be
distinguished: the CTX-M-producing enterobacteria (28) (clinical isolates and transformants) and non-
CTX-M-producing enterobacteria (24) (including susceptible strains and CMY-2, KPC-2 and TEM
producers). Furthermore, the 25 CTX-M-producing isolates were correctly classified into CTX-M-1 (6),
CTX-M-2 (15) and CTX-M-9 (7) groups. Conclusions: Our MALDI-TOF assay was able to identify the most
frequent CTX-M ß-lactamase groups reported in Argentina in different enterobacterial species.
Considering that our results could be obtained as early as bacterial identification time, and together
with previous communications on other β-lactamases, they may constitute the basis for designing a
database of β-lactamases peaks that could be detected in a single event, which may have deep impact
on resistance characterization.
Session Number: 395
Session Number: 395
Abstract Title:
Rapid Antibiotic Susceptibility Testing Based on Tmrna Detection
A. Khine, A. Samiei, A. Talebpour, T. Alavie; Qvella Corp., Richmond Hill, ON, Canada
Abstract Body:
Background: Conventional antimicrobial susceptibility testing (AST) typically delivers results 2 to 5 days
after sample collection, which is often too late to have a meaningful impact on treatment. This work
demonstrates a new, rapid, phenotypic approach to AST using transfer messenger RNA (tmRNA) to
assess pathogen susceptibility to antibiotic exposure. tmRNA was identified as a suitable marker for the
real-time assessment of pathogen viability and susceptibility to antibiotics based on control of the cell
cycle being tightly regulated by the timing of both synthesis and degradation of tmRNA. Methods:
Studies were performed to demonstrate the effectiveness of tmRNA as a suitable marker of pathogen
viability for rapid phenotypic AST. K. pneumoniae cells were spiked into 2 mL of EDTA-treated whole
blood at 100 CFU/mL. Blood cells were selectively lysed and microbial cells isolated using Qvella’s FAST™
Prep centrifugal separation process. Cells were re-suspended in 4 mL of TSB growth medium followed by
pre-incubation at 37° C for 2 hours. This cell suspension was divided into one control aliquot and three
aliquots having norfloxacin, tetracycline, and oxacillin at concentrations of 8, 8, and 2 ug/mL,
respectively. The aliquots were incubated at 37° C for 2 hours and then subjected to Qvella’s FASTTM ID
process involving centrifugal separation, electrical lysis (e-lysisTM), and RT-PCR for tmRNA targets.
Results: The levels of tmRNA markers in the aliquots incubated in the presence of norfloxacin and
tetracycline were approximately 500 and 50 times less than the control level, respectively, while the
tmRNA level for the aliquot incubated with oxacillin was unchanged. These levels are in agreement with
the expected mode of action for these antibiotics. For the bactericidal antibiotic norfloxacin, loss of
tmRNA content is expected during centrifugal separation due to cell wall damage. For the bacteriostatic
antibiotic tetracycline, diminished tmRNA content is also expected. However, for the antibiotic oxacillin,
to which K. pneumonia is not susceptible, little or no effect on tmRNA is expected. Conclusions: This
study highlights the utility of tmRNA as a marker of pathogen susceptibility during antibiotic exposure
for both bactericidal and bacteriostatic antibiotics, demonstrating the feasibility of tmRNA-based rapid
phenotypic AST. Further analysis would need to be performed to show correlation with standardized
techniques for routine susceptibility testing in the clinical laboratory setting.
Session Number: 395
Session Number: 395
Abstract Title:
Fully Automated Disk Diffusion Susceptibility Testing by Adagio Wasplab Expert Sys. Compared to Vitek2
and Manual Disk Diffusion in Urines from Daily Clin. Practice
R. Verschuijten1, T. Liebregts2, L. B. J. Velden van der2, A. R. Jansz2, N. L. A. Arents2; 1FONTYS
Hogeschool voor toegepaste natuurwetenschappen, Eindhoven, Netherlands, 2PAMM, Veldhoven,
Netherlands
Abstract Body:
Background: Decreasing reimbursements stimulate clinical laboratories to search for the most cost-
effective diagnostic approaches. Over the past years PAMM has implemented COPAN WASPLab which
provides automated streaking, dispersion of antibiotic disks, incubation and digital imaging of incubated
culture media. In this study, we investigated the next step: fully automated disk diffusion interpretation
by ADAGIO WASPLab Expert System (WASPLab DD) of urine cultures from daily clinical practice.
Methods: From october 2017 to the end of 2017 culture positive urine samples derived from daily
clinical practice were considerd for this study. If these cultures showed Gram negative rods (GNR),
Staphylococci (STAP) or Enterococci (ENCO), demanding antibiotic susceptibility testing (AST) according
to our laboratory SOP, the strains were enrolled in the study. GNR AST was performed by VITEK2 and
compared to WASPLab DD for amoxicillin-clavulanic acid, cefotaxim, ceftazidim, ciprofloxacin,
fosfomycin, nitrofurantoin, trimethoprim and thrimethoprim-sulfamethoxazol. STAP AST was performed
by VITEK2 and compared to WASPLab DD for cefoxitin, ciprofloxacin, clindamycin, erythromycin and
thrimethoprim-sulfamethoxazol. ENCO AST by manual disk diffusion was compared to WASPLab DD for
nitrofurantoin, norfloxacin, tetracyclin and fosfomycin. Ampicillin AST in ENCO was performed by agar
dilution and compared to WASPLab DD. All ASTs were interpreted according to EUCAST breakpoints
except for ENCO and tetracyclin, which was interpreted by CLSI breakpoints. A cost-effectiveness
analysis comparing hands-on time and material cost per sample is ongoing. Results: In total 886 strains
(810 GNR, 41 STAP, 35 ENCO) were derived from 862 urine samples. For GNR and STAP concordance
between VITEK2 and WASPLab DD was 95,2% and 99,5% respectively. For ENCO concordence between
manual disk diffusion/agar dilution and WASPLab DD was 98,9%. The most frequently encounterd
discrepancy was a very major error when testing Enterobacteriaceae for AMCL (127 strains; 2,4% from
total). These strains were retested by AMCL Etest (with a variable clavulanic acid ratio concentration)
and proved to be Etest sensitive. Conclusions: In conclusion, ADAGIO WASPLab Expert System disk
diffusion reading and interpretation showed excellent overall results (concordance >95%) compared to
VITEK2 and manual disk diffusion based AST. The well known discrepancy between AMCL AST with a
fixed and a variable concentration of clavulanic acid accounted for 40% of all errors.
Session Number: 395
Session Number: 395
Abstract Title:
Detection of Community Acquired Antibiotics from Cholera and Non-Cholera Patient Urine and Stool
Samples Using Mass Spectrometry
E. J. Nelson1, L. Alexandrova2, F. Haque3, V. Ramachandran2, P. Rodriguez1, A. Creasy1, C. Adams2, S.
A. Siddique4, A. I. Khan4, F. Qadri5, M. Rahman3, A. Chien2; 1Univ. of Florida, Gainesville, FL, 2Stanford
Univ., Stanford, CA, 3Inst. of Epidemiology D
Abstract Body:
Antibiotics impact infection and transmission of gastrointestinal pathogens like Vibrio cholerae.
However, it remains difficult to monitor antibiotic impact because self-reported medication use is
unreliable and analytic assays are often too expensive, or lack sensitivity and specificity. To address this
problem, we developed a unified liquid chromatography-mass spectrometry (LC-MS) method that
minimizes cost by qualitatively targeting sixteen clinically relevant antibiotic and non-antibiotic
medications for diarrheal disease treatment and streamlining preparation and analysis. We validated
the method by prospectively collecting and testing urine and diarrheal samples from a cohort of patients
in Bangladesh. Due to differences in analyte excretion patterns in urine and stool, the spectra were
analyzed for both parent and known metabolites. A total of 30 paired urine and diarrheal samples were
tested (10 non-cholera and 20 cholera). All samples contained at least one antibiotic and 80% (N=24/30)
had at least two antibiotics independent of self-reports. Metronidazole (80%), ciprofloxacin (60%),
ondansetron (Zofran; 36%), azithromycin (33%) and paracetamol (20%) were the most common
medications. The primary limitation is stool analysis showed less sensitivity due to filtration, dilution
because increased methanol was needed to accommodate for high protein concentration, and likely
different excretion and degradation. We advocate that this technique be deployed to monitor for
antibiotics in research designed to elucidate critical determinants of diarrheal disease severity and
transmission. This platform can also be a gold-standard to assess biologic antimicrobial detection assays
and diagnostic performance in the setting of antibiotics.
Session Number: 395
Session Number: 395
Abstract Title:
Evaluation of A Novel Microbiologic Rapid Diagnostic Sys. on Antimicrobial Selection for Gram-Negative
Bacillary Bloodstream Isolates
S. McCullough, A. J. Mathers, F. Brewster, M. D. Poulter, Z. Elliott, H. Cox; Univ. of Virginia Hlth.System,
Charlottesville, VA
Abstract Body:
The Accelerate Pheno™ system (AXDX) is a rapid diagnostic tool capable of performing identification (ID)
and antimicrobial susceptibility testing (AST) on positive blood cultures within 7 hours. Previous studies
regarding AXDX have shown significant reductions in time to ID and AST. However, the AXDX AST
currently lacks FDA-approval for narrow-spectrum agents such as cefazolin and ampicillin. Therefore, the
purpose of this study will be to assess the potential clinical impact of the utilization of AXDX on
antibiotic selection. This is a retrospective comparison of AXDX and standard of care (SOC) procedures
applied to monomicrobial blood cultures with Gram-negative bacilli on Gram stain. Blood cultures from
the institutional validation of the AXDX system between January and April 2017 were utilized in this
study. Organisms unidentified by AXDX were excluded. SOC included biochemical assays, Vitek 2, and
MALDI-TOF. Outcomes assessed included: time to ID/AST, rate of categorical (CA) and essential
agreement (EA), rates of very major, major and minor error, and the narrowest susceptible beta-lactam
from AST results of each method. Of the 33 samples screened, 22 met inclusion criteria. Escherichia coli
and Klebsiella spp. accounted for 41% (9/22) and 18% (4/22), respectively; Enterobacter aerogenes,
Pseudomonas aeruginosa, and Serratia marcescens were each isolated twice; Citrobacter freundii,
Citrobacter koseri, and Enterobacter cloacae were each isolated once. AXDX reduced the time to ID and
AST by 24.1 (p<0.001) and 29.3 hours (p<0.001), respectively. AST results demonstrated 92% CA and
91% EA between each method. Among beta-lactams, AXDX reported ceftriaxone and
ampicillin/sulbactam as the narrowest agent for 46% (6/13) and 39% (5/13), respectively, for all E. coli
and Klebsiella isolates. Narrower therapeutic options were available by SOC as compared to AXDX for
89% of E. coli isolates (8 cefazolin-susceptible, 5 ampicillin-susceptible). All Klebsiella spp. were
cefazolin-susceptible. AXDX provided faster time to organism ID and AST but limited the opportunity to
de-escalate to the narrowest spectrum beta-lactam in more than half of cases, particularly in the setting
of E. coli bacteremia. While AXDX complements antimicrobial stewardship activities by promoting earlier
active antibiotic administration; SOC methods remain necessary to optimize definitive antimicrobial
therapy.
Session Number: 396
Session Number: 396
Session Title: CPHM02- Antimicrobial Susceptibility Testing: Traditional Methods
Abstract Title:
Calculation of the Uncertainty of Measurement (Um) for Disk Diffusion (Dd) Results in A Clin.
Microbiology Laboratory
J. Romano, Q. Liu, L. Lang, P. Lo, T. Mazzulli, S. M. Poutanen; Univ. Hlth.Network/Sinai Hlth.System
Dept. of Microbiol., Toronto, ON, Canada
Abstract Body:
Background: Laboratory accreditors require UM data for quantitative results released by a clinical
laboratory. Some have proposed that microbiology laboratories calculate UM related to measuring
antimicrobial (ABX) DD inhibition zones. The purpose and utility of this has been challenged in light of
Clinical Laboratory Standards Institute (CLSI) breakpoint and quality control (QC) standards that take UM
into account. The purpose of this study was to calculate DD UM and compare it to CLSI QC DD ranges.
Methods: DD UM was calculated using data compiled from weekly ABX DD QC results using 4 ATCC
control strains [Streptococcus pneumoniae-SPN (5 ABX), Staphylococcus aureus-SA (11 ABX), Escherichia
coli-ECOL (17 ABX), and Pseudomonas aeruginosa-PSA (5 ABX)] from September 1, 2016 through August
31, 2017. The calculated UM range was determined using QC results for each ABX-organism
combination [UM=+/-rounded up(1.96*weighted standard deviation in mm]. UM results were compared
to corresponding CLSI QC DD ranges (=/-x mm) using paired T-test (GraphPad InStat). Results: All QC
results fell within acceptable CLSI QC ranges. Calculated UM for SPN, SA, and ECOL were not significantly
different from the corresponding CLSI QC ranges: SPN (P=0.43), SA (P=0.37), ECOL (P=0.44) with average
differences (UM-CLSI) being: SPN -0.3mm (95%CI-1.24 to 0.62); SA -0.18mm (95%CI-0.25 to 0.61); ECOL -
1.5mm(-0.52 to 0.25). The calculated UM for PA was significantly smaller than the CLSI QC range: PA
(P=0.03), -1.1mm (-2.0 to -0.17). Conclusion: Calculated DD UM using ATCC controls corresponded or
underestimated uncertainty measurements reflected by existing CLSI QC ranges. If laboratory’s DD QC
results lie within acceptable ATCC QC DD ranges, existing CLSI ATCC QC DD ranges can be used to
provide an estimate of DD UM to interested stakeholders saving laboratories time and resources that
would otherwise be used to calculate laboratory-specific UM.
Session Number: 396
Session Number: 396
Abstract Title:
Reproducibility of Reference Broth Microdilution Mics When Testing Commonly Encountered Bacterial
Pathogens
K. Spafford1, P. D. Stamper2, C. Zimmerman3, J. A. Hindler4, R. Humphries1; 1Accelerate Diagnostics,
Tucson, AZ, 2Johns Hopkins Univ., Baltimore, MD, 3MRI Global, Palm Bay, FL, 4UCLA, Los Angeles, CA
Abstract Body:
Background: Minimum inhibitory concentration (MIC) testing by broth microdilution (BMD) per Clinical
and Laboratory Standards M07-A10 is the gold standard method for commercial antimicrobial
susceptibility test system evaluation for clearance by the U.S. Food and Drug Administration. This study
evaluated the precision of BMD MICs measured during the Accelerate PhenoTest™ BC kit clinical trial.
Methods: BMD was performed according to CLSI M07-A10 using frozen panels prepared in-house. A
total of 3328 isolates of Enterobacteriaceae, Pseudomonas aeruginosa, Acinetobacter baumannii,
Staphylococcus spp. and Enterococcus spp. were received from 12 clinical sites. For BMD inocula
preparation, 3 separate McFarland 0.5 suspensions were prepared for each isolate from colonies on an
overnight BAP. These were combined into a single suspension, which was subsequently diluted and used
to inoculate 3 MIC panels. Two highly trained technologists daily performed manual reads (n=7
technologists in total performed reads). Results from a digital BMD imaging system (Biotek) were also
examined. Results for intrinsic resistance were excluded from the data set. Individual MIC results were
compared to the modal MIC obtained from the 3 results for each drug/isolate combination, and
absolute agreement (AA) and essential agreement (EA, i.e., MIC +/- 1 log2 dilution) were evaluated.
Results: From the 3,328 isolates, 100,061 individual MICs were assessed. For 4% of reads, a mode could
not be established. By organism, EA ranged from 90.2-98.5% and AA ranged from 77.9-97.6%. EA by
antimicrobial ranged from 91.5-99.3% and AA from 79.6-97.6%. When only isolates with modal MICs in
the intermediate interpretive category were evaluated (n=717), EA was 90.9-100% and AA was 70.0-
100%. Precision of technologists ranged from 93.3-97.4% EA and 86.5-92.7 AA. EA of instrument
readings was 95.0% and AA was 88.0%. Conclusions: Under the controlled environment of a
standardized reference laboratory using highly trained technologists, precision with both manual and
instrument MIC readings from BMD panels was within the expected >90% EA. However, EA and AA was
poorer for isolates with MICs in the intermediate category.
Session Number: 396
Session Number: 396
Abstract Title:
Assessing Clin. Lab. Capacity for Detection of Antibiotic Resistance in Low and Middle Income Countries:
Data from Pakistan and Georgia, 2017
S. Bollinger1, T. J. McKinney1, M. Omondi1, N. Macharashvili2, M. Salman3, J. Weiss1, B. Skaggs4, R.
Smith1; 1CDC, Atlanta, GA, 2Infectious Diseases, AIDS and Clinical Immunology Res. Ctr., Tbilisi, Georgia,
3NIH, Islamabad, Pakistan, 4CDC, Tbilisi, Geor
Abstract Body:
Control of antibiotic resistance (AR) is a global public health priority. Robust national AR surveillance
networks are critical to inform policy and control efforts, but the capacity of clinical bacteriology
laboratories to produce reliable, accurate data is poorly characterized in many low and middle-income
countries (LMIC). As a part of ongoing efforts to implement national surveillance systems, CDC and
partners developed and piloted a novel Laboratory Assessment of AR Capacity (LAARC). The LAARC
assesses bacteriology capacity through 250 questions in five domains: infrastructure, specimen
processing, quality assurance (QA), organism identification (ID), and antibiotic susceptibility testing
(AST). Individual questions receive a weighted value, and values are summed to present proportions
within domains. The LAARC was piloted in a convenience sample of laboratories in Pakistan and Georgia
in 2017. Trained assessors conducted staff interviews, reviewed relevant documents and observed
bench procedures; data were entered in a standardized EpiInfo database. Of the eight laboratories
assessed, three were private, two were national reference laboratories, one of which had substantial,
ongoing US support. One laboratory had current ISO:15189 accreditation. Median overall score was 69%
(range, 46-94%). Lowest median scores were in the QA and AST domains, 66% (range, 38-100%) and 63%
(range, 49-78%) respectively; median scores were highest for infrastructure (median 84%, range, 66-
100%), and specimen processing (median, 84%, range, 62-95%). Median score for ID was 73% (range, 17-
95%). Four laboratories participated in an external quality assessment (EQA) program for ID and AST;
one used an accredited EQA provider (ISO:17043). EQA scores were not available for review at any lab.
All laboratories performed disk diffusion testing and reconstituted their own Mueller Hinton agar.
Necessary ATCC quality control (QC) strains were present in 6 (75%) of the labs; of those, 5 (83%) failed
to perform AST QC at the required (weekly) frequency and 3 (50%) reported patient AST results even
when QC failures occurred. Evidence of troubleshooting AST QC failures could be found in 2 (25%)
laboratories. The LAARC provided a detailed description of bacteriology capacity in Pakistan and Georgia
and demonstrated that numerous gaps exist, particularly in QC processes. As a part of national AR
surveillance system efforts, capacity building in these laboratories should focus on strengthening QC
processes, particularly around troubleshooting QC failures.
Session Number: 396
Session Number: 396
Abstract Title:
Implementation of Custom Broth Microdilution Panels in the Antibiotic Resistance Lab. Network
E. Ransom1, A. Bhatnagar2, A. C. Brown3, J. Patel3; 1Association of Publ. Hlth.Lab., Silver Spring, MD,
2IHRC, Inc., Atlanta, GA, 3CDC, Atlanta, GA
Abstract Body:
Background: Antimicrobial resistance poses one of the greatest threats to human health. One way the
Centers for Disease Control and Prevention (CDC) has responded to this threat is by establishing the
Antibiotic Resistance Laboratory Network (ARLN). ARLN is a nationwide network of clinical and public
health laboratories devoted to the rapid detection of antibiotic resistance. A major challenge facing the
ARLN is the need to provide antimicrobial susceptibility testing (AST) using new antimicrobials when
pan-resistant or highly resistant bacteria cause serious infections. This type of testing typically requires
the reference broth microdilution method (BMD), which is rarely used in laboratories due to its
complexity, cost, and access to new antimicrobials. To address these concerns, ARLN is implementing a
simple and cost-effective digital dispensing method for making small batches of custom broth
microdilution panels. Here, we describe our internal validation for this method. Methods: Custom AST
panels were poured using the HP D300e digital dispenser. As a comparator, the reference CDC BMD
panels were tested in parallel. AST was performed per Clinical and Laboratory Standards Institute
reference method. Results: We evaluated 31 isolates from the CDC-FDA AR Isolate Bank and 4 isolates
from the American Type Culture Collection. Isolates were tested for susceptibility to 16 antimicrobials,
including all major drug classes. Of note, we also tested all combinations of ceftazidime, aztreonam, and
avibactam to assess synergistic effects on highly resistant isolates. Validation of the HP D300e digital
dispenser passed for all antimicrobials with >90% essential agreement, >90% category agreement, <5%
minor errors, <3% major errors, and <3% very major errors. Conclusion: Custom BMD panels made by
digital dispensing provided equivalent data as compared to the reference CDC BMD panels.
Implementation of this technology in the ARLN will fill an unmet need for susceptibility testing. This will
enable ARLN laboratories to test the most promising therapeutic options for a highly resistant isolate
while also ensuring antimicrobials are used judiciously.
Session Number: 396
Session Number: 396
Abstract Title:
Outstanding Abstract Award: Small Differences in Inoculum Within the Clsi Acceptable Range Have
Dramatic Effect on Minimal Inhibitory Concentration
K. P. Smith, J. E. Kirby; Beth Israel Deaconess Med. Ctr., Boston, MA
Abstract Body:
Background: MICs may vary depending on initial bacterial inoculum density, the so called "inoculum
effect". This effect is most apparent with β-lactams and is typically investigated using cell densities 100-
fold greater than the CLSI inoculum of 5 x 105 CFU/mL. We hypothesized that the inoculum effect may
occur even with small (≤2-fold) differences in inoculation density, including those within the CLSI
allowable range (2-8 x 105 CFU/mL) Methods: Here, we leveraged inkjet printing technology to
investigate such inoculum effects by generating fine dilution series of antibiotics and bacteria. First, we
evaluated the ability to inkjet print Escherichia coli, Pseudomonas aeruginosa, and Klebsiella
pneumoniae into plates containing Mueller-Hinton broth and quantified bacterial density by plate
count. We next combined inkjet dispensing of bacteria and antibiotics in orthogonal 2-fold titrations to
quantify the inoculum effect on meropenem, cefepime, and ceftazidime-avibactam susceptibility using
clinical isolates of Enterobacteriaceae and Pseudomonas. Finally, we investigated 8 carbapenemase
producing- Enterobacteriaceae (CPE) strains with MICs up to 2 dilutions above the CLSI resistance
breakpoint in a “high-resolution” assay using an orthogonal 1.19-fold dilution series for antibiotics and
1.1-fold dilution series for bacteria spanning the CLSI recommended inoculum range. Results: Inkjet
dispensing of bacteria was highly reproducible for both 2-fold (R2 > 0.99) and 1.1-fold (R2 > 0.98)
dilution series. The inoculum effect for meropenem was most apparent at low inocula (<1 x 105
CFU/mL) where a 2-fold reduction in inoculum resulted in a 0.72 Log2-fold reduction in MIC. Conversely,
cefepime showed a pronounced inoculum effect at elevated inocula (>1 x 106 CFU/mL) where a 2-fold
increase in inoculum resulted in a 1.28 Log2-fold increase in MIC. An inoculum effect for ceftazidime-
avibactam was not observed. Using inocula spanning the allowable CLSI recommended range,
meropenem MICs for CPE ranged from 4-fold lower to 2-fold higher than the MIC measured at the exact
5 x 105 CFU/mL inoculum. At the lower limit of the CLSI range, 52.2 and 43.5% of MICs were 2 and 4-fold
lower, respectively, than the MIC at the exact CLSI inoculum. Conclusions: The inoculum effect is evident
even with small changes in bacterial density. Even small inoculum differences within the CLSI allowable
range resulted in marked differences in meropenem MICs for CPE. These findings have implications for
reliability and reproducibility of susceptibility testing methods.
Session Number: 397
Session Number: 397
Session Title: CPHM03 - Diagnostic Bacteriology: Emerging Methods
Abstract Title:
A Rapid Single Molecule Counting Method Sensitively Detects Clostridium Difficile Toxin B Directly in
Stool Samples
D. Archambault1, D. Straus1, S. Gite1, J. Bowers1, M. Cappillino1, T. Shatova1, D. Tempesta1, B. Walsh1,
J. Walsh1, J. Kirby2; 1First Light BioSci.s, Bedford, MA, 2Beth Israel Deaconess Med. Ctr., Boston, MA
Abstract Body:
Background: We developed an ultrasensitive Clostridium difficile toxin B test based on a novel digital
imaging technology that counts single target molecules in stool samples little or no sample preparation.
Current tests for C. difficile gastrointestinal infection can be inaccurate. C. difficile toxin immunoassays
often lack clinical sensivity. Nucleic acid amplification tests have excellent clinical sensivity but can have
diminished clinical specificity due to their inability to distinguish patients with C. difficile infection from
patients that are carriers of C. difficile organisms. Because production of toxin is a hallmark of C. difficile
infection, an ultrasensitive C. difficile toxin test, such as the one presented in this report, could address
the issues with the current tests and offer improved accuracy for detecting patients with the devasting
C. difficile gastrointestinal infection. Materials/Methods: The MultiPath™ C. difficile Toxin B test uses
non-magnified digital imaging to count target-specific magnetic and fluorescent particles that have been
tethered together by toxin molecules. The method includes the use of a novel dye-cushion to eliminate
the need for sample preparation and wash steps. We tested clinical stool samples to estimate the limit
of detection, imprecision, and dynamic range. We assessed the potential for achieving good clinical
accuracy by comparing the results using the new toxin test to those of the sensitive cell cytotoxicity
reference method for toxin detection. Results: In 30 minutes using clinical stool samples with minimal
sample preparation, the MultiPath C. difficile Toxin B test generated a limit of detection of 45 pg/mL, a
precision profile with CV’s below 10%, and a dynamic range covering >4 orders of magnitude in stool
samples. With a training set of 320 clinical stool samples, the MultiPath C. difficile toxin B test showed
97.0% sensitivity, 95%CI [91.4-99.4%]; 98.3% specificity, 95%CI [96.8-99.2%]; and 98.1% accuracy, 95%CI
[96.7-99.0%] when compared to the cell cytotoxicity reference method. Conclusions: The data
presented demonstrate the potential of the ultrasensitive MultiPath technology to deliver rapid,
accurate, easy-to-use test for C. difficile Toxin B. The technology should also have value for a variety of
other important infectious disease applications.
Session Number: 397
Session Number: 397
Abstract Title:
Siderophores As Specific and Sensitive Markers of Microbial Infections
M. Petrik1, A. Skriba2, T. Pluhacek2, D. Luptakova2, A. Palyzova2, O. Benada2, K. Lemr2, G. Mitulovic3, J.
Novak2, V. Havlicek2; 1Inst. of Molecular and Translational Med., Olomouc, Czech Republic, 2Inst. of
Microbiol., Prague 4, Czech Republic, 3Med. Un
Abstract Body:
Background: Mixed infections represent a diagnostic challenge for any microbiology laboratory. In this
work high mass resolution spectrometry is introduced as extremely sensitive tool for early detecting
Aspergillus and Pseudomonas infections in rat models. Microbial siderophores were used as specific
disease biomarkers [1] and detected in urine, serum and tissues of infected animals. Methods: Using a
model of experimental aspergillosis in Lewis rats, the fungal siderophores ferricrocin (FC) and
triacetylfusarinine C (TAFC) were identified as markers of Aspergillus fumigatus infection [2].
Analogously, bacterial pyoverdine E (PyE) was quantified in urine, serum and lung tissues in rats infected
with Pseudomonas aeruginosa. Molecular biomarkers were analyzed by matrix-assisted laser desorption
ionization (MALDI) or electrospray ionization (ESI) using a 12T SolariX Fourier transform ion cyclotron
resonance (FTICR) mass spectrometer (MS). Non-invasive diagnoses were performed with animal urine.
MS imaging (MSI) experiments on tissues and liquid chromatography (LC)-ESI analyses of rat sera
represented an invasive armory. Results: In experimental aspergillosis, the mean concentrations of TAFC
and FC in the infected rat urine were 0.37 and 0.63 µg/mL, respectively. The limits of detection of the
ferri-forms of TAFC and FC in the rat urine were 0.02 and 0.03 ng/mL, respectively. As an example, the
initial FC signal reflecting the infection appeared two days post-infection. The MALDI FTICR MSI
illustrated the actual microbial ferricrocin distribution in the lung tissue and resolved the false-positive
results obtained by the light microscopy and histological staining. In an analogous experiment with
Pseudomonas aeruginosa, PyE was quantified in rat urine by LC-FTICR approach by application of PyE
non-isobaric analogues. Separation of desferri and ferriforms of siderophores was achieved on biphenyl
LC column. In parallel, PyE was visualized by MALDI-MSI in infected lung and muscle tissues. Lateral
distribution of PyE correlated with bacterial bodies visualized by scanning electron microscopy.
Conclusion: Ferricrocin, triacetylfusarinine C and pyoverdine E detection in urine represents an
innovative non-invasive indication of Aspergillus and Pseudomonas infections in a host.
Session Number: 397
Session Number: 397
Abstract Title:
Novel Point-Of-Care Electricity-Free Centrifugation of Blood for Resource Challenged Diagnostic
Microbiology Lab. Using Ultra-Low Cost Hand-Powered Plastic/Paper Centrifuge
A. Gautam1, B. Subedi2, A. K. Gupta1, N. Koirala1; 1Dr.Koirala Res. Inst. for Biotechnology, Kathmandu,
Nepal, 2Pokhara Univ., Pokhara, Nepal
Abstract Body:
Background: We are witnessing a great paradigm shift from central laboratory-based diagnosis to point-
of -care based diagnosis. The currently used centrifuges are heavy, large, expensive and impractical for
field clinics, with no electricity access. The design and fabrication of hand-powered centrifuge is based
on principle of an ancient whirligig string toy operated by spinning. We evaluated the electricity-free
centrifugation potential of the hand-powered centrifuge using human blood at point-of-care level.
Methods: We designed and fabricated plastic/paper centrifuge of 12-cm diameter size. It was composed
of two circular 1-mm thick plastic/paper discs used for stationery purpose which was bound by an
adhesive tapes with two small holes in the center. Two sample capillary holders were glued horizontally
to the discs. An extra-strong 62-cm long fishing line was used for spinning. Phlebotomy was performed
aseptically during remote field clinics and 2-3 ml blood was transferred to small tube. Then it was placed
inside the sample holder and rotated by hand for 3-4 minutes. This resulted in a good separation of
plasma from the cellular blood components. Results: We found that hand-powered centrifuge can
adequately separate the plasma from anti-coagulated blood within 2-3 minutes. The overall cost and
weight of centrifuge is $ 0.5 USD and about 25-30g respectively, which is more practical and cost-
effective than modern electric centrifuges. We were able to show good qualitative agreement in terms
of centrifugation and separation of pure plasma by the hand-powered centrifuge Conclusion: Re-
purposing of a simple toy has resulted in a novel point-of-care electricity-free blood centrifuging device
for remote diagnostic laboratory. Additionally the device and methodology provides a practical
alternative when the serum or plasma is required for point-of-care field testing or analysis by diagnostic
kits and device (e.g. testing for a number of infectious diseases.<br /><p><a
href="http://files.abstractsonline.com/CTRL/d1/c/6ae/f32/ddf/4bd/9ab/7b3/1fc/e48/3f8/0a/g2188_1.j
src="http://files.abstractsonline.com/CTRL/d1/c/6ae/f32/ddf/4bd/9ab/7b3/1fc/e48/3f8/0a/g2188_1.jp
Session Number: 397
Session Number: 397
Abstract Title:
Role of Glutathione in Neutralization of Oxidative Stress and Antibiotic Susceptibility in Pseudomonas
Aeruginosa
S. Alsaleh, D. Kwon; Long Island Univ., Brooklyn, NY
Abstract Body:
Pseudomonas aeruginosa is a major causative agent of hospital- and community-acquired infections.
Antibacterial treatment of the infections is often difficult due to presence of antibiotic resistant P.
aeruginosa. A number of intrinsic and acquired antibiotic resistance mechanisms are reported in P.
aeruginosa. Recently, scavenging intracellular reactive oxygen species (ROS) has been suggested as an
intrinsic antibiotic resistance mechanism to all bacterial species since antibiotics induce oxidative stress
in the bacterial species. However, this intrinsic resistance mechanism is currently controversial and
further clarification requires. Glutathione is a sulfuhydryl (-SH)-containing tri-peptide intracellular
antioxidant and serves as a scavenger of the intracellular ROS. In this study, a mutant P. aeruginosa
knocked-out a gene (gshA) encoding glutathione synthetase, a gshA-complemented mutant P.
aeruginosa, and their parental wild type (MPAO1) were used to understand the role of glutathione in
the neutralization of oxidative stress (H2O2) and antibiotic susceptibility. Bacterial killing assays showed
that the mutant strain (gshA::Tn-Tc) was completely killed at 0.005% of H2O2 while the gshA-
complemented and their parental strains were both completely killed at 0.01% of H2O2. Antibiotic
susceptibility testing showed that the mutant strain was at least 2-fold more susceptible to all tested
antibiotics than that of its parental strain. The gshA-complemented strain fully restored the
susceptibility to the same antibiotics as the same levels of the parental strain. The results indicate that i)
glutathione is associated with neutralization of oxidative stress, ii) antibiotics induce the oxidative stress,
and iii) the antibiotic-induced oxidative stress in the mutant strain may have more ROS than its parental
strain which results in the increased-susceptibility to antibiotics. Overall, the results suggest that
glutathione is one of the intrinsic antibiotic resistance mechanisms and may be a possible drug target to
treat the untreatable multidrug resistant P. aeruginosa.
Session Number: 397
Session Number: 397
Abstract Title:
Unravelling Core Functional Microbiota in Traditional Solid-State Fermentation by High-Throughput
Amplicons and Metatranscriptomics Sequencing
Z. Song, H. Du, X. Wang, H. Zhang, Y. Tan, Y. Xu; Jiangnan Univ., Wuxi, China
Abstract Body:
Background: Fermentation microbiota is particular microorganisms that generate multiple metabolites
in different productions. In traditional solid-state fermentation, the structural composition and
functional capacity of the core microbiota determine the quality and quantity of products. As a typical
food fermentation, Chinese soy sauce aroma type liquor production involves complicated microbes and
various metabolites. However, the microbial succession and functional shift of the core microbiota in
this traditional food fermentation remain unclear. Methods: high-throughput amplicons (16S rRNA gene
and internal transcribed space amplicon sequencing) and metatranscriptomics sequencing technologies
were combined to reveal the structure and function of the core microbiota in Chinese soy sauce aroma
type liquor production. In addition, ultra-performance liquid chromatography and headspace-solid
phase microextraction-gas chromatography-mass spectrometry were employed to provide qualitative
and quantitative analysis of the major flavor metabolites. Results: A total of 10 fungal and 11 bacterial
genera were identified as the potential core microbiota. In addition, metatranscriptomic analysis
illustrated pyruvate metabolism in yeasts (genera Pichia, Schizosaccharomyces, Saccharomyces and
Zygosaccharomyces) and lactic acid bacteria (genus Lactobacillus) classified fermentation process into
different stages in the production of flavor components. Stage I involved high-level alcohol (ethanol)
production, with the genus Schizosaccharomyces serving as the core functional microorganism. Stage II
involved high-level acid (lactic acid and acetic acid) production, with the genus Lactobacillus serving as
the core functional microorganism. The functional shift from the genus Schizosaccharomyces to
Lactobacillus drives flavor component conversion from alcohol (ethanol) to acid (lactic acid and acetic
acid) in liquor production. Conclusions: Our findings provide insight into the effects of the core
functional microbiota in Chinese soy sauce aroma type liquor production and the functional
characteristics of the fermentation microbiota under different environmental conditions.
Session Number: 397
Session Number: 397
Abstract Title:
Psmβ As Potential Diagnostic Biomarker for Monitoring Staphylococcus Epidermidis Biofilm on Dermal
Fillers
X. Tan1, J. Wu1, Y. Wang2, Y. Wang1, K. S. Phillips2, C. Xi1; 1Univ. of Michigan, Ann Arbor, MI, 2United
Sates Food and Drug Admin., Silver Spring, MD
Abstract Body:
Injectable dermal fillers use in aesthetic clinics are now widespread. Although they are considered
material-safe, soft-tissue filler injections are also always associated with infections. These infections are
reported to be associated with biofilm presence on dermal fillers [1]. As the diagnostics remains the best
option for prevention and treatment of biofilm infections, the goal of this work was to search for
potential markers which could monitor biofilms developed on dermal fillers. For this purpose,
Staphylococcus epidermidis RP62A was used as the model strain to mimic biofilm development on
surface of polyacrylamide hydrogels (PAAm) in vitro. Microscopic imaging and RNA-seq techniques were
applied for monitoring biofilm development and the profile of whole genomic expression. The
microscopic fluorescent imaging showed that RP62A on PAAm surface formed micro-colonies at 6 h,
then developed mushroom-like structure at 12 h, and at 24 h mushroom-like structure disappeared,
remaining a thin bacterial lawn. RNA-seq data analysis revealed that the expression levels of genes
mapped to “agr quorum sensing” and “valine, leucine and isoleucine biosynthesis” were significantly
higher in biofilms (log2foldchange between 2.96 and 5.41) than in planktonic forms, especially the
expression of psmβ (β-type phenol-soluble modulins, log2foldchange between 4.00 and 5.41), which are
strictly regulated by agr quorum sensing [2, 3]. In addition, expression of those genes related to
“quorum sensing (agrB, agrD, and psmβ3)” and “valine, leucine and isoleucine biosynthesis (ilvA and
leuA)” was further monitored during biofilm formation using RT-qPCR assay. The results revealed that
their expressions were higher at maturation than accumulation stage, especially psmβ3 (log2foldchange
of 5.72 at maturation, -1.18 at accumulation). Therefore, all the data suggests that PSMβ plays an
important role in S. epidermidis biofilm formation on dermal fillers, which would serve as potential
marker to detect infection by S. epidermidis biofilms on dermal fillers. Further studies on hemolytic
activities and quantification of PSMs from planktonic and biofilm cultures are ongoing to develop a
simple, fast and sensitive method targeting PSMβ.
Session Number: 397
Session Number: 397
Abstract Title:
Accurate and Expedited Detection of Bacterial Pathogen Presence in Human Bronchoalveolar Lavage
Specimens Using Laser Light-Scattering
A. Tomaras1, K. Rand2; 1BacterioScan, Inc., St. Louis, MO, 2Univ. of Florida, Gainesville, FL
Abstract Body:
Background: Bacterial lower respiratory tract infections, in particular ventilator-associated pneumonia
(VAP), carry high rates of mortality and are often caused by antibiotic-resistant pathogens. Despite the
dire circumstances for VAP-afflicted patients, methods to expedite diagnosis over standard, quantitative
culturing are not readily available, leaving clinicians with little choice but to administer broad-spectrum,
empiric antibiotics. In uninfected patients, this approach adds to the risk of antibiotic resistance and
thwarts antibiotic stewardship efforts. The BacterioScan 216Dx, a laser light-scattering instrument,
offers more rapid detection of bacterial growth in human specimens, as evidenced by its recent utility in
screening urine specimens to facilitate the diagnosis of urinary tract infections. Here, we extended those
detection capabilities to patient bronchoalveolar lavage (BAL) specimens to determine the improvement
in performance compared to standard methods. Methods: A total of 55 de-identified clinical BAL
specimens were analyzed by first diluting them into brain heart infusion broth and subsequently
incubating them in the 216Dx for 20 hours, during which optical measurements were collected every 3-5
minutes. Standard culture data (recovered pathogen density and identity) served as the reference to
assess 216Dx performance. Bacterial pathogen presence ≥10,000 CFU/ml was considered clinically
significant, whereas lower pathogen densities and flora/fungal presence was regarded as insignificant.
Results: Using the parameters described above, 25% of all BAL specimens evaluated were positive by
plate-based culturing. No bacteria were recovered from 13% of the specimens, with the remaining 62%
harboring commensal flora only. Based on optical signal increases over five consecutive data points, the
216Dx demonstrated sensitivity and specificity values of 85.7% and 87.8%, respectively, after six hours
of analysis. After eight hours in the 216Dx, these values changed to 92.9% sensitive and 73.2% specific,
resulting in more rapid negative classification of BALs in 54.5% of all specimens tested. Conclusions:
Laser light-scattering provided a rapid and effective screening method for BAL specimens with clinically-
relevant densities of respiratory pathogens. Based on these results, processing in the 216Dx would
expedite BAL classification by at least 16 hours relative to conventional methods, and promote the
withholding or prompt de-escalation of antibiotic therapy to uninfected patients.
Session Number: 397
Session Number: 397
Abstract Title:
Electric Cell Substrate Impedance Sensing As A Means of Determining the Virulence Potential of
bacterial Isolates
M. A. Nahid1, C. E. Campbell1, M. B. Lustik1, M. A. Washington2; 1Tripler Army Med. Ctr., Honolulu, HI,
2U.S. Army Med. Res. Unit, Tbilisi, Georgia
Abstract Body:
Background: The ability of bacterial pathogens to disrupt and invade cell monolayers is a surrogate
measure of virulence. This is due to the fact that virulent pathogens tend to degrade tissue barriers and
disperse throughout the body. It has been shown that the ability of pathogens to invade host tissues is
responsible for the spread and dissemination of these pathogens within the host. The purpose of this
study was to demonstrate that Electric Cell-substrate Impedance Sensing, ECIS can be used to detect
invasive properties of pathogens in vitro. Methods: Using ECIS we evaluated 20 archived isolates of 4
bacterial species: E. coli, S. aureus, E. faecalis, and P. aeruginosa. Arrays consisting of 8 wells with 10
electrodes on the planar surface of each well were used for all assays. To form cell monolayers, poly L
lysine treated ECIS wells were seeded with 250,000 A549 cells in complete media. Cell adhesion and
spreading was registered prior to and post pathogen introduction as AC impedance changes at the
electrode over 11 frequencies. Cell adhesion and spreading was registered for at least 24 hours prior to
introduction of pathogen. Bacteria suspension, OD 1.0 was added to established cell monolayers in
antibiotic free media. Controls included electrode and media only or pathogen free cell monolayers. The
ECIS analysis software was used to extract barrier function, alpha, a measure of cell surface distance,
and cell membrane capacitance values. Results: There was a significant difference in impedance values
between A549 cells only and A549 cells exposed to pathogens. Each bacterial strain showed unique
impedance changes. Serial dilution series demonstrated a correlation between pathogen load and
impedance changes. Additionally, heat killed pathogens did not show significant change of impedance
over 24 hours in the A549 cells monolayer. The impedance response also reflected differences between
pathogenic strains invading A549 cells. Initially, in samples containing P. aeruginosa, the impedance
increased significantly before subsequently decreasing. In contrast, E. coli, S. aureus, and E. faecalis
samples induced a significant decrease in impedance. Barrier function, cell membrane capacitance, and
alpha, were extracted from the impedance values obtained over a range of frequencies. Conclusions:
Our findings indicate that ECIS provides a novel, automated, and quantitative method to distinguish
pathogens rapidly in the clinical laboratory.
Session Number: 397
Session Number: 397
Abstract Title:
Rapid and Reagent-Free Typing of Vancomycin-Resistant Enterococcus faecium by Attenuated Total
Reflectance-Fourier Transform Infrared (Atr-Ftir) Spectroscopy As A New Tool for Outbreak
Investigations
T. Tsutsumi1, L. Lam1, C. Frenette2, N. Doherty2, J. Sedman1, A. Ismail1; 1McGill Univ., Sainte-Anne-de-
Bellevue, QC, Canada, 2McGill Univ. Hlth.Ctr., Montreal, QC, Canada
Abstract Body:
Whole-organism fingerprinting by ATR-FTIR spectroscopy is a rapid, reagent-free technique for bacterial
identification and classification with subspecies-level discriminatory capabilities. ATR-FTIR methods for
identification of nosocomial pathogens developed by our group include the identification of
vancomycin-resistant (VR) Enterococcus faecium, based on the capability of ATR-FTIR spectroscopy to
discriminate between antibiotic-resistant and susceptible strains in the absence of antibiotic. The
present study extends this work to examine the possibility of employing ATR-FTIR spectroscopy as a
rapid method for discriminating among VRE clonal groups. As pulsed-field gel electrophoresis (PFGE) is
the gold-standard method for strain typing in VRE outbreak investigations, a set of 93 clinical isolates of
VRE. faecium previously typed by PFGE was employed in this study; these isolates were distributed
between two pulsotypes. FTIR spectra were acquired by transferring isolated colonies directly from
blood agar plates onto the sampling surface of a portable ATR-FTIR spectrometer; spectral acquisition
time was 1 min. Triplicate spectra were collected per isolate, each from different colonies on the same
culture plate. Spectral data analysis was performed by hierarchical cluster analysis and principal
component analysis (PCA) in conjunction with the use of a feature selection algorithm. The spectra of all
isolates were correctly identified as VRE. faecium using an ATR-FTIR spectral database for identification
of Gram-positive pathogens associated with nosocomial infection, developed in our previous work. PCA
showed successful discrimination between the two clonal groups, yielding 99% concordance with PFGE
results and indicating that ATR-FTIR spectroscopy may provide a rapid and easily implemented typing
method suitable for intrahospital surveillance of VRE outbreaks. Furthermore, this study illustrates that
the ATR-FTIR spectrum of a clinical isolate can be used to identify it, classify it as antibiotic resistant, and
assign it to a clonal group, ultimately reducing the time and cost of labor and reagents required,
compared to current laboratory procedures that require multiple analyses.
Session Number: 397
Session Number: 397
Abstract Title:
Biospectrrix: Rapid and Sensitive Bacterial Identification Directly from Blood
R. Krishnamurthy, J. Janicki; 3i Diagnostics, Inc., Germantown, MD
Abstract Body:
Background: It is widely acknowledged that this empirical antibiotic treatment leads to misuse &
overuse of antibiotics. Significant reductions in costs, complications, and mortality are possible, if the
infection-causing bacteria can be identified sooner than currently possible. We report the development
and evaluation of a system (BiospectrixTM) that does not require culturing and identifies a broad range
of bacteria in < 1 hour directly from blood.  Methods: The system includes a high throughput
microfluidic system that selectively breaks down blood cells but not bacteria such that the largest debris
is smaller than intact bacteria. The highly selective lysis is achieved by leveraging the differential
response of bacterial and human cells to mechanical stresses imposed on them by passing the sample
through a finely tuned porous network. Intact bacteria are then separated from the lysis debris and
concentrated by filtration. The bacteria on the surface of the filter are analyzed by Fourier-Transform
Infrared Spectroscopy (FTIR) to determine their identity on the basis of their unique spectral profile. Ten
different bacterial species (5 gram-negative and 5 gram-positive) were cultured and spiked into 5 mL of
whole blood at concentrations ranging from 10 - 1000 CFU/mL. The seeded samples were passaged
through the microfluidic system at flow rates ranging from 0.05 - 0.3 mL/min followed by filtration using
a 0.4 micron filter. The isolated bacteria were identified by comparing the sample’s FTIR profile against
those in a reference database.  Results: 99.999% of the blood cells were lysed by passage through the
porous network. The size of the largest debris was < 500nm. Bacterial recovery, measured by culturing,
was > 93%. Recovery of bacteria during the filtration step was > 95%. The FTIR profile could clearly
identify the ten species of bacteria. The LoD of the method is < 100 CFU/mL. Conclusions: Our findings
indicate the feasibility of a culture-independent, rapid, sensitive, and specific diagnostic approach to
identify bacteria directly from blood. Such a system can provide much-needed information to the
physician - whether bacteria are present in the sample and their identity - aiding the early and
appropriate use of targeted antibiotics.
Session Number: 397
Session Number: 397
Abstract Title:
Utilization of Laser Light Scattering for Rapid Detection and Subsequent Antimicrobial Susceptibility
Testing Directly from Urine Specimens
S. Collier1, A. Tomaras2, A. Skinner1, F. Albarillo1, A. Harrington1; 1Loyola Univ. Med. Ctr., Maywood,
IL, 2BacterioScan, Inc, St Louis, MO
Abstract Body:
Background: The high volumes and lengthy turnaround times required to process urine specimens
present a workflow challenge and prompt the use of empiric therapy, including to patients whose
specimens may be uninfected. Methods that return diagnostic information more rapidly would improve
patient care and facilitate antibiotic stewardship efforts. The BacterioScan 216Dx is a laser light
scattering device that detects urinary tract infections (UTIs) more rapidly than current standard of care
(SOC) methods. Our lab evaluated the 216Dx’s ability to provide rapid antimicrobial susceptibility testing
(AST) data using samples deemed positive during the UTI screening protocol. Methods: Ninety-five urine
specimens were analyzed using the 216Dx UTI detection protocol according to the manufacturer’s
instructions. Cuvette contents from positively flagged specimens were spread onto pre-warmed blood
and MacConkey agar plates and incubated at 37°C for 4 hours. MALDI-TOF MS (Bruker) identification
was performed on the resulting agar surface film. Direct-from-positive specimen AST was also evaluated
on 22 samples containing Gram negative pathogens using a panel of 4 antibiotics tested at clinical
breakpoint concentrations. Cuvette contents were diluted to ~5x105 CFU/ml into cation-adjusted
Mueller Hinton Broth prior to dispensing into antibiotic-containing cuvettes. AST runs in the 216Dx were
conducted at 35°C for 16 hours, after which optical profiles were compared to a corresponding no drug
control. Results: Fifty-four specimens possessed ≥10,000 CFU/ml of a UTI pathogen, 51 of which were
accurately detected by the 216Dx after 190 minutes. Despite contaminant presence in many of the
specimens, MALDI-TOF MS analysis of positively-flagged cuvette material showed ~75% agreement with
SOC data, with the majority of failed samples harboring lower density infections, Gram positive
pathogens, or possessing >1 UTI pathogen. The SOC AST method (MicroScan) indicated non-
susceptibility for 36.3, 36.3, 4.5, and 0% of isolates to Ciprofloxacin (CIP), Levofloxacin (LVX), Cefepime
(FEP), and Meropenem (MER), respectively. After 3-4 hours of analysis, the 216Dx demonstrated
categorical agreement at 100% for CIP, FEP, and MER, and 95.4% for LVX. Conclusion: Based on these
data, the BacterioScan 216Dx has the potential to offer accurate UTI detection and, when coupled with
MALDI-TOF MS, robust pathogen ID/AST for at least 75% of Gram negative infected urine specimens,
within as little as 8 hours post-collection. Further validation is needed and ongoing.
Session Number: 397
Session Number: 397
Abstract Title:
Beyond Maldi-Tof's Score Values...Use of Bionumerics Software to Overcome Maldi-Tof Limitations.
Identification of Classical Bordetella Species
J. C. Zintgraff, C. S. Lara, M. Arias, G. A. Ayala, M. Santos; INEI-ANLIS Dr Carlos G Malbran, Ciudad
Autonoma de Bs As, Argentina
Abstract Body:
Background: Differentiation of classical Bordetella species by MALDI-TOF MS is well known to be
challenging. This study used the Bruker MALDI Biotyper system in addition to mass spectra model
analysis generated with the BioNumerics software package. Methods: This study used Bruker MALDI
Biotyper system in addition to a mass spectra model analysis generated by reference strains of
Bordetella pertussis, Bordetella parapertussis andBordetella bronchiseptica in the BioNumerics software
package (Applied Maths, Belgium) to identify clinical isolates. The results were compared with those
generated by MALDI Biotyper system alone.Different classifiers were evalated such as SVM and Basic
Similarity among others. PCA plotters were also performed. Results: The percentages of correct species
level identification using MALDI Biotyper system alone and following manufacturer recommendations
was 62.2% (66/106) with the additional Bionumerics mass spectra analysis the percentage of correct
identification increased to 99.1% (105/106) according the use of different classifiers. Conclusions: This
new workflow may improve the accuracy of classical Bordetella identification significantly. However
validation and verification procedure for accuracy and precision need to be evaluated in further
analysis.<br /><p><a
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src="http://files.abstractsonline.com/CTRL/55/b/113/c1b/d90/42d/a81/5e0/767/c0e/fd8/38/g2091_1.p
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Session Number: 397
Session Number: 397
Abstract Title:
An Improved In-House Maldi-Tof Ms Protocol for Direct Cost-Effective Identification of Pathogens from
Blood Cultures
M. Zhou, Q. Yang, Y. Xu; Peking Union Med. Coll. Hosp., Beijing, China
Abstract Body:
Background: Bloodstream infection is a major cause of morbidity and mortality in hospitalized patients
worldwide. Delays in the identification of microorganisms often leads to a poor prognosis. The
application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF
MS) directly to blood culture (BC) broth can potentially identify bloodstream infections earlier, and
facilitate timely management. Materials/Methods: We developed an “in-house” (IH) protocol for direct
MALDI-TOF MS based identification of organisms in positive BCs. The IH protocol was initially evaluated
and improved with spiked BC samples, and its performance was compared with the commercial
Sepsityper™ kit using both traditional and modified cut-off values. We then studied in parallel the
performance of the IH protocol and the colony MS identifications in positive clinical BC samples using
only modified cut-off values. All discrepancies were investigated by “gold standard” of gene sequencing.
Results: In 54 spiked BC samples, the IH method showed comparable results with Sepsityper™ after
applying modified cut-off values. Specifically, accurate species and genus level identification was
achieved in 88.7% and 3.9% of all the clinical monomicrobial BCs (284/301, 94.4%), respectively. The IH
protocol exhibited superior performance for Gram negative bacteria than for Gram positive bacteria
(92.8% versus 82.4%). For anaerobes and yeasts, accurate species identification was achieved in 80.0%
and 90.0% of the cases, respectively. For polymicrobial cultures (17/301, 5.6%), MALDI-TOF MS correctly
identified a single species present in all the polymicrobial BCs under the Standard mode, while using the
MIXED method, two species were correctly identified in 52.9% of the samples. Comparisons based on BC
bottle type, showed that the BACTECTM Lytic/10 Anaerobic/F culture vials performed the best.
Conclusions: Our study provides a novel and effective sample preparation method for MALDI-TOF MS
direct identification of pathogens from positive BC vials, with a lower cost ($1.5 vs $ 7) albeit a slightly
more laborious extracting process (an extra 15 minutes) compared with Sepsityper™ kit.
Session Number: 397
Session Number: 397
Abstract Title:
Media Makes A Difference in the Detection of Surveillance Isolates of Methicillin Resistant
Staphylococcus aureus Using the Bruker Maldi-Tof Mass Spectrometry Mrsa Psm-Mec Detection Module
D. Boulton; Grand River Hosp., Kitchener, ON, Canada
Abstract Body:
Background: Methicillin resistant Staphylococcus aureus (MRSA) is a major nosocomial pathogen
worldwide and associated with high rates of mortality and morbidity. Recently, Bruker developed a
software package to be used with their MALDI Biotyper System (MBT) allowing for direct detection of
phenol soluble moduline (PSM-mec), a staphylococcal toxin produced by a part of MRSA strains. This
toxin, coupled with antibiotic resistance genes allows a simultaneous species identification and MRSA
positivity alert by detection of the PSM-mec signal in MALDI-TOF mass spectra. Methods: Stored MRSA
isolates from surveillance samples were used to investigate the performance of the PSM detection
module. Isolates were originally confirmed from typical morphological growth on BioRad MRSASelect
Agar culture media and/or a positive Alere PBP2a SA Culture Colony Test. Each strain was plated onto
Columbia agar with 5% sheep blood (BA), BioRad MRSASelect Agar, and Alere Colorex MRSA Agar plates
and incubated at 35°C for 18-24 hours. Typical colonies from each plate were inoculated onto a target
plate before adding formic acid and matrix for protein extraction and run in the Bruker MALDI Biotyper
System. Ability to detect PSM-mec between chromogenic media was compared, using the BA as a non-
chromogenic comparison. Results: A total of 200 confirmed MRSA isolates from March to October 2017
were analyzed. Of the 200 samples, 78 (39%) detected PSM-mec when run from the BA plate, all 200
samples (100%) detected PSM-mec when run from the Alere Colorex MRSA Agar plates, and none of the
samples detected PSM-mec when run from the Bio-Rad Select Chromogenic plates. Conclusion: The
performance of the PSM MBT Subtyping module is influenced by the type of chromogenic media from
which isolates are run. Alere Colorex MRSA Agar plates outperformed both MRSASelect Agar (BioRad)
and BA plates.
Session Number: 397
Session Number: 397
Abstract Title:
Viability of Candida Auris and Other Candida Species after Three Maldi-Tof Preparation Protocols
A. Bateman, A. Sterkel, A. Valley, D. Warshauer; Wisconsin State Lab. of Hygiene, Madison, WI
Abstract Body:
Background: Candida auris is an emerging, often multidrug-resistant, yeast of public health concern. This
organism can persist in the environment, which complicates efforts to prevent transmission in health
care settings. Commercially-available identification techniques can mis-identify this species. Matrix-
assisted, laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry is one of the most
accurate methods for identifying this species. However, there is a lack of data about C. auris viability
after various preparation protocols for MALDI-TOF testing. Methods: We tested 10 C. auris isolates and
20 isolates of various other Candida species with three different MALDI-TOF preparation protocols: on-
plate extraction with formic acid (extended direct method), CDC 1:1 ethanol:water extraction, and
Bruker tube extraction with formic acid and acetonitrile. Viability was assessed after various steps in the
Bruker tube extraction by removing a small aliquot after each step. After spotting the Bruker MALDI-TOF
target plate in duplicate with each preparation and overlaying with matrix, a flocked swab dampened
with RPMI broth was used to remove the dried spots, and growth in RPMI broth and on Sabouraud
Dextrose plates was assessed. Positive controls consisted of spotting yeast on the MALDI target plate,
followed by removing with a dampened flocked swab and assessing growth. Results: No growth in RPMI
broth or on Sabouraud Dextrose plates was observed after 48 hours with any Candida species treated
with the 1:1 ethanol:water extraction or after any step in the Bruker tube extraction. The extended
direct method greatly decreased the viability; with C. auris, there were no colonies on any of the 10
isolates, and only 5/10 RPMI broths showed growth. With the extended direct method for C. albicans,
every RPMI broth showed growth and colonies were present on Sabourad Dextrose plates, but the
number of colonies was <1% of the positive control. Conclusions: These data indicate that the CDC 1:1
ethanol:water and Bruker tube extraction methods are sufficient to kill Candida species, including C.
auris, prior to MALDI-TOF analysis. This information can help inform risk assessments at clinical and
public health microbiology laboratories performing MALDI-TOF yeast identification.
Session Number: 397
Session Number: 397
Abstract Title:
Maldi-Tof Ms for Automated Detection of Kpc-Producing Enterobacteria - Beyond Klebsiella Pneumoniae
M. Cordovana1, M. Kostrzewa2, M. Bienia3, S. Ambretti1, A. Pranada3; 1Univ. Hosp. of Bologna -
Policlinico Sant'Orsola-Malpighi, Bologna, Italy, 2Bruker Daltonik GmbH, Bremen, Germany, 3MVZ Dr.
Eberhard & Partner Dortmund, Dortmund, Germany
Abstract Body:
Background: Carbapenem resistant enterobacteria are a major public health concern. Initially, resistance
mediated by blaKPC evolved only in Klebsiella pneumoniae, but reports of KPC-production in other
enterobacteria, especially in Escherichia coli, are increasing worldwide.Recently, we described the
automated detection of the KPC-specific peak at 11,109 m/z in the MALDI-TOF MS spectra of K.
pneumoniae, enabling the instant detection of KPC-producing strains during identification process. In
this study we have extended the automated real-time detection of the KPC-specific peak to further
clinically relevant species of enterobacteria. Methods: Mass spectra of n=8801 enterobacteria isolates
(n=3502 E. coli, n=2663 Enterobacter spp., n=1460 K. oxytoca, n=639 Citrobacter spp. and n=537 S.
marcescens) were investigated for the presence of the KPC-specific peak.Spectra were acquired for
strains collected from 2009 to 2017 in two laboratories, located in Italy (S.Orsola-Malpighi hospital,
Bologna) and Germany (MVZ Dr. Eberhardt & Partner Dortmundincluding carbapenem-resistant (n=521)
and -susceptible isolates (n=8280) with a MALDI Biotyper system (Bruker Daltonik, Germany). KPC
producers were identified by disc-diffusion synergy and/or immunochromatographic test. For each
species, a specific algorithm for the automated detection of the KPC peak was developed and evaluated,
in order to be implemented into the Biotyper software. Results: The number of KPC-producing E. coli
rose from a single isolate in 2011 to 66 in 2017. The new algorithm detected the specific peak in
126/146 (86.3.%) of the strains.The KPC peak was also detected in 5/5 E. cloacae complex, 9/11 E.
aerogenes, 3/5 K. oxytoca, 8/9 C. freundii, and 4/6 S. marcescens KPC-producers.The peak was not
detected in the 8280 non-KPC strains but one non-KPC C. freundii strain resulted as positive.
Conclusions: We have developed specific algorithms for automated detection of KPC-producing
enterobacteria other than Klebsiella pneumoniae. Our study shows a high sensitivity (85%) and excellent
specificity (99.9%), similar to the established detection of KPC in K. pneumoniae. The sensitivity is likely
related to the prevalence of the specific plasmid harboring clones among all the KPC-producing
circulating strains. Integration of these novel algorithms into the MALDI species identification routine
could allow fast detection of these worrisome resistance markers, accelerating respective infection
control measures.
Session Number: 397
Session Number: 397
Abstract Title:
Multilocus Sequence Typing and Maldi-Tof Ms Fingerprinting for Discrimination of Cronobacter Sakazakii
Isolates from Environmental Surveillance Samples
I. Sulaiman, N. Miranda, S. Simpson, K. Kerdahi; FDA, Atlanta, GA
Abstract Body:
Background: Cronobacter species are considered as opportunistic foodborne pathogenic bacteria
causing acute meningitis and necrotizing enterocolitis in neonates, and linked to powdered infant
formula worldwide. Of these, three Cronobacter species (C. sakazakii, C. malonaticus and C. turicensis)
have been reported to be more virulent, and isolated often from infant meningitis cases. Species
identification of Cronobacter is critical for the detection of foodborne pathogen and source tracking of
contaminated foods Methods: A total of 314 environmental surveillance samples were analyzed to
isolate Cronobacter, initially by two-step enrichment followed by streaking on a selective agar. Initial
identification of recovered isolates was completed using bioMerieux VITEK 2 System and real-time PCR
analysis. Afterwards, the isolates were analyzed on the bioMerieux VITEK MALDI-TOF MS system.
Multilocus sequence typing (MLST) was performed based on the seven housekeeping genes (atpD, fusA,
glnS, gltB, gyrB, infB, and pps) using ABI 3500XL Genetic Analyzer. Results: All ten recovered Cronobacter
isolates were identified as Cronobacter sakazakii with a high confidence value (99.9%) by the VITEK MS
system. The MLST analysis identified three distinct clonal complex (2/CC1, 2/CC4 and 8/CC64) for the
recovered Cronobacter sakazakii isolates. All the three-clonal complex have been previously recovered
from Powdered Infant Formula, Infant Formula Production Factory, and patient with Neonatal
Meningitis. Conclusions: Results of this study suggest that MALDI-TOF MS and MLST based analysis can
be used for species-identification of Cronobacter isolated from environmental surveillance samples of
public health importance.
Session Number: 397
Session Number: 397
Abstract Title:
Identification (Maldi-Tof Ms) and Susceptibility Testing Directly from Positive Blood Culture Broth
M. Pham, L. Ortiz, K. Van Horn; Southern California Permanente Med. Group, Chino Hills, CA
Abstract Body:
Rapid identification and susceptibility testing of blood culture pathogens is critical for patient
management. Positive blood culture broth may be used for direct identification by MALDI-TOF mass
spectrometry (MS) and for direct automated susceptibility testing. In this study, we used a simplified
non-lysis centrifugation method to obtain organism essentially free of blood cells to use for
identification by VITEK® MS and susceptibility testing by VITEK® 2 (bioMerieux). Briefly, 2 mL of positive
blood culture broth was centrifuged at low speed (400 rpm) to pellet and remove human cells. The
supernatant was then centrifuged at high speed (4000 rpm) to pellet organisms, washed with saline and
recentrifuged to obtain an organism pellet. A 1 μl loop was used to spot the VITEK MS target, then
processed according to manufacturer’s instructions with 2 spots for matrix and 2 additional spots for
formic acid (FA)/matrix. For susceptibility testing, VITEK 2 susceptibility cards, chosen based on the
Gram stain, were inoculated with the pelleted organism, incubated and resulted according to
manufacturer’s instructions. Identification and susceptibility results were compared to results obtained
with the standard methods used in our laboratory. Direct VITEK MS was performed on 62 Gram-negative
rods, 100 Gram-positive cocci, and 2 anaerobes. Overall, 95 (58%) organisms were identified with matrix
extraction and 119 (73%) with FA/matrix extraction. The VITEK MS accurately identified 53 (85%) of the
Gram-negatives with matrix extraction and 55 (89%) with FA/matrix. The addition of FA/matrix for
Gram-positives yielded best results compared to matrix alone with 64 (64%) identified: 46 staphylococci
(n=72); 3 streptococci (n=10); 15 enterococci (n=18). One of two anaerobic Gram-negative rods was
accurately identified. There were a total of 1878 susceptibility data points evaluated. For Gram-positives
there were 1054 data points with 9 discrepancies (99.1% accuracy): 2 very major errors; 1 major error; 6
minor errors. For Gram-negatives there were 824 data points with 7 discrepancies (99.2% accuracy): 1
very major error; 1 major error; 5 minor errors. A simplified centrifugation method for rapid
identification of positive blood cultures by VITEK MS is accurate for Gram-negative rods, but may require
supplemental testing for Gram-positives. Direct susceptibility testing of positive blood cultures by VITEK
2 yielded accurate results when compared to standard laboratory methods.
Session Number: 397
Session Number: 397
Abstract Title:
Accurate Differentiation of Novel Staphylococcus Argenteus from Staphylococcus aureus Using Matrix-
Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry
H. Lee1, S-Y. Chen1, S-H. Teng1, X-M. Wang1, T-F. Lee1, Y-C. Huang1, C-H. Liao1, L-J. Teng2, P-R. Hsueh1;
12Natl. Taiwan Univ. Hosp., Taipei, Taiwan
Abstract Body:
Background: Staphylococcus argenteus, a newly discovered staphylococcal species, has been
increasingly reported globally. Failure to identify S. argenteus in routine microbiology laboratories
prevents the evaluation of actual disease burden caused by S. argenteus. We sought to develop a S.
argenteus-specific diagnostic profile for matrix-assisted laser desorption ionization-time of light mass
spectrometry (MALDI-TOF MS) system to facilitate rapid and accurate identification of this novel
bacterium. Methods: We re-identified 97 S. argenteus isolates from 915 methicillin-susceptible S. aureus
(MSSA) bloodstream isolates based on the absence of the crtM gene and multilocus sequence analysis.
Cluster analysis of MALDI-TOF MS results for 25 S. argenteus and 25 matched MSSA isolates was
performed. A classification model was generated to differentiate S. argenteus from S. aureus. The
performance of the classification model was validated against the remaining 72 S. argenteus and 72
MSSA bloodstream isolates. Results: All S. argenteus isolates were negative for crtM gene and 74.2%
(72/97,) were multilocus sequence type 2250. MALDI-TOF MS analysis based on the existing database
failed to differentiate the 97 S. argenteus from the 97 matched S. aureus bloodstream isolates, with
median identification score distribution of 2.149 (range, 2.260-1.855) and 2.397 (2.545-2.228),
respectively. However, with cluster analysis and classification model, differentiation of S. argenteus from
S. aureus had 100% accuracy in chemical extraction method and had 87.5% sensitivity and 100.0%
specificity in direct smear method. Conclusions: With the new database and classification model created
for S. argenteus, MALDI-TOF MS could rapidly and accurately differentiate S. argenteus from S. aureus
isolates.
Session Number: 397
Session Number: 397
Abstract Title:
Mass Spectrometry-Based Mis-Identification of Klebsiella Variicola and K. pneumoniae Resolved
Through Average Nucleotide Identity by Blast (Anib) Analysis
R. J. Cybulski, Jr., M. Stewart, S. Turner, L. Cummings, M. Sims, S. J. Salipante, B. T. Cookson; Univ. of
Washington Med. Ctr., Seattle, WA
Abstract Body:
Background: A recently updated Bruker Biotyper RUO database includes Klebsiella pneumoniae 37924
PFM Main Spectra (MSP) reassigned as Klebsiella variicola, a plant-colonizer (1) that shares 96-97%
sequence identity at the rDNA level to K. pneumoniae and K. quasipneumoniae. Questions remain
regarding the prevalence (2,3) and virulence (4) of Klebsiella variicola as compared to K. pneumoniae, as
well as the underlying genetic bases of Klebsiella variicola virulence (2,4). Methods: We used MALDI-TOF
to retrospectively re-identify inpatient bloodstream isolates previously identified as K. pneumoniae.
These isolates also underwent whole genome sequencing (WGS) in order to confirm their identity,
establish their sequence and capsular type, assess their relatedness and search for K. pneumoniae
associated virulence determinants. Results: 561 K. pneumoniae isolates located in the UWMC
Microbiology repository represented 243 unique cases of bacteremia between 2008-2017. 197 isolates
were confirmed as K. pneumoniae by Matrix-Assisted Laser Adsorption and Ionization, Time of Flight
(MALDI), while 55 were presumptively re-identified as Klebsiella variicola based on the reassignment of
MSP 37924. Subsequent WGS-based Average Nucleotide Identity, by BLAST (ANIb) analysis yielded 9
discrepancies among the MALDI-based Klebsiella variicola identities. Clustering based upon ANI
identified 8 isolates as K. pneumoniae. One isolate was more consistent with K. quasipneumoniae.
Adonitol fermentation testing (1) was performed as discrepant analysis and was consistent with ANIb
assignments for all 9 isolates. MALDI scores that generated the incorrect Klebsiella variicola identity had
an average run score (2.00) that was significantly lower (p < 0.0001) than those with a properly assigned
Klebsiella variicola identity (2.37). Passaging of these strains at 37C for 24 hours led to identification of
all nine as K. pneumoniae, with average run scores of 2.52. Conclusion: K. variicola is an emerging
pathogen that is misidentified as K. pneumoniae by many commercially-available identification
methods. Matrix-Assisted Laser Adsorption and Ionization, Time of Flight (MALDI-ToF) offers enhanced
discriminatory power for these species. However, the specificity of the K. variicola identity is dependent
upon the establishment of a higher threshold than is typically required for isolate identification when
using the Bruker Biotyper RUO database.
Session Number: 397
Session Number: 397
Abstract Title:
Reproducibility of the New bioMérieux® VITEK MS v3.2* Database for Identification of Brucella
C. Bradford1, N. Tolli2, R. Lloyd2, K. Clem2, K. Kiss2, S. Blamey1, D. H. Pincus1; 1bioMérieux, Inc.,
Hazelwood, MO, 2ATCC, Manassas, VA
Abstract Body:
Background: VITEK® MS v3 / KB3.2.0 is a microbial identification system which uses matrix-assisted laser
desorption /ionization time of flight (MALDI-TOF) mass spectrometry. The system is used routinely in the
clinical microbiology laboratory for the identification of aerobic and anaerobic Gram-positive and Gram-
negative bacteria and fungi. In this study, the reproducibility of the new VITEK MS version 3.2 database
to identify Brucella species was evaluated. Methods: A Brucella panel of three well-characterized
blinded samples were tested at ATCC using the VITEK MS system. The panel was tested in duplicate on
each of five days by two technologists. Three separate batches of solvent mixture were prepared for
each day of testing and used by each technologist to inactivate samples. For each day of testing, samples
were positioned on the disposable slide in sequential order by sample number by one technologist. The
second technologist randomly positioned samples on the disposable slide using a specific pattern. The
organism specific and overall rates of correct identification results were determined. Results: Each
technologist collected 30 results for each reproducibility organism, for a total of 60 results per strain.
Identifications were reproducible at 100% (60/60) for each organism tested, with combined agreement
at 100% (180/180). Conclusion: This study showed the VITEK MS to be a reproducible method for
identification of Brucella. *VITEK MS v3 / KB v3.2.0 has not yet been submitted to the United States FDA
for clearance and is not available for commercial sale in the United States
Session Number: 397
Session Number: 397
Abstract Title:
Validation of the Vitek Ms Inactivation Kits and Vitek Ms V3.0 for Mycobacteria, Nocardia and Mold
Identification
D. Contreras, L. Mortimer, R. Mirasol, K. Gih, O. B. Garner; UCLA, Los Angeles, CA
Abstract Body:
Background: Infections caused by invasive molds, Mycobacteria and Nocardia can be the cause of high
morbidity and mortality within the clinical setting, especially among the immunocompromised patient
population. Species level identification is crucial for the appropriate administration of antimicrobial
therapy and patient management because some strains exhibit intrinsic resistance to antimicrobial
agents. Identification of these organisms in the clinical laboratory has been slow and time consuming,
often relying on phenotypic and genotypic characteristics. Matrix-assisted laser desorption ionization-
time of flight mass spectrometry (MALDI-TOF MS) is now a reliable tool for identifying clinically
important molds, Mycobacteria, and Nocardia allowing for rapid and accurate identification to the
species level. Currently, the FDA has cleared the bioMerieux VITEK MS v3.0 for identification of 19
Mycobacteria, 47 molds and 12 Nocardia species, as well as two inactivation kits for organism
inactivation and protein extraction. It is critical to lab safety to confirm the inactivation of these
organisms before placing them on the MALDI-TOF for identification. In this study, we evaluated the
performance of the VITEK MS Inactivation Kit to verify a lack of biological risk before the subsequent
assessment of the VITEK MS v3.0 system. Methods: In total, 15 Mycobacteria, 33 molds and 5 Nocardia
derived from a mixture of clinical isolates and commercial strains were tested. Mycobacteria were
inactivated from both fresh liquid and solid cultures, while Nocardia and molds were inactivated from
solid culture. Briefly, Mycobacteria and Nocardia were subjected to mechanical disruption for 15 min
with 0.5 mm glass beads in 70% ethanol using a vortex mixer with horizontal adapter, followed by 10
min incubation at room temperature. Mold inactivation in 70% ethanol was done when conidia were
present to ensure the process was effective on these more hardy structures. For all organisms, proteins
were extracted using 70% formic acid and acetonitrile. Results: The resulting supernatants were plated
onto appropriate solid media and incubated in conditions for recovery of these organisms. Mycobacteria
and Nocardia were monitored for 42 days and molds for 8 weeks. Conclusions: In conclusion, our
viability studies have rendered no viable Mycobacteria, Nocardia and molds at the specified time points
after performing the provided kit inactivation protocols. We are currently assessing the extracted
protein for identification on the VITEK MS v3.0.
Session Number: 397
Session Number: 397
Abstract Title:
Performance of Bacterial Identification Directly from Blood Cultures by Maldi-Tof Vitek Ms
I. L. Thomas, K. A. Heath, M. Oethinger; Providence St. Joseph's Hlth., Portland, OR
Abstract Body:
Background: Rapid identification of bloodstream pathogens is important for selection of appropriate
antibiotic therapy. Rapid, inexpensive bacterial identification is possible by MALDI-TOF (MALDI) directly
from blood cultures (BC). The goal of this study was to develop the best method for rapid bacterial
identification directly from BCs using MALDI. Materials/Methods: After an initial pilot study, we used the
following method which comprised several steps at which bacteria were spotted to a MALDI slide
(“spotted”): (A) 5 ml content of the BC bottle was transferred to a serum separator tube, spun and the
bacterial pellet spotted; (B) the remaining pellet from (A) was re-suspended with 200 μl of sterile water,
centrifuged and the pellet spotted to a MALDI slide; (C) the rest of the suspension from (B) was pipetted
to pre-warmed blood agar and chocolate plates to form a thick spot, without streaking for colonies, and
incubated for 4 h at 5% CO2. Visible growth after 4 h was spotted to MALDI slides from both plates, and
- depending on the identification result - rapid spot tests were performed (indole, oxidase, PYR, catalase,
and Streptex(TM) latex agglutination if growth showed β-hemolysis on blood agar). Gold standard was
identification by culture by conventional methods, including MALDI. Results: A total of 318 consecutive
positive BC bottles were evaluated. 152/318 (47.8%) of bloodstream pathogens were Gram-negative
(GN) bacteria, 166/318 (52.2%) Gram-positive (GP) bacteria. Most of GN bacteria were bacilli (97.4%),
and most GP bacteria were cocci (89.8%). Overall, 85.2% of bacteria were correctly identified to species
level. Success was notably better for GN bacteria (98%) than for GP bacteria (73.5%). The rate of correct
species identification at different steps, as described above, for GN vs. GP bacteria was as follows: (A)
39.5% vs. 9.6%, (B) 65.8% vs. 18.7%, and (C) 98% vs. 73.5%. Interestingly, all 7 anaerobe GNB were
successfully identified, either from the pellet or after 4 h of microaerophilic incubation. Conclusion: In
this study, we describe the optimum method for direct bacterial identification directly from blood
cultures by MALDI. About two thirds of all GN bacteria were successfully identified within minutes after
blood cultures turned positive, and almost all of them were identified 4 h later. GP bacteria did better
when cultured for 4 h at which point about three quarters of them were correctly identified. This
indicates that a different workflow for Gram-negative and Gram-positive bacteria may save time and
labor going forward.
Session Number: 398
Session Number: 398
Session Title: CPHM05 - Diagnostic Mycobacteriology: Detection, Species Differentiation, and Drug
Resistance
Abstract Title:
Clinical Characteristics of the Delayed Diagnosis and Treatment in Immunocompromised Patients with
Paucibacillary Pulmonary Tuberculosis
J. Park, M. Bae, S. Choi, K. Jung, M. Kim, Y. Chong, S-O. Lee, S-H. Choi, Y. Kim, J. Woo, K. Jo, T. Shim, S-H.
Kim; Asan Med. Ctr., Univ. of Ulsan Coll. of Med., Seoul, Korea, Republic of
Abstract Body:
Background: Delayed diagnosis and treatment of pulmonary tuberculosis (TB) may lead to the
transmission of TB and increase the severity, morbidity, and mortality of the disease. Paucibacillary
pulmonary TB especially in immunocompromised patients has potential risk for delayed diagnosis and
treatment because the radiologic features of pulmonary TB in immunocompromised patients are
various. We thus evaluated the clinical characteristics of delayed diagnosis and treatment in
immunocompromised patients with paucibacillary pulmonary TB in a TB-intermediate burden country.
Methods: Immunocompromised adult patients with paucibacillary pulmonary TB were retrospectively
enrolled in a tertiary hospital, Seoul, South Korea, during a 5-year period. Paucibacillary pulmonary TB
was defined as pulmonary TB with negative AFB smear results and positive M. tuberculosis culture
results from respiratory specimens. We defined “missed TB” patients as those who started anti-TB
therapy after confirming positive mycobacterial culture results and diagnostic delay as the interval
between the acquisition of sputum or BAL samples and the start of anti-TB therapy. Results: A total of
258 immunocompromised patients with paucibacillary pulmonary TB were finally analyzed. Of these 258
patients, 134 patients (52%) were classified as missed TB group and the remaining 124 (48%) as not
missed TB group. Of 106 molecular test results including MTB PCR and/or Xpert TB/RIF in the 124
patients with not missed TB, 55 (52%) revealed positive results, while 60 molecular test results in the
134 patients with missed TB all revealed negative results. In the missed TB group, the median diagnostic
delay (IQR) was 30 days (24-42), which was significantly longer than the not missed TB group (6 days (1-
14))(p <0.001). In the missed TB group, the most common working diagnoses before the diagnosis of TB
were pneumonia (46 patients, 34%) and lung metastasis of malignancy (40 patients, 30%). Old age (OR
1.03), solid organ transplantation (OR 3.46), solid tumor (OR 3.83), and hematologic malignancy (OR
4.04) were independently associated with missed TB. Conclusions: The diagnosis of paucibacillary TB is
delayed until the availability of mycobacterial culture results in about half of immunocompromised
patients. A cautious approach to differentiate paucibacillary TB is needed especially in
immunocompromised patients with old age, organ transplantation, hematologic malignancy, or solid
tumor. Further rapid diagnostic tests to rule out paucibacillary TB are urgently needed.
Session Number: 398
Session Number: 398
Resistance
Abstract Title:
Index Cases of Buruli Ulcer Disease in Four States of Southwest, Nigeria
A. Oke1, I. Komolafe1, O. Akinwale2, P. Gyang2, T. Nwafor2, E. Henry2, S. Festus3; 1Coll. of Natural Sci.,
Redeemer’s Univ., Ede, Nigeria, 2Nigerian Inst. of Med. Res., Lagos, Nigeria, 3Tuberculosis, Leprosy and
Buruli Ulcer Control Programme, Ogun State
Abstract Body:
Buruli ulcer (BU), a neglected tropical disease caused by Mycobacterium ulcerans is an indolent
necrotizing disease of the skin, cutaneous tissue and occasionally bones, occurring in tropical and
subtropical countries of the world especially in Central America, Australia, South-East Asia and Africa.
West Africa is now the epicenter of the disease. The causative organism is closely related to the
mycobacteria that cause tuberculosis and leprosy, making Buruli ulcer the third most common
mycobacterial disease of humans. The mode of transmission of M. ulcerans is poorly understood. There
are reported cases of BU in neighbouring countries however it is not a reportable disease in Nigeria due
to the poor awareness of the disease among healthcare professionals and the populace and lack of
adequate public health facilities for early detection thus making diagnosis a challenging exercise. The
probability of being mistaken it for other forms of skin ulcer is high. A recent publication of the
retrospective data of PCR-confirmed Nigerian patients with Buruli ulcer treated in the neighbouring
Benin Republic gives an indication it may be more prevalent than had been previously thought. Thus the
need for this study to investigate the disease prevalence. This study is a part of an ongoing research
project aimed to determine BUD prevalence in six states of SW Nigeria. Swab and FNA samples were
collected from forty-eight (48) BU suspected cases at community level during active case search after
sensitization visits in four states of south-west Nigeria. Nested PCR protocol was carried out on the
samples to diagnose the disease. The results showed that of the 48 samples collected and analyzed so
far 15(31.3%) were PCR positive to IS2404 of Mycobacterium ulcerans. Most of the positive cases were
adults: 9(60%) comprising 4(44.4%) females and 5(55.6%) males. The remaining 6(40%) were children
below 20 years of age and all were from one of the states. While 6(40%) were above 40 years of age,
3(20%) were between 18 and 40 years while only 6(40%) were below 15 years. 11 (73.3%) of the ulcers
were located on the lower limbs and 4 (26.7%) on the hand. The 6(26.1%) children positive were all from
one of the states. 10 (71.4%) of the ulcers were located on the lower limbs and 4 (28.6%) on the hand.
5(35.7%) of the patients were above 40 years of age, 3(21.4%) were between 18 and 40 years while only
6(42.9%) were below 15 years. Deploying the nested PCR (IS2404) technique, index cases of Buruli ulcer
disease were confirmed in four states (Ekiti, Lagos, Ogun, and Ondo) of south-west Nigeria.
Session Number: 398
Session Number: 398
Resistance
Abstract Title:
Subspecies Distribution of Mycobacterium Abscessus in Adult Cystic Fibrosis and Non-Cystic Fibrosis
Patients
M. Salfinger, N. Helstrom, R. Rodger, A. Sherwood, C. Daley, J. Nick; Natl. Jewish Hlth., Denver, CO
Abstract Body:
Background: In 2015, of the 14,225 individuals in the United States Cystic Fibrosis (CF) Foundation
Patient Registry who had a mycobacterial culture performed, 1,692 (11.9%) had a nontuberculous
mycobacterium (NTM) isolated ≥1 time. M. abscessus (MABS) and its subspecies (subsp. abscessus,
massiliense and bolletii) are the second most cultured NTM species in respiratory samples after M.
avium complex. Methods: The National Jewish Health laboratory information system was queried for
cultures positive with MABS for 2015 and 2016. Results: During 2015 to 2016, of a total of 297 patients
with MABS. 15 patients were excluded from evaluation: 6 were <18 years of age, 8 had > 1 subspecies
identified, and 1 patient’s isolate could not be subspeciated. Of the remaining 282 patients (241 Non-CF
and 41 CF), 210 (74.5%) had MABS subsp. abscessus (abs), 58 (20.6%) MABS subsp. massiliense (mass)
and 14 (5.0%) MABS subsp. bolletii (boll). The distribution of the subspecies for 241 Non-CF versus 41 CF
are as follows: abs-178 (73.9%) vs. abs-32 (78.0%); mass-52 (21.6%) vs. mass-6 (14.6%); boll-11 (4.6%)
vs. boll-3 (7.3%), respectively. The female/male ratio for Non-CF vs. CF are as follows: abs 4.74 vs. 0.78;
mass 1.6 vs. 2.0; boll 0.8 vs. 2.0, respectively. The mean age for patients with abs is 64.5 years for Non-
CF and 34.5 years for CF. Conclusions: The subspecies distribution is similar between the Non-CF and CF
patients. The 30-year difference in the mean age between Non-CF and CF patients is not surprising.
Female Non-CF patients are 4 times more likely to harbor MABS isolates versus males. This is in stark
contrast to the CF patients, where the sex distribution is not different.
Session Number: 398
Session Number: 398
Resistance
Abstract Title:
Natiowide Subtyping of Human Isolates of Mycobacterium Kansasii In Slovenia
U. Kuzmič1, T. Jagielski2, S. Truden1, M. Žolnir-Dovč1; 1Univ. Clinic of Respiratory and Allergic Diseases
Golnik, Golnik, Slovenia, 2Univ. of Warsaw, Faculty of Biology, Inst. of Microbiol., Warsaw, Poland
Abstract Body:
Background: Mycobacterium (M.) kansasii is an opportunistic human pathogen whose environmental
reservoirs include tap water, soil and animals. Phylogenetic and molecular studies have demonstrated
M. kansasii to be a heterogeneous species with 7 major subtypes identified by the PCR-restriction
enzyme analysis (PCR-REA) of the hsp65 gene. This heterogeneity could have an important clinical and
epidemiological implication. According to current reports, the M. kansasii involved in human disease
belongs almost exclusively to subtypes I and II. Other subtypes (III-VII) are generally from environmental
sources with no clinical relevance. The aims of the present study were to determine, for the first time in
Slovenia, the distribution of M. kansasii subtypes and to find out any correlation of the particular
subtype with human disease. Methods: The study included 64 M. kansasii isolates from as many patients
(21 women, 43 men). The isolates were collected over a 7-year period (2010-2017) and identified using
the GenoType Mycobacterium CM/AS® test. Subtyping was performed with the PCR-restriction enzyme
analysis (PCR-REA). Briefly, a 740-bp fragment of the tuf gene was PCR-amplified and the resulting
amplicons were subjected to digestion with the MvaI restriction endonuclease. DNA fragments were
visualized on 4% agarose E-gels®. The restriction profiles of individual strains were assigned to individual
types according to the restriction profiles of the reference strains. Results: Among the 64 patients, 36
(56.2%; 27.8% women, 72.2% men) had, while 22 (34.4%) did not have true non-tuberculous
mycobacterial disease (NMD) according to the criteria of the American Thoracic Society (ATS). Six (9.4%)
patients could not be classified as per the ATS criteria. Among patients with NMD, 21 (58.3%) were
smear-positive and 15 (41.7%) were smear-negative. NMD manifested as a pulmonary (30; 83.3%)
and/or an extrapulmonary disease (6; 16.7%). The most prevalent M. kansasii subtype responsible for
human disease was subtype I (29/36; 80.5%), followed by subtype II (7/36; 19.4%). Patients with no
NMD excreted M. kansasii subtype I (14), subtype II (6), subtype III (1), and subtype V (1). Conclusions: In
Slovenia, M. kansasii is the second, after M. avium, most frequently isolated species from patients with
NMD in both immunocompetent and immunocompromised patients. The findings of this study indicate
that subtypes I and II are most frequently involved in the causation of human M. kansasii disease in
Slovenia. Our study also shows that this disease is more likely in men.
Session Number: 398
Session Number: 398
Resistance
Abstract Title:
Outstanding Abstract Award: Detection of Mixed Mycobacterial Infections in Paraffin Embedded Tissues
from Patients Using Laser Microdissection-Based Molecular Assays
V. W. Rowlett, A. K. Paulino, M. DeLeon-Carnes, G. L. Hale, W-J. Shieh, S. Zaki, J. Bhatnagar; CDC,
Atlanta, GA
Abstract Body:
Diagnosis and characterization of mycobacterial infections from tissues is challenging because of lack of
sensitivity and specificity of conventional tissue-based methods, including acid-fast bacilli (AFB) and
immunohistochemistry (IHC) testing. PCR performed on formalin-fixed, paraffin-embedded (FFPE) tissue
is a sensitive and specific method to identify pathogens; however, the presence of commensal
colonizers and environmental microorganisms in the tissue and the occurrence of potential co-infections
can hamper identification of the etiologic pathogen. In addition, co-infections of mycobacteria are more
common in patients that are immune compromised and it is important that they are identified to
provide the appropriate treatment. The laser microdissection (LMD) technique allows separation and
recovery of target pathogens under microscopic visualization directly from areas of granulomatous
inflammation, which is a histopathologic hallmark of mycobacterial infection. We developed LMD-based
molecular assays for detection and characterization of mycobacteria and evaluated various FFPE tissues
from 10 case-patients with clinical and histopathological suspicion of mycobacterial infection. Whole
FFPE tissue sections from these cases were also analyzed by a routine conventional mycobacterium
genus PCR assay targeting the 16S rRNA gene (MB16S PCR). Of 10 case-patients, by whole FFPE tissue
section PCR assays, Mycobacterium avium complex (MAC) was detected in 1 (10%), commensal or
environmental organisms were detected in 7 and 2 were negative. LMD was performed on areas of
granulomatous inflammation in hematoxylin-eosin, AFB and IHC stained slides, and DNA was extracted
using a DNA Micro kit (QIAGEN). The MB16S PCR assay was performed on DNA, followed by Sanger
sequencing of positive amplicons. This analysis resulted in the identification of mycobacteria in 9 cases
(90%): M. tuberculosis complex (MTBC) in 5 cases and MAC in 4 cases. In 4 of these cases, co-infections
of MTBC, MAC and other non-tuberculous mycobacterium (NTM) species were detected. Interestingly,
MTBC was identified in the 1 previously positive case where only MAC was identified by routine MB16S
PCR in the whole tissue section. This data demonstrates that LMD-based molecular assays improve the
detection of mycobacteria in FFPE tissues, allows for differentiation of MTBC from NTM, and can be a
valuable diagnostic adjunct for mixed infections.
Session Number: 398
Session Number: 398
Resistance
Abstract Title:
Molecular Characterization of Mycobacterium Bovis Isolated from Cattle Lymph Nodes in Theeastern
Cape Province, South Africa
N. L. Bhembe1, L. V. Mabinya1, A. I. Okoh1, E. Green2; 1Univ. of Fort Hare, Alice, South Africa, 2Univ. of
Johannesburg, Johannesburg, South Africa
Abstract Body:
Background: Tuberculosis (TB) is a disease caused by the members of the Mycobacterium tuberculosis
complex (MTBC), which infects both animals and humans. This study is aimed at determining the
prevalence of the disease, characterizing the MTBC isolates and increasing the understanding of the
genetic diversity MTBC in the Eastern Cape Province of South Africa. Methods: A total of 376 cattle
lymph nodes were collected from two commercial abattoirs for investigations. All collected samples
were cultured, and isolates were confirmed with a multiplex polymerase chain reaction (PCR) targeting
the mpb64 and IS6110 genes. Positive isolates were tested for resistance to anti-TB drugs using the
standard Lowenstein Jensen proportion method. Isolates from cattle lymph nodes were further
characterized to species level, using the region of deletion 1 (RD1) and oxyR restriction fragment length
polymorphism analyses. MIRU-VNTR typing and spoligotyping assays were used to further examine the
genetic diversity of all strains. Results: MTBC was detected in 162 (43.09%) cattle lymph nodes and some
isolates 25.9% (42/162) were resistant to rifampicin (RIF). All the isolates exhibited the RD1 gene, and
the oxyR analysis classified 96.9% of the isolates as Mycobacterium tuberculosis complex and 3.1% as
Mycobacterium bovis. Twenty-seven spoligotype patterns were, with 10 shared types (SIT) consigned to
5 lineages, including Bov_4-Caprea (1.9%), Microti (0.6%) and Beijing (17.9%) constituting East Asian,
Latin-American-Mediterranean (LAM) (3.7%), X (4.3%), MANU (3.1%), S (0.6%) and 67.9% were orphans.
Spoligotyping showed a higher clustering rate of 82.1%, with the lowest (HGDI) = 0.485. The 12 MIRU-
VNTR clustering rate was 64.8%, showing a higher HGDI of 0.671. However, a combination of both
methods showed a decreased clustering rate of 53.7%. Conclusions: The results of this study show the
diversity of MTBC strains in the Eastern Cape Province and the low clustering rate indicates continuing
transmission in the province. The detection of MTBC strains from slaughtered cattle lymph nodes in
abattoirs confirms the risk of providing TB infected meat in markets which has serious implications for
the control of TB in South Africa. The continuing spread of MTBC in South Africa threatens both the
public and economic health.
Session Number: 398
Session Number: 398
Resistance
Abstract Title:
Magnetic Bead Based Concentration and Fluorescence Staining in A Single Tube for Capturing
Mycobacterium Tuberculosis in Sputum Samples
M. Kumar1, R. K. Verma1, S. Verma2, T. N. Dhole3; 1UP Univ. of Med. Sci., Etawah, India, 2King George's
Med. Univ., Lucknow, India, 3Sanjay Gandhi Post Graduate Inst. of Med. Sci., Lucknow, India
Abstract Body:
Background: Direct sputum smear microscopy is the mainstay of TB diagnosis in most low and middle
income countries, and is highly specific for Mycobacterium tuberculosis (M. Tuberculosis) in such
settings. However it is fast and inexpensive technique but limited by low sensitivity. Concentration by
centrifugation has been reported to be more sensitive than direct smear preparation, but is only
suitable for referral laboratories. An easier concentration methods are urgently needed to improve M.
Tuberculosis detection rates at peripheral center. Methods: The feasibility of a ligand-coated magnetic
bead technology to concentrate M. tuberculosis in single tube (decontamination, concentration and
staining) prior to detection by fluorescence microscopy was observed in this study. We compared the
sensitivity of this method with concentrated Ziehl-Neelsen (ZN) and fluorescence microscopy (FM) in
sputum samples. The kappa coefficient (Kc) analysis was performed for positive correlation between the
tests and LJ culture was taken as a gold standard for evaluation to sensitivity and specificity of
performed tests. Results: By active screening in all performed tests, the concentrated magnetic bead-FM
possess showing higher positivity rate (61%), concentrated FM microscopy (46.5%) and ZN microscopy
(40.5%) among all 200 sputum samples. The concentrated magnetic bead-FM having significantly higher
sensitivity (91.3%) over the concentrated FM (75.7%) and ZN microscopy (72.8%) in compare with LJ
culture. The specificity of magnetic bead FM, FM and ZN microscopy were 71.1%, 84.5% and 93.8% were
observed. The fair correlation found between culture and concentrated ZN microscopy (Kc = 0.66,),
concentrated magnetic bead-FM (Kc = 0.63) and Kc = 0.60 with concentrated FM. Conclusion: Magnetic
bead concentration of M. tuberculosis from sputum led to significant improvement in the sensitivity of
microscopy. Therefore, the national programs in high TB burden countries may consider incorporating
the technique into their guidelines at least in the district and higher level laboratories to improve case
finding strategy.
Session Number: 398
Session Number: 398
Resistance
Abstract Title:
Magnetic Bead Assay A Rapid Cost Effective Approach for Diagnosis of Tuberculous Meningitis (Tbm) in
Low Resource Settings
K. Sharma, M. Modi, M. Sharma, A. Sharma, P. Ray; PGIMER, Chandigarh, India
Abstract Body:
Background: Rapid and specific diagnosis of tubercular meningitis (TBM) is of paramount importance to
decrease associated morbidity and mortality. Although there have been many recent advances in MTB
detection technologies, there still remains a major need to develop simpler point-of-care techniques. In
an effort towards such a diagnostic test for resource-poor settings, we have designed a test based on
detecting amplified DNA via magnetic beads bridging flocculation technique. The assay is inexpensive
and rapid (60 minutes), with a sensitivity approaching a single cell of Mycobacterium tuberculosis.
Methods: In the present study we evaluated magnetic beads bridging flocculation technique on 110
culture confirmed CSF samples and 200 clinically suspected TBM patients, who were culture and smear
negative but responded to ATT, in these patients composite reference standard as described in previous
studies were taken as reference for evaluation of test. We also evaluated this test from 100 non TB
control CSF samples. DNA extracted from H37RV was used as positive control. Test was also evaluated
with DNA extracted from various NTM and other bacteria for specificity. Results: Analytical sensitivity of
test is: single copy of M.tuberculosis . Analytical specificity: it is highly specific as there is no cross
reactivity with DNA extracted from various NTMs and other bacteria. In culture positive samples 104 out
of 110 (94.54%) were positive by this new test, in clinically suspected cases 179 out of 200 (89.5%) were
positive by this test. Specificity of the test was100% as it was negative in all the control patients and
DNA extracted from NTM and other bacteria. Conclusion: The assay is an affordable test (less than 1
dollar), with a sensitivity approaching a single cell of Mycobacterium tuberculosis. Magnetic bead assay
(MBA) is robust and cost effective method for diagnosis of TBM in low resource and high endemic
settings..
Session Number: 398
Session Number: 398
Resistance
Abstract Title:
Use of Maldi-Tof Mass Spectrometry for Identification of Non-Tuberculous Mycobacteria
E. Sodja1, I. Perko1, H. Ribič2, M. Žolnir-Dovč1; 1Univ. Clinic of Respiratory and Allergic Diseases Golnik,
Golnik, Slovenia, 2Natl. Lab. of Hlth., Environment and Food, Kranj, Slovenia
Abstract Body:
Background: Matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS) has
been recently proven to effectively identify mycobacteria directly from cultures. Aim of the study was to
compare and evaluate MALDI-TOF MS Biotyper system (Bruker Daltonics, Bremen, Germany) with
GenoType Mycbacterium CM/AS (GTM; Hain Lifescience, Nehren, Germany). Methods: Between 2014
and 2016, we retrospectively tested 160 isolates from various clinical samples and 9 veterinary NTM
isolates. NTM subcultures grown on solid media (Middlebrook 7H10 or Lowenstein-Jensen) at 37°C or
30°C as appropriate were identified with GTM assays. For MALDI-TOF MS analysis, the NTM colonies
were heat inactivated at 100°C and 0.5mm zirconia/silicate beads were used for mechanical disruption.
Protein extractions were elicited with acetonitrile and formic acid. The samples were identified with
Bruker MALDI Biotyper using automatic spectral acquisition and Bruker Mycobacteria Library 3.0.
Results: 149/169 (88.2%) mycobacterial isolates were identified using GTM to species level. The Bruker
Mycobacteria Library 3.0 gave log (score) values higher than 1.7 for 163 (96.4%) NTM isolates. When
comparing GTM assay and MALDI-TOF MS analysis, 122/137 (89.1%) NTM isolates were comparably
identified to the species level. 12 NTM isolates (7.1%) identified as M. fortuitum 2/M. mageritense with
GTM were identified as M. senegalense (10), M. mageritense (1) or M. fortuitum complex (1) using
MALDI-TOF MS analysis. Furthermore, NTM isolates not identified with GTM (20; 11,8%) were identified
with MALDI-TOF MS analysis to species level as M. novocastrense (4), M. neoaurum (2), M. elephantis
(2), M. bohemicum (1), M. mageritense (1), M. kumamotonense (2), M. algericum (2), M. arupense (3),
M. nonchromogenicum (1) or yielded in no reliable identification (2). Conclusions: According to our data,
MALDI-TOF MS analysis has potential for identifying mycobacteria in the clinical laboratory practice, by
reducing identification turnaround time and laboratory costs.
Session Number: 398
Session Number: 398
Resistance
Abstract Title:
Use of Maldi-Tof Ms for the Identification of Non-Tuberculous Mycobacteria and Subspeciation of
Mycobacterium Abscessus
J. Schneider, J. Manaloor, C. Hehman, R. Relich, C. Emery, T. Davis, B. Schmitt; Indiana Univ. Sch. of
Med., Indianapolis, IN
Abstract Body:
Background: Rapid identification of nontuberculous mycobacteria (NTM) is necessary for appropriate
clinical management and timely intervention, yet current routine clinical methods for identification
remain complex and tedious. In particular, differentiating subspecies of Mycobacterium abscessus
(abscessus, massiliense and bolletii) is clinically useful given recently described macrolide resistance in
certain subspecies of Mycobacterium abscessus with the erm(41) gene. Methods: We evaluated matrix-
assisted laser desorption/ionization time-of-flight mass spectrometry (Bruker Biotyper MALDI-TOF MS)
for the rapid identification of NTM from solid media (Middlebrook 7H10 agar) using a modified Bruker
extraction procedure which involved 0.5 mm silica beads (Biospec), 70% formic acid, acetonitrile, and
100% EtOH. These results were then compared with those obtained by high performance liquid
chromatography (HPLC) or AccuProbe. Results: Of the 163 NTM isolates tested, 152 (93.2%) were
reliably identified when comparing MALDI-TOF MS to standard methods (HPLC or Accuprobe). 11
discrepant identifications were shown to be in 100% agreement with MALDI-TOF MS species level
identification after rpoB sequencing was performed. Of the 34 Mycobacterium abscessus isolates
included, 22 (64.7%) were reliably identified to the subspecies level by MALDI-TOF MS [2/22: subspecies
bolletii; 20/22: subspecies abscessus]. 10 of these 22 isolates for M. abscessus isolates had confirmed
subspecies data for comparison (e.g., rpoB sequencing). Conclusion: Our study supports MALDI-TOF MS
analysis as an alternative for clinical microbiology laboratories to accurately identify mycobacterial
species from solid growth media, and also highlights its rapid capability for providing subspecies level
identification for M. abscessus isolates, which may assist in predicting the disease process and outcomes
of infected patients
Session Number: 398
Session Number: 398
Resistance
Abstract Title:
Identification of Mycobacteria Using Maldi-Tof Mass Spectrometry in the Clin. Microbiology Laboratory
J. Vitale, D. S. S. Wijetunge, D. Myers, D. Craft; Penn State Hlth., Hershey, PA
Abstract Body:
Introduction: Identification of mycobacteria to the species level is important for antimicrobial therapy
and precautions associated with infection control and patient isolation. Methods for identifying
mycobacteria in our laboratory include growth rate, colony morphology, RNA hybridization, and send
out to reference labs for definitive speciation by proteomics or sequencing. Extended turn-around times
for send-outs may result in sub-optimal patient outcomes due to delays in appropriate therapy. MALDI-
TOF is a recent and robust technology used for the rapid identification of bacteria and this study
evaluates its accuracy for the speciation of mycobacteria. Methods: 51 isolates of Mycobacterium spp.,
including 41 from our medical center (Nov 2014 - Dec 2017) and 10 from CAP proficiency surveys were
used in the study. Proteins were extracted according to protocol (Bruker Daltonics). Briefly, a10µl
loopful of bacteria in 300μl of HPLC water was heat inactivated by boiling for 30 minutes followed by
bead beating and ethanol-formic acid extraction. Mass spectra were obtained using a 1µl protein extract
and analyzed using the Bruker Mycobacteria Library 4.0 with a cut-off score of ≥1.70 for species level
identification. Species results were compared to culture, hybridization (Hologic AccuProbe), and
reference laboratory results. Results: All isolates were correctly identified into 12 different
species/groups: M. kansasii (n=3), M. nebraskense (n=1), M. avium (n=10), M. chimera-intracellulare
(n=7), M. heckenshornense (n=1), M. abscessus (n=11), M. chelonae (n=5), M. fortuitum (n=6), M.
mucogenicum-phocaicum group(n=3), M. brisbanense (n=1), M. neoaurum (n=1), and M. tuberculosis
complex (n=2). 4 of 5 M. chelonae isolates were identified as M. salmoniphilium, a non-human pathogen
in the M. chelonae group. Conclusions: MALDI-TOF results were 100% concordant with current
identification protocols. Turnaround times to results (2 hours) was significantly decreased compared to
hybridization (4 hours) and send-out (weeks), and MALDI-TOF was able to differentiate M. avium from
M. chimera-intracellulare and M. abscessus from M. chelonae. For the latter, 2 isolates identified by
reference labs as M. abscessus/chelonae group and 2 identified as M. chelonae were identified as M.
salmoniphilium, a fish pathogen not reported in human infections. Further investigation of MALDI-TOF
speciation in M. chelonae group is needed. Overall, our study indicates that MALDI-TOF is a rapid and
reliable method to identify mycobacteria to the species level.
Session Number: 398
Session Number: 398
Resistance
Abstract Title:
Surface-Enhanced Raman Scattering with Sensible Functional Linear Discriminant Analysis Effectively
Discriminates Mycobacterium Species
W-C. Cheng1, L-H. Chen2, C-R. Jiang3, C-H. Lin4, R. Jou5, J-K. Wang1, Y-L. Wang1; 1Inst. of Atomic and
Molecular Sci., Academia sinica, Taipei, Taiwan, 2Natl. Chung Hsing Univ., Taipei, Taiwan, 3Inst. of
Statistical Sci., Academia sinica, Taipei, Taiwan,
Abstract Body:
Tuberculosis caused by Mycobacterium tuberculosis complex (MTBC) is one of major infectious diseases.
Timely diagnosis of MTBC and nontuberculous mycobacteria (NTM) could guide proper treatment and
management. This study aimed to develop a surface-enhanced Raman scattering (SERS)-based
metabolites detecting method for differential identification of mycobacteria. This work tested 4 MTBC
and 8 NTM strains. The bacilli were suspended in water and centrifuged. The supernatant of each
sample was measured on a SERS-active substrate made of Ag nanoparticle array. Besides scrutiny with a
new sensible functional linear discriminant analysis (SLDA), the acquired SERS spectra were analyzed
with conventional linear discriminant analysis (LDA) and principal component analysis (PCA) for
comparison. We found that LDA and PCA analyses could distinguish the SERS spectra of MTBC from
those of NTM, but they failed to discern NTM species. In contrast, the SLDA analysis perfectly
differentiated the SERS spectra of 8 NTM strains. The process of finding optimal projections with SLDA
would effectively capture the unique propensities of random fluctuations inherent in SERS spectra and
separate them from that of the spectral characteristics of bacterial metabolites, allowing for accurate
speciation. The test could be completed within 1 h. Our findings successfully demonstrated that the
SERS-based method assisted by SLDA opens unprecedented opportunities for rapidly differentiating
mycobacteria.
Session Number: 398
Session Number: 398
Resistance
Abstract Title:
Usefulness of Rapid and Accurate Identification by Pyrosequencing in Nontuberculosis Mycobacteria
N. Akamatsu, N. Kaku, J. Matsuda, K. Kosai, Y. Morinaga, K. Yanagihara; Nagasaki Univ. Hosp., Nagasaki,
Japan
Abstract Body:
Background: In recent years, nontuberculosis mycobacteria (NTM) infection and disease has been
increased in many different parts of the world. However, most of the identification of NTM except
Mycobacterium avium complex (MAC) is not established in clinical laboratory test. Therefore, we
established rapid and accurate identification of NTM method using pyrosequencing. Methods: A total of
17 NTM species (M. avium, M. peregrinum, M. intracellulare, M. szulgai, M. chelonae, M. shimoidei, M.
celatum, M. simiae, M. gordonae, M. nonchromogenicum, M. malmoense, M. marinum, M. abscessus,
M. scrofulaceum, M. xenopi, M. fortuitum, M. kansasii) containing of the ATCC strains and clinical
specimens were used in this study. The PCR amplification primer was designed for the 16S rRNA gene
containing of the hypervariable region A. Sequencing primer was designed for the most effective
position of hypervariable region A. The confirmation of PCR products was done with agarose gel
electrophoresis. Pyrosequencing was performed with 24 cycles of T, G, C, A dispensations according to
the protocol. The obtained sequences were compared with sequences from Gene Bank database and
identified the species. Results: In agarose gel electrophoresis after the PCR amplification, specific
products of the 140-bp were found in all of NTM 17 species and clinical specimens (fig.1). The 30-bp
sequences of the NTM 17 species which we determined by pyrosequencing were completely (100%)
accorded with the species of Gene Bank database. However, M. scrofulaceum and M. simiae were not
identified in this method. In clinical specimens, M. genavense were identified from paraffin embedded
tissue. Conclusion: This method is rapid, accurate, and useful for identification of NTM, especially, the
species which it is impossible to culture. In addition, we identified the species from paraffin embedded
tissue. We think that this method has potential for the routine test in clinical laboratory.<br /><p><a
href="http://files.abstractsonline.com/CTRL/c3/5/b1a/0de/5dc/4e2/d93/cc3/07a/46a/507/36/g4932_1.
src="http://files.abstractsonline.com/CTRL/c3/5/b1a/0de/5dc/4e2/d93/cc3/07a/46a/507/36/g4932_1.j
Session Number: 398
Session Number: 398
Resistance
Abstract Title:
High Resolution Melting Curve Analysis (Hrm) for Differentiation of Mycobacterium Tuberculosis And
Common Non Tuberculous Mycobacteria (Ntm)
K. Sharma, M. Modi, A. Sharma, M. Sharma, P. Ray; PGIMER, Chandigarh, India
Abstract Body:
Background: India constitutes nearly 25% of the world’s tuberculosis burden. Non-tuberculous
mycobacteria (NTM) are also gaining importance as opportunistic pathogens responsible for a variety of
clinical diseases. There is need for rapid, accurate and cost effective identification of Mycobacterium
species to aid in diagnosis and for initiating appropriate therapy. We aimed to evaluate high-resolution
melting curve (HRM) analysis to identify and differentiate clinical isolates of Mycobacterium spp. To
evaluate Heat shock protein 65 (hsp65) gene HRM analysis for identification of M.tuberculosis (MTB)
and NTM from clinical mycobacterial isolates. Methods: A total of 170 mycobacterial isolates were
evaluated. MPT64 antigen detection was performed to identify MTB while the identification of the
MPT64 antigen negative isolates (NTM) was confirmed by hsp65 sequencing. All isolates were subjected
to hsp65 real time PCR- HRM analysis for species identification. Results: Among the 170 isolates, 100
were MPT64 positive (MTB) while 70 were MPT64 negative (NTM). There was 100% concordance
between HRM analysis and MPT64 test. Amongst the 70 MPT 64 negative isolates 100% concordance
was observed between hsp65 HRM analysis and hsp65 sequencing results. Of the 70 NTM isolates, the
majority was constituted by M. intracellulare 20 (28.57%) followed by M. avium 18 (25.71%), M.
fortuitum 13 (18.57%), M. abscessus 11 (15.75%), M. gordonae 6 (8.57%) and M. kansasii 2 (2.85%).
Conclusions: By analyzing both the melting temperature and melting profile of the hsp65 gene, were
able to discriminate 7 different mycobacterial species simultaneously in 90 minutes. The hsp65 real-time
HRM assay is therefore a rapid, sensitive and cost effective method for identifying commonly prevalent
NTM and MTB in the clinical laboratory with subsequent utility in patient management.
Session Number: 398
Session Number: 398
Resistance
Abstract Title:
Point of Care Diagnosis of Tuberculosis Using Loop Mediated Isothermal Amplification
M. Nimesh, D. Joon, M. Varma-Basil, D. Saluja; Univ. of Delhi, Delhi, India
Abstract Body:
Background: Rapid detection of tuberculosis (TB) is essential to improve management of infected
patients and avoid transmission in the community. Automated real-time PCR based Xpert MTB/RIF assay
has been endorsed by WHO for detecting the presence of Mycobacterium tuberculosis. There is need
for sensitive diagnostic test with minimal infrastructure, cost and training. Therefore, present study was
carried out to design and evaluate the diagnostic performance of loop-mediated isothermal
amplification (LAMP) assay combined with lateral flow dipstick (LFD) as point of care method. Methods:
LAMP assay targeting sdaA gene for detection of M. tuberculosis was modified for sequence specific
detection by lateral flow dipstick. The LAMP-LFD assay was evaluated in a cross-sectional study using
respiratory specimens collected from patients visiting Vallabhbhai Patel Chest Institute, Delhi, India.
Pulmonary TB diagnosis using sputum smear microscopy, culture, GeneXpert MTB assay and sdaA LAMP
assay combined with LFD was carried out. LAMP assay targeting rpoB gene of M. tuberculosis was also
standardized to amplify the rifampicin resistance determining region and combined with LFD to develop
multiplex assay. Results: The LAMP assay was standardized for sequence specific detection of LAMP
amplified products in user friendly and rapid format. The LAMP-LFD assay was tested in 18 culture
confirmed specimens for pulmonary tuberculosis. All the specimens showed positive result with LAMP-
LFD assay. The diagnostic accuracy of the method was also evaluated in comparison with GeneXpert
MTB/RIF assay using 107 clinical specimens from patients with clinical symptoms of pulmonary
tuberculosis. Out of 107, 15 specimens were positive with both the methods showing high concordance.
LAMP assay targeting rpoB gene combined with LFD in multiplex format was successfully tested as
proof-of-concept for diagnosis of TB and screening for drug resistance. Conclusions: Lateral flow dipstick
method has provided an excellent detection format with LAMP method. The sdaA LAMP-LFD assay
showed high diagnostic accuracy in comparison to other methods and can be used as point of care test.
The increasing resistance to drugs used in anti-tubercular treatment is cause of concern to global TB
control efforts. The rpoB LAMP assay multiplexed with sdaA LAMP assay for simultaneous amplification
of both targets leads to diagnosis of tuberculosis as well as screening for drug resistance.
Session Number: 398
Session Number: 398
Resistance
Abstract Title:
Amplification and Melt Curve Analysis of the Hsp65-Gene for Identification of Mycobacteria Species
from Tissue Samples
J. A. Diaz-Perez, E. McElvania, K. A. Mangold, S. Das, K. L. Kaul; NorthShore Univ. Hlth.System, Evanston,
IL
Abstract Body:
Background: The use of molecular methods for mycobacteria species identification from formalin fixed,
paraffin embedded tissue is suboptimal. Conventional microbiologic culture based methods are highly
sensitive but require long times for incubation and species-level identification. Methods: Patients that
received culture for mycobacteria from biopsies between January-2011 to October-2017 were studied.
Culture was performed using the automated BACTEC Mycobacterial Growth Indicator Tube (MGIT) broth
incubation system. Real-time PCR melt curve assay of the hsp65 gene was performed on positive broth
confirmed to contain acid-fast bacilli (AFB). Consensus primers amplified the hsp65-gene, and
fluorescence resonance energy transfer probes were used for genus and species level identification.
Conventional identification was performed from growth on solid media. Results: Positive culture for
mycobacteria were seen in 106 of 4177 (2.54%) unique patients. The number of tissue specimens sent
for culture per patient ranged from 1-28. Mean age of patients with positive culture was 61.7±17.5 years
with a female male ratio of 1.25. Samples from positive individuals were collected most commonly from
lung 42.4%, skin 25.4%, lymph node 14.1%, and large joints 7.5%. Only 7 patients were positive for AFB
on direct smear examination. Rapidly growing species identified were M. chelonae (n=8), M. abscessus
(n=7), M. fortuitum (n=7), M. mucogenicum (n=4) and M. goodii (n=1). Slowly growing species were M.
avium-intracellulare complex (n=36), M. tuberculosis complex (n=34), M. marinum (n=5), M. kansaii
(n=2), M. gordonae (n=1), and Mycobacterium xenopi (n=1). Conclusions: Identification of mycobacteria
using PCR melt curve assay can provide identification for most commonly isolated species within 8-24h.
Additionally, identification can be achieved using growth from MGIT broth, unlike traditional methods
which are performed after colonies appear on solid media. Rapid species identification using PCR melt
curve assay is a useful adjunct to traditional methods that assists in initiation of early therapy.
Session Number: 398
Session Number: 398
Resistance
Abstract Title:
Genotyping by Spoligotyping, Miru-Vntr Typing and Rflp Typing on Myanmar Mycobacterium
Tuberculosis Isolates
P. Ei1, W. Aung1, W. Nyunt2, T. Swe2, S. Aung3, M. Htwe1, S. Win1, H. Lee4, J. Lee5, C. Chang6; 1Dept.
of Med. Res., Yangon, Myanmar, 2Natl. Tuberculosis Reference Lab., Compound of Tuberculosis Hosp.,
Yangon, Myanmar, 3, Natl. Tuberculosis Control Progr
Abstract Body:
Background: Determining the genetic diversity of Mycobacterium tuberculosis (MTB) strains allows
identification of the distinct MTB genotypes in different regions and also provides an invaluable tool for
the study of epidemiology of tuberculosis (TB). The present study was carried out to determine the
genetic diversity of MTB strains isolated from pulmonary tuberculosis patients in Myanmar by three
genotyping methods; spoligotyping, mycobacterial intersperse repetitive unit- variable number tandem
repeat (MIRU-VNTR) and IS6110 restriction fragment length polymorphism (RFLP) typing. Methods:
Genotyping by spoligotyping, MIRU-VNTR typing and IS6110 RFLP typing was performed on 105 isolates
from sputum samples of new and retreatment cases. Results: Spoligotyping differentiated the strains
into 8 major lineages and 13 orphans while MIRU-VNTR showed 10 different lineages with one each of
unknown and multiple matches. Among 105 strains, 64 were Beijing and 21 were EAI. Other genotypes
include, CAS/Delhi, LAM, Uganda 1, MANU2, West African 1, URAL, S, X. There were 30 different types of
spoligotypes with 9 clusters of 2 to 62 isolates and 21 unique types. By RFLP method, there were 82
different IS6110 pattern with 13 clusters of 2 to 5 isolates and unique pattern of 66 isolates. MIRU-VNTR
typing showed no cluster and all isolates have unique patterns but the Phylogenetic tree by UPGMA
revealed groups of similar patterns by MIRU-VNTR were concordance with clusters by spoligotyping. The
largest cluster by spoligotyping was Beijing genotype which was diverse into small clusters by RFLP
typing and unique patterns by MIRU-VNTR typing. The mode number of IS6110 was 18 and 20 (11
numbers each). Higher band numbers were found in Beijing genotype compare to non-Beijing genotype
(p<0.0002). One isolate without IS6110 was found in U type according to spoligotyping but it was shown
as an unknown strain by MIRU-VNTR method. Conclusions: Diverse genotypes were detected in
Myanmar TB isolates with the highest proportion of Beijing genotype. So the prevailing genotypes in
Myanmar seems to be shifted from EAI to Beijing when compare to studies from previous decade.
Session Number: 398
Session Number: 398
Resistance
Abstract Title:
Performance of Rgm Medium for the Isolation of Nontuberculous Mycobacteria from Respiratory
Specimens from Non-Cystic Fibrosis Patients
S. Rotcheewaphan1, O. Odusanya1, C. Henderson1, D. Stephenson2, K. Olivier1, J. Perry2, A. Zelazny1;
1NIH, Bethesda, MD, 2Freeman Hosp., Newcastle upon Tyne, United Kingdom
Abstract Body:
Background: Nontuberculous mycobacteria (NTM) are significant lung pathogens in patients with cystic
fibrosis (CF) as well as non-CF structural lung conditions such as bronchiectasis, chronic obstructive
pulmonary disease (COPD), or prior TB infection. This study evaluated a new selective agar medium for
rapidly-growing mycobacteria (RGM medium), for the isolation of NTM from respiratory specimens from
non-CF patients compared to Mycobacterial Growth Indicator Tube (MGIT) system and Middlebrook
7H11 solid medium. Methods: A total of 46 respiratory specimens collected so far (39 mucolyzed sputa,
7 BAL) were inoculated on RGM medium without NaOH/NALC decontamination and incubated at both
30°C (RGM30) and 35°C (RGM35) over a 28-day period. NaOH/NALC decontaminated specimens were
inoculated into MGIT and Middlebrook 7H11 medium and incubated at 35°C for 6 weeks. NTM were
identified by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF
MS) and if needed gene sequencing. Results: NTM were recovered from 22 samples (47.8%, 27 NTM
isolates) by RGM30, 18 samples (39.13%, 19 NTM isolates) by RGM35, 21 samples (45.65%, 21 NTM
isolates) by MGIT, and 17 samples (36.96%, 17 NTM isolates) by Middlebrook 7H11 medium. RGM30 led
to growth of NTM within 21 days of incubation (average 8.6 days) in the absence of any non-
mycobacterial contaminants. All 4 culture systems in this study grew NTM from all 14 AFB smear
positive specimens (Auramine-rhodamine stain). For 32 AFB smear negative specimens, RGM30,
RGM35, MGIT, and Middlebrook 7H11 allowed recovery of NTM from 8, 3, 7, and 3 specimens,
respectively. Moreover, RGM30 recovered additional isolates of M. chelonae, M. immunogenum, M.
mucogenicum group, M. gordonae and M. paragordonae. However, MGIT was superior to RGM30,
RGM35 or Middlebrook 7H11 medium for recovery of M. avium complex (MAC). Conclusion: RGM
medium at 30°C offers a convenient and effective culture method for isolation of NTM, particularly
rapidly growing mycobacteria, from respiratory samples from non-CF patients. MGIT system may still be
needed to assure optimal recovery of MAC. Further evaluation of the increased NTM isolation from
RGM medium and correlation with clinical status is warranted.
Session Number: 398
Session Number: 398
Resistance
Abstract Title:
Characterization of Genes Resistance by Using Genotype Mtbdr Plus Ver 2.0
C. L. Yehouenou; Teaching Hosp. of Pneumology, Cotonou, Benin
Abstract Body:
Objectives: To characterise genes resistance in patients who were be recruited in teaching Dakar
hospital and to compare genotype MTBDRplus VER 2.0 plus results with conventional DST test.
Methods: A total of 1560 isolates of Mycobacterium tuberculosis complex from pulmonary or extra
pulmonary tuberculosis were analysed. After Ziehl Neelsen staining, Lowenstein-Jensen medium was be
used for culture and MGIT 960 SIRE for drug susceptibility testing. Genotype MTBDR plus was performed
to characterize genes resistance in study population. Results: MGIT 960 showed 26,47% of Isoniazid
mono-resistance. MDR were 15 among 102 positive cultures. Genotype MTBDR plus VER 2.0 confirmed
9/15 MDR. We had one indeterminate result and 5 MDR strains, which are definitively sensitive with this
method. Mutations were carried by D516V for rpo B gene, C15T for InhA, and S315T for KatG gene.
Conclusion: Genotype MTBDR plus VER 2.0 was performed to detect different mutations associated to
rpoB, kat G or Inh A gene in Senegal. However, sequencing DNA must be completed to confirm
discrepancy between MGIT 960 and MTBDR plus about five strains.
Session Number: 398
Session Number: 398
Resistance
Abstract Title:
Outstanding Abstract Award: Single-Cell Characterization and Isolation of Antibiotic Tolerant
Mycobacterium Tuberculosis Directly from Human Samples
H. L. Glasgow1, M. R. O'Donnell2, M. H. Larsen1, W. R. Jacobs, Jr1, A. Pym3; 1Albert Einstein Coll. of
Med., Bronx, NY, 2Columbia Univ. Med. Ctr., New York, NY, 3Africa Hlth.Res. Inst., Durban, South Africa
Abstract Body:
Background: Mycobacterium tuberculosis (Mtb) persistence, or the phenotypic ability of Mtb
subpopulations to survive extensive antibiotic exposure, underlies the necessity for prolonged antibiotic
treatment to cure tuberculosis (TB). Experimental models have identified heterogeneous populations of
Mtb persisters, but it is unknown if these recapitulate clinical persistence. We developed a method to
characterize and isolate viable Mtb in sputum from TB patients using fluorescence activated cell sorting
(FACS) to understand clinically relevant persister phenotypes, including non-acid-fast and viable but
non-culturable Mtb. Methods: Combining FACS with single-cell bacterial cultures, qPCR, and microscopy,
we tested protocols to fluorescently stain and single cell sort Mtb directly from sputum. We compared
the efficiency of a viability dye (calcein AM) and FITC-trehalose to label viable Mtb from decontaminated
TB patient sputum. To determine if antibodies to lipoarabinnomannan (LAM) and Mtb minus LAM, could
distinguish Mtb from mixed populations, we compared labeling of pure Mtb and Escherichia coli (E. coli)
cultures by flow cytometry and microscopy. Results: Staining with calcein AM dye or FITC-trehalose after
decontamination labeled 50% of all culturable Mtb within TB patient sputum. The calcein-stained
population was enriched in viable Mtb, confirmed by qPCR and culture of sorted cells. In pure bacterial
cultures, anti-LAM staining fluorescently labeled 60% of Mtb and 0% of E. coli cells, while anti-Mtb
minus LAM labeled 53% of Mtb and 26% of E. coli cells. By microscopy, anti-LAM staining was more
associated with whole bacterial cells, while the anti-Mtb antibody also stained acellular debris.
Combining anti-LAM and auramine-acid fast staining of exponentially growing aerobic and hypoxic Mtb
cultures revealed acid-fast and non-acid-fast phenotypes. Conclusion: Viability dye staining and FACS
selects a population enriched for viable Mtb, confirmed by culture and qPCR. This approach coupled
with anti-LAM antibody and acid-fast staining allow us to detect and study heterogeneous Mtb
subpopulations at the single cell level from humans to determine their relevance to clinical outcomes.
Ongoing studies will quantitate these Mtb subpopulations in sputum from a cohort of patients on TB
chemotherapy and determine their association with clinical outcomes. These methods can be used to
identify the molecular pathways that Mtb uses to retard killing by the antibiotics during TB treatment.
Session Number: 398
Session Number: 398
Resistance
Abstract Title:
Molecular Characterization of the Linezolid Resistant Mdr, Xdr Mycobacterium Tuberculosis Clin.
Isolates
J. Jung1, S-H. Park1, J. Kim1, M. Chung2, H. Kang1, N. Sung1, S. Ryoo1; 1Masan Natl. Tuberculosis Hosp.,
Changwon-si, Korea, Republic of, 2Korea NIH, Cheongju-si, Korea, Republic of
Abstract Body:
Background: Tuberculosis (TB) remains a major threat to global public health. Since appearance of
multidrug-resistant (MDR) and extensively drug-resistant (XDR) M. tuberculosis strains, the application
of reserve antibiotics has become more important. Linezolid (LZD), the first member of the
oxazolidinones approved for clinical use, is a promising antimicrobial agent for the treatment of MDR-
and XDR-TB. LZD acts on the 50S ribosomal subunit, specifically, the peptidyl-transferase center (PTC),
and inhibits the translation process Methods: While examining the characterization of LZD resistant
among MDR- and XDR-TB, we’ve selected 33 clinical isolates from 5 patients with failed LZD treatment in
Masan National Tuberculosis Hospital. The patients who we designated their specimen serial within a
three-month interval continued to be detected with TB bacilli from sputum after LZD prescription. The
drug susceptibility tests, including LZD, of 33 clinical isolates were determined by the minimal inhibitory
concentration (MIC) method. Moreover, whole genome sequencing (WGS) of culture isolates can
generate a complete genetic drug resistance profiled of the 33 isolates. Results: Out of 33 isolates, 20
were LZD resistant (MICs ≥ 1 μg/ml), and most of them were LZD resistant after 3 months of treatment.
Twenty isolates of LZD resistant mostly had mutations in 23S rRNA (rrl) or ribosomal protein L3 (rplC)
gene. Four isolates had G2814T mutation in rrl. G2814T stacked on top of the active site nucleotide
G2743, therefore, the mutation in G2814 were likely to disrupt the LZD binding site. And 11 isolates had
a T460C mutation in rplC, it was the dominant mutation observed in LZD-resistant clinical isolates. The
G2814T mutant in rrl or T460C mutant in rplC were resistant to LZD MICs up to 8 μg/ml. One isolate,
mutated in both G2814T and T460C, was resistant to LZD MICs above to 16 μg/ml. Whereas 5 isolates
had not observed genetic mutant of rrl or rplC gene. Conclusions: In conclusion, LZD-treatment failed
patients showed LZD resistant within 3 months after prescription. In addition, mutations in the 23S rRNA
(rrl) and the ribosomal protein L3 (rplC) are associated with resistance to the linezolid. Consistent with
previous reports, mutation in the 23S rRNA gene were, in combination with rplC gene mutation, cause
high-level LZD resistance (above to 16 μg/ml). The unsatisfactory correlation between mutant genotypes
and phenotypes needs to be investigated with another mechanism for LZD resistance that has not yet
been unfolded.
Session Number: 398
Session Number: 398
Resistance
Abstract Title:
Genexpert Mtb/Rif Assay and Culture Reveal High Prevalence of Pulmonary Tuberculosis and Rifampicin
Resistance among Patients Co-Infected with Hiv Presenting At A Healthcare Facility in Central Nigeria
M. P. Adoga1, A. M. Peter1, G. M. Salihu2; 1Nasarawa State Univ., Keffi, Nigeria, 2Dalhatu Araf
Specialist Hosp., Lafia, Nasarawa State, Lafia, Nigeria
Abstract Body:
Background: One third of the world’s population has tuberculosis (TB), with 10.4 million new cases
globally; 11 % (1.2 million) of which is from HIV-infected individuals. The emergence of drug-resistant
strains of Mycobacterium tuberculosis (MTB) poses a serious threat to effective treatment and the
WHO’s End TB Strategy (2016 - 2035). We evaluated the prevalence of pulmonary tuberculosis (PTB) and
rifampicin (RIF) resistance among HIV/AIDS and HIV-MTB co-infected patients respectively, presenting at
a healthcare facility in central Nigeria. Methods: Prior to the study, ethical clearance was obtained from
the Health Research Ethics Committee at Dalhatu Araf Specialist Hospital (DASH), Lafia, central Nigeria.
Informed consent was also obtained from each subject and structured questionnaires administered. In a
cross-sectional prospective study, 503 sputum samples were obtained from HIV/AIDS patients accessing
care at DASH between 2016 and 2017. MTB infection and resistance to rifampicin were detected with
GeneXpert MTB/RIF assay (Cepheid, Sunnyvale, CA, USA) following manufacturer’s standard operating
procedure. MTB-positive samples were further confirmed on Lowenstein-Jensen medium and drug
susceptibility test performed on cultures of RIF-resistant MTB. Data was analysed using SPSS version
16.0. Pearson’s chi-squared tests were performed at 95 % confidence interval with P values ≤ 0.05
considered significant. Results: Out of the 503 participating subjects, 310 (61.6%) were female and 193
(38.4%) were male. Overall, the prevalence of PTB was 13.9 % (n=70), and 21.4% (n=15) of these were
RIF-resistant. Although infection with MTB was more likely in subjects who were male (16.1%), aged 38 -
47 years (17.3%), rural dwellers (16.7%), retirees (23.8%) and had post-secondary education (14.4%),
these were not significant predictors (P>0.05). However, RIF resistance was significantly more likely in
female (23.1%) and married (23.5%) subjects compared to their male (19.4 %) and single (15.8%)
counterparts respectively (P < 0.05). Conclusions: The prevalence rates of PTB and rifampicin resistance
were high among patients co-infected with HIV accessing care in a facility in central Nigeria; with age,
gender, geographical location, occupation and educational level not being significant predictors of MTB
infection. However, gender and marital status were significantly associated with RIF resistance. These
findings have critical implication for TB treatment and drug resistance especially among patients co-
infected with HIV.
Session Number: 398
Session Number: 398
Resistance
Abstract Title:
Spectrum of Rapid Molecular Testing Gene Xpert Mycobacterium Tuberculosis/ Rifampicin (Mtb/Rif)
Assay in Detection of Multi-Drug Resistant Tuberculosis (Mdrtb) in Khartoum State, Sudan
M. A. O. Habeeb, M. M. E. Ebrahim, O. M. A. Abdelhafiz; Publ. Hlth.Lab., Lab. Adminstrtion, Khartoum,
Sudan
Abstract Body:
Background:Tuberculosis (TB) due to Mycobacterium tuberculosis (MTB) remains a major public health
issue: the infection affects up to one third of the world population. However, in many areas of the
world, TB diagnosis still relies on insensitive, poorly standardized sputum microscopy methods.
Antibiotic resistance is a growing problem with increasing rate of drug-resistant tuberculosis (MDRTB)
which is a serious public health problem in many developing countries .Treatment of MDTB takes longer
and requires more expensive drugs. GeneXpert assay is a molecular technique that simultaneously
detects (MTB) and Rifampicin resistant Tuberculosis. Objectives: To assess the performance of
GeneXpert MTB/ RIF, an automated molecular test for Mycobacterium tuberculosis (MTB) and
resistance to rifampin (RIF) among MTB patients, by determining the disease’s epidemiology and
calculating the proportion of cases infected just with TB and those with a resistance to rifampicin .
Methods: A retrospective cross sectional review of patients screened for TB with GeneXpert assay at TB
Reference Laboratory ,Khartoum State ,Sudan from 20th June 2017 and 30th November 2017 (n=583).
Results: Respiratory samples from patients suspected of MTB infection received by State TB Reference
Laboratory from different TB Management, were processed using the GeneXpert MTB/Rif assay
machine. The samples were (Sputum, Pleural fluids, Gastric fluids, Ascetic fluids, Lymph node
Aspiration).The total number of specimens collected from patients was 583 (Males 402/Females
181).One hundred and fifty nine (159) (27.3%) of the screened samples had positive results for MTB ,
(Males 123 /Females 36) of which 63 has history of past TB treatment , 71 New and 25 has un known
past history of TB treatment. Among the positive MTB there is one case positive for HIV. Out of 159
there were 26 (4.4%) had rifampicin resistant MDR TB (22 males /4 females), all of MDR TB were more
than 14 years,16 has previous treatment of TB , 9 new and 1 has un known history of TB treatment. The
male patients were more resistant to rifampicin when compared to the female patients.MTB was not
detected in 424 (279 males 145 females). Conclusions: Tuberculosis is widely distributed throughout
Khartoum State, with slightly more males infected than females. GeneXpert MTB/RIF assay is efficient
and reliable technique for the rapid diagnostic of TB. It's simplicity, high sensitivity and specificity for RIF
resistance detection make this technique a very attractive tool for diagnostic of MTB and RIF resistance
in MDR suspects.
Session Number: 398
Session Number: 398
Resistance
Abstract Title:
Genexpert® Technology; Validity for the Diagnosis of Hiv-Associated Tuberculosis: is Scale-Up Worth It?
M. Saeed1, S. Hussain1, S. Riaz1, F. Rasheed2; 1Punjab Univ., Lahore, Pakistan, 2Allama Iqbal Med. Coll.,
Lahore, Pakistan
Abstract Body:
Background: Recent evaluations of GeneXpert MTB/RIF Assay that in less than two hours simultaneously
detects Mycobacterium tuberculosis and tests for drug resistance have stimulated tremendous
enthusiasm. Is this the breakthrough that TB control has been waiting for? Present study was designed
to evaluate the validity of GeneXpert technology among HIV positive/negative patients for the detection
of Mycobacterium tuberculosis. Methods: The present cross sectional study was directed at the
Mycobacteriology laboratory, Allama Iqbal Medical College, Lahore, from 2011-2015. A total of 3784
(HIV-ve n=3568, HIV +ve n=216) sputum samples were collected with complete history (Gender, age, HIV
status, ATT history) from strong pulmonary TB (PTB) suspects attending multidrug resistance (MDR) and
PACP clinic Jinnah Hospital Lahore. Every sample was processed by performing ZN smear, LJ culture and
GeneXpertAssay. Only HIV positive status patients were selected for HIV PCR, 03 ml blood sample in
EDTA tube was collected and processed for HIV-PCR. Results: Out of total n=3784 PTB, The prevalence of
HIV was 5.7% (n=216/3784). Of 216 HIV +ve TB suspects, 15.7% (n=34) were HIV&TB co-infected, of
which males, females and trans-genders were 67.6% (n=23), 20.5% (n=7) and 11.7% (n=4) respectively.
Dramatically reduced sensitivities were observed in Zn smear and GeneXpert for HIV&TB co-infected
patients. The sensitivity and specificity,of ZN smear was 63.7%, and 100%,[HIV-ve 64.7% and
100%],[HIV+ve 23.5% and 100%] respectively. GeneXpert showed sensitivity and specificity of 94.5% and
100% [HIV-ve 95.0%, and 100%], [HIV+ve 76.4%,and 100%] respectively. Additionally, in case of smear
negative samples GeneXpert showed remarkable and noticeable sensitivity and specificity 85.8%,
100%and 69.2%, 100%, respectively for HIV negative and positive patients.MDR rate was also very high
30.7% (n=8/26) in HIV&TB co-infected patients as compare to HIV negative patients 9.5% (n=117/1220).
Conclusions: Although GeneXpert is WHO endorsed technology, but it attained less sensitivity for
HIV&TB co-infected patients as compare to HIV-negative patients. However, it seems to be close to the
gold standard for TB testing. Key words: Tuberculosis,HIV&TB co-infection, GeneXpert,
Session Number: 398
Session Number: 398
Resistance
Abstract Title:
Genexpert® Technology: A Breakthrough for the Diagnosis of Tuberculous Pericarditis, Pleuritis in Less
Than Two Hours
M. Saeed; DHQ Hosp. Mandi Bahauddin, Mandi Bahauddin, Pakistan
Abstract Body:
Background: Diagnosis of Tuberculous Pericarditis and Pleuritis remains the greatest challenge for
clinicians. Material and Methods: A cross sectional study was conducted at Mycobacteriology
laboratory of Allama Iqbal Medical College Lahore. A total of 286 (158 pleural & 128 pericardial fluids)
samples were received from strong TB suspects, during the period of January 2014 to August 2016.
Every sample was processed for Zn smear, LJ culture, GeneXpert MTB/RIF assay according to standard
protocols. Validity of GeneXpert assay for the detection of MTB was evaluated keeping LJ culture as gold
standard. Results: Out of 286 effusions samples, MTB was isolated by LJ culture in 51 (17.8%) samples
followed by GeneXpert in 43 (15.0%) and AFB was detected by Zn smear microscopy in 11 (3.8%)
samples. GeneXpert showed high sensitivity 84.3%, specificity 100%, with Positive predictive value
100%, and Negative predictive value 96.7%, while Zn smear showed sensitivity 18.3% , specificity , 99.1%
, Positive predictive value 81.8% , Negative predictive value 85.4 %. A strikingly high sensitivity of 72.2%
was observed for pericardial fluid by GeneXpert. Conclusion: GeneXpert assay is an innovative tool, for
prompt detection of MTB and drug resistance. It is definitely an attractive point of care test, with high
sensitivity and specificity along with turnaround time of two hours which facilitates timely diagnosis and
appropriate management of Tuberculous pleuritis and pericarditis. Keywords: GeneXpert MTB/RIF
assay, Pleural effusions, pericardial effusion,LJ culture.
Session Number: 398
Session Number: 398
Resistance
Abstract Title:
Xpert Mtb/Rif Assay for Detection of Rifampicin-Resistant M. Tuberculosis From Presumptive Drug
Resistance Tuberculosis Patients in Ethiopia
M. Tadesse, Z. Bonsa, G. Balay, N. Negash, G. Abebe; Jimma Univ., Jimma, Ethiopia
Abstract Body:
Background: Rifampicin is a potent first line drug for treatment of M. tuberculosis and acts by inhibiting
bacterial DNA-dependent RNA polymerase, encoded by the RNA polymerase gene (rpoB). Resistance to
rifampicin has mainly been associated with mutations in the rpoB gene. Xpert® MTB/RIF (Cepheid,
Sunnyvale, CA, USA) is real-time PCR technology that uses molecular beacons to detect rifampicin
resistance in the rpoB gene. In the current study, we aimed to asssess the accuracy of Xpert MTB/RIF for
detection of rifampicin-resistance M. tuberculosis in pulmonary tuberculosis (TB) patients in Ethiopia.
Methods: A cross-sectional study was carried out at Mycobacteriology Research Center of Jimma
University in Southwest Ethiopia. A total of 67 smear-positive sputum specimens collected from
pulmonary TB patients with increased suspicion of drug resistance were tested by GenoType MTBDRplus
line probe assay and Xpert MTB/RIF tests. Results: Of 67 pulmonary TB patients, 21 (31.3%) were
multidrug resistant TB (MDR-TB) (resistant to both rifampicin and isonizide). Xpert MTB/RIF detected
rifampicin-resistance in 23 (34.3%) pulmonary TB patients, of these 22 were also confirmed to be
rifampicin-resistant by line probe assay. In addition, 21 (91%) Xpert MTB/RIF-rifampicin-resistant cases
were confirmed as MDR-TB cases, making rifampicin-resistance a good surrogate marker of MDR-TB in
Southwest Ethiopia. Probe E related mutations (codon 447-452) was the most common rpoB genetic
mutation observed in 87% of rifampicin-resistant strains, suggesting sucessful transmission of these
strains in Ethiopia. Compared to line probe assay, Xpert MTB/RIF detected all rifampicin-resistance cases
correctly with 100% sensitivity and 97.8% specificity. Conclusions: The high sensitivity and specificity of
Xpert MTB/RIF for rifampicn-resistance detection support its use as an initial diagnostic test for drug
resistance TB. Implementation of Xpert MTB/RIF for direct diagnosis of rifampicn-resistance would be of
great benefit in adapting treatment regimens and limiting transmission of drug resistant strains.
Session Number: 398
Session Number: 398
Resistance
Abstract Title:
Xpert Mtb/Rif Assay for the Diagnosis of Mycobacterium Tuberculosis and its Rifampicin Resistance At
Felege Hiwot and Debre Tabor Hospitals; Northwest Ethiopia: A Preliminary Implementation Research
A. Habteyohhanes; Bahir Dar Univ., Bahir Dra, Ethiopia
Abstract Body:
Background: The World Health Organization in 2010 indorsed Xpert MTB/RIF (Xpert) assay for the
diagnosis of tuberculosis and multidrug resistant tuberculosis. However, the use of this novel diagnostic
method is still limited in a high TB and human immunodeficiency virus burden settings including
Ethiopia. Therefore, we conducted this study to describe the first implementation result of Xpert assay
in the diagnosis of TB, TB/HIV and MDR-TB at Felege Hiwot Referral Hospital (FHRH) and Debre Tabor
General Hospital (DTGH), Northwest Ethiopia. Methods: We analyzed the records of 1922 (FHRH=544
and DTGH=1378) presumptive TB patients diagnosed using Xpert test from 2015 to 2016 at FHRH and
DTGH, Northwest Ethiopia. Information on the demographic and clinical data was collected. Data were
entered, cleared, and analyzed using SPSS statistical software package; p < 0.05 was considered to be
significant. Results: Overall Xpert assay properly diagnosed 14.6% of the cases (258/1922). Among this
rifampicin (RIF) resistance was detected at 9.3% (24/258) of the cases. In the studied region, clinical data
reported that 81.0% (1556/1922) of the cases were MDR- TB. Among the study subjects, 888 (46.2 %) of
them were HIV positive. TB-HIV coinfection rate was at 41.9% (108/258). Of the total patients
registered, 1005 (52.3%) of whom were males. The mean age of patients was 31.1 years with SD of 17.5.
Significant predictors of the Xpert test were: age (p=0.000), sex (p=0.009), HIV (p=0.003) and
presumptive MDR-TB (p=0.000). Conclusions: In the studied areas, large proportion of clinically TB
suspected patients were wrongly diagnosed with multidrug resistant TB. Therefore, the use of Xpert
assay in health settings with no culture facility will decrease the unnecessary use of anti-TB drugs and
improve rapid TB, TB/HIV and MDR-TB detection and proper management of the cases.
Session Number: 398
Session Number: 398
Resistance
Abstract Title:
Xpert Mtb/Rif Assay for Detection of M. Tuberculosis and Rifampicin Resistance in Extrapulmonary
Specimens
M. Tadesse1, A. Bekele1, M. Bezabih1, D. Yilma1, G. Abebe1, L. Rigouts2; 1Jimma Univ., Jimma,
Ethiopia, 2Inst. of Tropical Med., Antwerp, Belgium
Abstract Body:
Background: Ethiopia has an extremely high rate of extrapulmonary tuberculosis (EPTB). However, the
diagnosis of EPTB is often made on clinical suspicion alone, and many people receive the wrong
diagnosis leading to unnecessary TB treatment or poor outcomes from untreated EPTB. In this study, we
evaluated the clinical utility of the Xpert MTB/RIF (real time PCR technology) for detection of M.
tuberculosis and rifampicin resistance in routinely collected extra-pulmonary specimens in Ethiopia.
Methods: This study was carried out at Jimma University Specialized Hospital, Southwest Ethiopia from
September 2015 to June 2017. Extrapulmonary specimens were collected from 572 patients with
clinically presumed of EPTB. These comprised 250 fine-needle aspirates (FNA), 45 cerebrospinal fluid
(CSF), 248 other fluids and 29 pus specimens. All specimens were tested for M. tuberculosis using liquid
culture (BACTEC 960 MGIT) and Xpert MTB/RIF at Mycobacteriology Research Center of Jimma
University. The sensitivity and specificity of Xpert MTB/RIF was calculated compared to culture. If
rifampicin resistance was detected by the Xpert MTB/RIF, further drug susceptibility testing by the
GenoTypeMTBDRplus line probe assay was performed on DNA extracted from a positive culture.
Results: Overall, 226 (39.5%) specimens were positive for M. tuberculosis by culture and 242 (42.3%) by
Xpert MTB/RIF. Xpert MTB/RIF gave positive results in 17 culture negative and in 9 culture contaminated
cases and in all these cases, TB was clinically confirmed. The pooled sensitivity and specificity of Xpert
MTB/RIF were calculated to be 91% and 90.6% respectively. Xpert MTB/RIF has the highest sensitivity
(94%) in FNA and moderate sensitivity (75%) in CSF. The sensitivity of Xpert MTB/RIF was lowest (60-
66%) in pleural or peritoneal fluids, with only around 35% of probable TB cases being detected. A
negative Xpert MTB/RIF on fluid specimens does not exclude the diagnosis of pleural or abdominal TB.
Xpert MTB/RIF detected rifampicin resistance in 13 patients in perfect agreement with line probe assay.
Conclusions: Our study showed that Xpert MTB/RIF is likely to be of greatest utility when testing FNA
and CSF specimens. Moreover, Xpert MTB/RIF can directly detect rifampicin resistant strains in extra-
pulmonary specimens and improves management of drug resistant EPTB patients.
Session Number: 398
Session Number: 398
Resistance
Abstract Title:
Evaluation of the Genexpert Mtb/Rif Assay on Sputum and Cultured Samples from the Tb Hosp. in Korea
N. Lee, S-H. Park, N. Sung, H. Kang, S. Ryoo; Masan Natl. Tuberculosis Hosp., Changwon-si, Korea,
Republic of
Abstract Body:
Background: In Korea, the incidence of tuberculosis is the highest among the OECD countries (77 per
100,000), and the number of TB-resistant tuberculosis is 4.8 per 100,000. There is a continuing need for
rapid drug-resistant diagnose in accordance with the increasing trend of global multidrug-resistant
tuberculosis. There have been several studies that have already applied Korean’s pulmonary specimens
to Xpert and DRplus, but they have not yet compared the two methods. In addition, previous studies
have put their focus on MTB diagnosis rather than on diagnosis of resistance. Methods: In this study, we
evaluated two molecular methods that could be applied to Korean pulmonary TB specimens. One
hundred sputum specimens and 104 cultured isolates from Masan National Tuberculosis Hospital
(MNTH), which represents a high percentage of patients with MDR-TB were applied to two methods.
Specimens were collected from the years 2009 to 2014 and stored at Specimen Biobank of MNTH.
Sequencing of the samples with inconsistent results was performed to find the cause of the
discrepancies. Results: As result of applying 100 sputum specimens to Xpert and DRplus, the sensitivity,
specificity and agreement of two methods were 97.7%, 98.2% and 98.0%, respectively. In the case of
cultured isolates, the sensitivity of Xpert was 93.4% and 94.7% for DRplus. The specificity was 100%
identically. The concordance rates of Xpert and DRplus were 95.2% and 96.2%, correspondingly.
Through the sequencing, of the 8 mismatched samples, 6 samples are considered to have mutations
outside the RRDR region. One of those samples has I572F (ATC/TTC) mutation and the other has both
I527F (ATC/TTC) and V643A (GTG/GCG) mutations. No mutation was found in the remaining 4 samples.
The other two samples were diagnosed as false resistant and false susceptible with DRplus,
subsequently. Conclusions: In conclusion, applying Korean pulmonary TB specimens to two different
molecular methods, both methods showed excellent diagnostic efficacy over 95%. Mutations in I527F
(ATC/TTC) and V643A (GTG/GCG) occurred simultaneously in one of the mismatched specimens. The
I527F mutation has been reported in previous articles, but the V643A mutation is a new mutation that
has never been reported. Although a small number, it is necessary to continue to research on the RIF
resistant associated mutation sites.
Session Number: 398
Session Number: 398
Resistance
Abstract Title:
Isolation and Antimicrobial Susceptibility Pattern of Non-Mtb Pathogens from Patients with Respiratory
Problems in Karachi, Pakistan
F. Wahab, S. U. Kazmi; Dadabohy Inst. of Higher Ed., Karachi, Pakistan
Abstract Body:
Background: Respiratory infections in young children and elderly immunocompromised individuals are a
great cause of public health concern resulting in high mortality and morbidity . Emerging antibiotic
resistance in pathogens is a serious health problem. In this study we identified Non-TB pathogens in
patients with respiratory problems , established their frequency and determined antimicrobial
susceptibility patterns. Methods : A total of 205 clinical samples of URT tracheal aspirate, sputum,
tracheal tip, bronchial alveolar lavage, bronchial tap and body fluids collected from patients reporting to
local hospitals were processed for isolation and identification of pathogens by conventional culture &
Rap ID system. The bacterial and fungal isolates were tested for susceptibility by disc diffusion method
on Mueller Hinton agar and with Thermo-Fisher Scientific Sensititre Yeast ONE Y010 Panels. Results : Out
of 205 , sputum samples (n: 117) ,trecheal lines samples (N: 50 ), bronchial alveolar lavage ( 07)
bronchial tap (n -6), trecheal tip( 5 ), ear swab (n-4) and body fluid ( 3 ), we isolated 220 different
potential pathogens . Most common isolate was Moraxella catarrhalis (25.8%) followed by Hemophilus
species Candida albicans , Acinetobacter species , Pseudmonas aeruginosa, , Staph aureus ,
Entereobacter species Kebsiella pneumonaie, (3-16%).Most pathogens exhibited susceptibility to
Amoxycillin/clavulanic acid , followed by Vancomycin , Colistin , Ceftriaxone, Polymyxin B , Tigecyclin ,
Ceftazidime. to Clindamycin Cefoxitin. , Cefoperazone/sulbactam , Chloramphenicol, Meropenem ,
Fusidic acid, Azithromycin, to Linezolid Amikacin (92- 52 %). Majority of Candida species were
susceptible to Micafungin followed by Voriconazole , Posaconazole & Caspofungin , Anidulafungin ,
Itraconazole (90- 74%). More than 50 % of the pathogens showed resistance to broad spectrum
antibiotics like Levofloxicin , Tetracycline, Ciprofloxacin , Ampicillin and most of the Candida species
were found to be resistant to Fluconazole and 5- Flucytosine .Conclusions : Our results indicate that Non
MTB pathogens like Moraxella catrrhalis , earlier known as a normal flora of URT and is now a
recognized pathogen , both in children and adults as well as and H, influenzae and Candida species are
increasing and inappropriate use of antibiotics is resulting in emergence of resistance against quinolones
and other antibiotics which needs to be controlled to combat the menace of antibiotic resistance .
Session Number: 398
Session Number: 398
Resistance
Abstract Title:
Species Distribution of Nontuberculous Mycobacteria In Peking Union Med. Coll. Hospital, China, 2013-
2017
J-J. Huang, M. Xiao, W-H. Yang, Y-C. Xu; Peking Union Med. Coll. Hosp., Chinese Academy of Med. Sci.,
Beijing, China
Abstract Body:
Background: To investigate the species distribution of nontuberculosis mycobacteria (NTM) in Peking
Union Medical College Hospital, China, and provide a reference for clinicians. Methods: From January
2013 to August 2017, species identification was carried out by DNA microarray chip on 116 clinical
specimens from Peking Union Medical College Hospital, China, which the results of Mycobacterium
tuberculosis or NTM testing were positive. Results: A total of 1031 specimens of Mycobacteria were
isolated from 1028 patients, among which NTM accounted for 31.5% (325/1031), increasing from 16.5%
in 2013 to 42.6% in 2017. The trends of decreasing proportion of MTB and increasing proportion of NTM
were statistically significant (P<0.001). Among the 116 specimens used to identification, 81.9% were
from respiratory tract, and 5.2% and 4.3% were from lymph nodes and pus respectively. NTM included
as many as 10 kinds. Mycobacterium intracellulare and Mycobacterium chelonae / Mycobacterium
abscessus were the most frequently isolated pathogens, both accounting for 28.4%, followed with
Mycobacterium gordonae, Mycobacterium avium and Mycobacterium kansasii accounting for 12.9%,
10.3% and 9.5% respectively. Among the 113 patients, 43.4% were male and 56.6% were female;
Patients at ages over 45 years accounted for 67.3%. Conclusions: The proportion of NTM in
Mycobacteria in Peking Union Medical College Hospital was increasing rapidly during five years. Middle-
aged and elderly patients are more likely to be infected, especial female. Mycobacterium intracellulare
and Mycobacterium chelonae / Mycobacterium abscessus were the main pathogens. The identification
of NTM is of vital importance to diagnosis and treatment.
Session Number: 399
Session Number: 399
Session Title: CPHM06 - Diagnostic Mycology: Identification and Clinical Relevance
Abstract Title:
Does Antigenuria Help Diagnosis of Blastomycosis and Histoplasmosis in Endemic Areas?
S. Das, I. Dusich, E. McElvania, R. B. Thomson, Jr.; NorthShore Univ. Hlth.System, Evanston, IL
Abstract Body:
Antigen detection in urine is considered to be a rapid, sensitive and non-invasive method for diagnosis
of blastomycosis and histoplasmosis, the two most common endemic mycoses encountered in North
America. Clinical presentation being diverse, suspicion often arises after failure of routine therapy.
Culture confirmation is slow requiring invasive procedures, delaying appropriate therapy. A
retrospective study was performed to assess the role of antigenuria as a diagnostic and prognostic aid
for endemic mycoses. All culture confirmed histoplasmosis and blastomycosis cases for a six year period
(2012-2017) were included. Commercially available histoplasmosis and blastomycosis antigen enzyme
immunoassay (EIA) and serum beta-D-glucan were sent out to be performed at MiraVista and Mayo
Medical reference laboratories, respectively. Antigen EIA and fungal culture results were analyzed. The
utility of antigenuria alone, was assessed by performing a subgroup analysis of a subset of patients
during this time period where antigen EIA results were available and fungal culture was negative or not
performed. We identified 58 culture positive unique patients. Median age at diagnosis was 56.4 years
(range 19.8-92.7), 16 patients (27.5%) were female and 77% specimens were from a respiratory source.
Urinary antigen levels were available for only 37 patients; results are shown in the table. Overall urinary
antigen was detected in 58% of culture confirmed patients. Serum beta-D-glucan was negative for all
patients. When tested serially, reduction in antigenuria indicated successful therapy. In the subgroup
analysis, 4 of 102 patients had positive EIA results; Histoplasma (n=3), Blastomyces (n=1) and both (n=1);
where antigenuria confirmed diagnosis in the absence of positive fungal culture. In our patient
population, diagnosis of endemic mycoses was achieved primarily by culture and cytopathology or
histopathology. Antigenuria, if present, was a useful adjunct to therapy and prognosis, but was not
sensitive in either pulmonary or disseminated disease. Table: Results of antigenuria in patients with
positive culture.<table border="1" cellpadding="1" class="DisplayTable" id="{E8BFE9BD-D57B-403E-
98F2-2E2914BD49C8}"><caption></caption><tr><td rowspan="1" colspan="1"><br></td><td
rowspan="1" colspan="1"><br></td><td rowspan="1" colspan="1"><br></td><td rowspan="1"
colspan="1"><br></td><td rowspan="1" colspan="1"><br></td><td rowspan="1"
colspan="1"><br></td><td rowspan="1" colspan="1"><br></td></tr><tr><td rowspan="1"
colspan="1"><br></td><td rowspan="1" colspan="6">Antigen EIA Results*</td></tr><tr><td
rowspan="1" colspan="1">Site of Infection</td><td rowspan="1" colspan="3">Blastomyces</td><td
rowspan="1" colspan="3">Histoplasma</td></tr><tr><td rowspan="1" colspan="1"><br></td><td
rowspan="1" colspan="1">Positive</td><td rowspan="1" colspan="1">Negative</td><td rowspan="1"
colspan="1">Not Done</td><td rowspan="1" colspan="1">Positive</td><td rowspan="1"
colspan="1">Negative</td><td rowspan="1" colspan="1">Not Done</td></tr><tr><td rowspan="1"
colspan="1">Pulmonary<br>(n=30)</td><td rowspan="1" colspan="1">17</td><td rowspan="1"
colspan="1">Bone/Skin/Joint (n=7)</td><td rowspan="1" colspan="1">4</td><td rowspan="1"
rowspan="1" colspan="1">2</td><td rowspan="1" colspan="1">4</td></tr></table> *15 patients with
results for both antigens; 8 positive for both and 5 negative for both, 2 positive for Blastomyces only.
Session Number: 399
Session Number: 399
Abstract Title:
Assessing the Clin. Relevance of Fungal Genotype in Blastomycosis Infections
K. L. Laux1, S. P. Bentivenga1, J. L. Anderson2, G. M. Gauthier3, J. K. Meece2; 1Univ. of Wisconsin
Oshkosh, Oshkosh, WI, 2The Marshfield Clinic Res. Inst., Marshfield, WI, 3Univ. of Wisconsin Madison,
Madison, WI
Abstract Body:
Background: Blastomycosis is a fungal disease caused by infection with the fungus Blastomyces which
grows as a mold at ambient temperatures and produces infectious spores which, when inhaled by
mammalian host, transform into yeast. Blastomyces yeast cause fungal infections such as pneumonia,
skin lesions, meningitis, bone infections, and sepsis. In 2013, multi-locus sequence typing revealed two
distinct species of Blastomyces were responsible for blastomycosis—Blastomyces dermatitidis and
Blastomyces gilchristii. Despite demonstrated differences in clinical presentation between these two
species, there is currently no diagnostic assay to distinguish between them. The goal of this study was to
assess whether this species distinction is relevant to care providers. Materials and Methods: Clinical
isolates (N=112) were obtained from patients in Wisconsin diagnosed with blastomycosis from 2008-
2016. The ITS region of the fungal genomes were sequenced and a single nucleotide polymorphism was
used to distinguish Blastomyces dermatitidis from Blastomyces gilchristii. Fungal genotype was
compared to patient demographics, sensitivity and specificity of the urine antigen test, and patient
treatment. Results: In keeping with previous studies, patients infected with B. dermatitidis infections
were 15 years older on average, were more likely to have a pre-existing condition, and were likely to be
male than those with B. gilchristii infections (p<0.05). Patients infected with B. gilchristii were diagnosed
earlier than those with B. dermatitidis (p=0.05) which may indicate differences in disease severity. In
terms of diagnosis and post-diagnostic monitoring, the urine antigen test was found to display a false
negative result 38% of the time in B. dermatitidis infections as opposed to 18% false negatives in B.
gilchristii infections. A false negative result was defined as no positive result at any time during the
course of blastomycosis disease. While 97% of physicians made use of itraconazole to treat
blastomycosis, only in 50% of cases physicians used the itraconazole serum concentration assay. We
found evidence to support use of therapeutic drug monitoring regardless of fungal genotype as 61% of
patients displayed at least one value that merited dose modification or patient education. Conclusion:
Fungal genotype is relevant in terms of patient demographics, clinical features, and urine antigen test
performance. Knowledge provided by this study will improve clinical decision making and patient care in
cases of blastomycosis.
Session Number: 399
Session Number: 399
Abstract Title:
Histoplasma Antigen Testing of Bronchoscopy Samples: A Retrospective Examination of Clin. Value
M. Patel1, J. J. Farrell2; 1Northwestern Univ., Evanston, IL, 2Univ. of Illinois Coll. of Med., Peoria, IL
Abstract Body:
Background: Histoplasma capsulatum is a dimorph endemic to midwestern U.S. river valleys that causes
disseminated infection when cell-mediated immunity is compromised. H capsulatum is the most
common endemic mycosis found in immunocompromised patients, but diagnosis of pulmonary infection
in immunocompetent individuals can be a challenge. Diagnostics for pulmonary histoplasmosis include
fungal culture, serologic & antigen assays, and pathologic exam. Fungal cultures require incubation for
weeks and have poor sensitivity, serologic testing cannot distinguish acute from prior infection, and
pathologic examination is dependent on recognition of narrow-necked budding yeast in specially stained
tissue samples. An FDA approved urine histo antigen test for diagnosis of disseminated infection in
immunocompromised patients is commercially available. There are no commercially available tests for
respiratory samples obtained by bronchoscopy (eg, washes and lavages), but a histoplasma reference
laboratory (MiraVista Diagnostics, Indianapolis, IN) offers histo antigen testing on these samples for
$119. Method: All patients who underwent bronchoscopy with histo antigen testing of bronchoscopic
samples between 4/15/15 - 4/15/17 were enrolled in an IRB approved review. Fungal cultures,
pathologic examination, and urine antigen testing were performed at physician discretion. Result:
Bronchoscopy samples were submitted to MiraVista from histo antigen testing for 658 patients. 11
patients with pulmonary histoplasmosis meet clinical/radiographic criteria of infection. Histo antigen
tests were positive in 6 patients (2 other patients were detected but below quantification). Infection was
confirmed in 5/6 cases: culture & urine antigen (2/6), urine antigen & biopsy (1/6), culture & biopsy
(1/6) or urine antigen (1/6). Three patients had positive urine antigen or grew H capsulatum, with
negative bronchial histo antigen results (including a patient with a positive urine histo antigen who died
before treatment was initiated). 6/7 patients with positive lymph node biopsies for H capsulatum and 1
patient with a positive lung biopsy were histo antigen negative. Conclusion: Histo antigen tests were
positive in 0.9% of patients (6/658) at cost=$13,050/positive. One false positive from cross reaction in a
fatal case of pulmonary aspergillosis, 3 false negatives in patients with culture or biopsy proven
pulmonary histoplasmosis, and the cost/positive test all suggest that indiscriminant testing of bronchial
samples should be discouraged.
Session Number: 399
Session Number: 399
Abstract Title:
Utility of Frozen Section in the Identification of Invasive Fungal Rhinosinusitis
R. Marrero Rolon, T. Scognamiglio; New York Presbyterian Hosp. - Weill Cornell Med., New York, NY
Abstract Body:
Background: Fungal infections are an important cause of morbidity and mortality in
immunocompromised hosts. Angioinvasive disease and dissemination leading to thrombosis, infarction,
and tissue destruction carries a high mortality rate and early diagnosis is critical. Culture recovery of the
most common causal organisms of the Mucorales order and Aspergillus spp. takes time and is not
always successful, making tissue diagnosis the most accessible diagnostic tool. Frozen sections (FS) are
used in some instances for timely diagnosis and monitoring of margins during surgical debridement but
significant rates of false negatives have been reported. This study aims to evaluate the usefulness of FS
in the diagnosis fungal infections. Methods: The surgical pathology archives of our institution from 2002
to 2017 were searched to identify cases in which FS was performed for the evaluation of fungus. A total
of 96 samples were identified from 40 patients, most of them from sinonasal and oral cavities. The FS
diagnoses were compared with final diagnoses (FD). All FS diagnoses were rendered after H&E
examination and FD were rendered after evaluation with H&E stained sections and in some cases, GMS
and PAS stains. The results of concurrent culture results were recorded when available. Results: An
overall concordance of 87.5% was identified between FS diagnoses and FD. Twelve of 96 samples were
discordant (12.5%). In 10 of the discordant cases, fungal organisms were not identified on FS and were
detected on permanent section. Of these, 5 were invasive, one of them angioinvasive. The FD in 8 of
these cases was achieved with the use of special stains. The two remaining discordant cases were false
positives. Fifty five cases were negative on both FS and FD and 29 cases were positive on both. FS
diagnosis correctly identified 20/29 (69%) samples of invasive infection. When comparing FS diagnosis to
the FD, including use of special stains, FS had a sensitivity of 74% and a specificity of 96%. Concurrent
cultures were available for 60 cases with Rhizopus spp. being most commonly isolated followed by
Aspergillus spp. Conclusions: In this study, the sensitivity of FS diagnosis for the detection of fungal
organisms is comparable with prior studies. False negative results can be a result of technical challenges
such as frozen artifact, obscuring inflammation and necrosis, and sampling. FS is a useful tool for
diagnosis of fungal infections including invasive fungal infections allowing for a rapid and early diagnosis.
Session Number: 399
Session Number: 399
Abstract Title:
Combined T2candida and Blood Culture Results Predict Mortality among Patients with Candidemia
C. Clancy, W. Pasculle, M. Nguyen; Univ. of Pittsburgh, Pittsburgh, PA
Abstract Body:
Background: Approximately 50% of patients (pts) with candidemia clear infection without complications
in the absence of antifungal treatment. However, all pts with candidemia receive full-course antifungal
treatment because clinicians are unable to identify this low-risk cohort. T2Candida (T2) is an FDA-
approved nanodiagnostic panel, which utilizes T2 magnetic resonance to detect Candida within
unprocessed whole blood samples. In multi-center studies (DIRECT2, STAMP), T2 was significantly more
likely to remain positive than blood culture (BC) in pts with candidemia. We hypothesized that
T2Candida results, alone or in combination with BC shortly after initial diagnosis, would predict 28 day
mortality in pts with candidemia. Methods: 32 pts (17 men) from our center enrolled in DIRECT2 were
studied. Pts were identified by positive diagnostic BC (dBC). Subsequent blood samples were collected
concurrently for testing by T2 and companion BC (cBC). T2 results are reported qualitatively for Candida
albicans/C. tropicalis (CA/CT), C. glabrata/C. krusei (CG/CK), and C. parapsilosis (CP). Results: Pts were
infected with CP (11/32, 34%), CA (10/32, 31%), CG (8/32, 25%) and CT (3/32, 9%). Median age was 50
yrs (23-86). Underlying conditions included diabetes (7/32, 22%), hemodialysis (6/32, 19%), organ
transplant (14/32, 44%) and abdominal surgery (7/32, 22%). 29/32 (91%) had IV devices; 22/32 (69%)
were receiving antifungal therapy when T2 and cBC were collected. Median time between dBC and
T2/cBC collections was 62 hrs (21-115). 6/32 pts (19%) died at 28 days: T2+/cBC+, 2/4 (50%); T2+/cBC-,
2/6, (33%); T2-/cBC+, 1/2 (50%); T2-/cBC-, 1/20 (5%). Mortality was 4/10 (40%) if T2 was + vs. 2/22 (9%)
if T2 was - (p=0.06), and 3/6 (50%) if cBC was + vs. 3/25 (12%) if cBC- was - (p=0.06). Mortality was 5/12
(42%) if either T2 or cBC was + vs. 1/20 (5%) if both were negative (p=0.02). T2 results were not
impacted by receipt of antifungal agents. Other clinical factors were not significantly associated with
mortality. Conclusions: T2Candida and BC results predicted mortality among patients with candidemia,
if considered together shortly after the initial diagnosis. Use of these tests within the first few days of
candidemia may identify low-risk patients in whom shorter course antifungal treatment may be
possible.
Session Number: 399
Session Number: 399
Abstract Title:
Using Microsatellite Cai Patterns to Analyze Invasive Candida Albicans Blood Isolates
Y-M. Wu1, S-H. Wang2, J-J. Lu1; 1Chang-Gung Mem. Hosp., Linkou, Taoyuan, Taiwan, 2Natl. Chiayi
Univ., Chiayi, Taiwan
Abstract Body:
Background: Invasive Candida albicans is a major cause of bloodstream infection, which may cause
morbidity and mortality in immunocompromised patients. Multilocus sequence typing (MLST) is highly
discriminatory method for analyzing the population structure and diversity of pathogens. However, the
cost of MLST is very high. Microsatellite PCR analysis is less expensive and is also highly discriminative in
microbial epidemiology. The CA (Candida albicans) microsatellites have been used for molecular typing
of C. albicans isolates. The purpose of this study is to analyze whether microsatellite analysis is
comparable to MLST in the molecular typing of C. albicans. Methods: One hundred eighty-eight C.
albicans blood isolates were collected from adult patients (≥ 18 years) registered in Chang-Gung
Memorial Hospital at Linkou in northern Taiwan from 2003 to 2011, and their genomic DNA was purified
for MLST and microsatellite analysis. MLST genotypes of those isolates were used for eBURST clustering
analysis and UPGMA phylogenetic analysis. Genotyping with microsatellite CAI, CAIII, CAV, CAVI and
CAVII were applied to those C. albicans isolates with electrophoresis analysis. Besides, demographics,
comorbidities, risk factors, clinical outcomes of these patients were reviewed. Results: The major MLST
genotypes of 186 C. albicans isolates are DST693 (19 isolates), DST659 (16 isolates), DST443 (13 isolates)
and DST766 (10 isolates), which were classified as clade 3/CC20, clade 4/CC8, clade 17/CC17, and clade
1/CC9, respectively. All DSTs displayed unique major CAI type, such as 25-27 in DST693, 11-20 in DST659,
18-41 in DST443, and 16-40 in DST766 isolates. Within four common clades in CGMH, clade 1 isolates
with alleles more than 29 repetitions contributed to higher 30-day mortality. Conclusions: CAI analysis is
a good alternative method in the molecular phylogenetic of C. albicans and epidemiologic studies. Clade
1 isolates with alleles more than 29 repetitions are related to higher 30-day mortality in our hospital.
Keywords: Candida albicans, Microsatellite PCR, CAI patterns, DSTs
Session Number: 399
Session Number: 399
Abstract Title:
Fungal Blood Cultures for the Detection of Candidemia: Time to Retire?
S. Altamimi, M. Jamal, O. Rayes, L. Samuel, G. Alangaden; Henry Ford Hlth.System, Detroit, MI
Abstract Body:
Background: Blood cultures (BC) have sensitivities of 40-70% for detection of bloodstream infection due
to Candida (BSI-CA). The poor sensitivity has led to the use of the isolator fungal blood culture (IFC)
system (Alere, Waltham, MA, USA) that utilizes lysis-centrifugation to improve recovery of candida. IFC
has not been extensively evaluated and is labor-intensive compared to automated BC (Trek Diagnostic
Systems, Oakwood, OH, USA). The aim of this study is to determine if IFC provide significant information
compared to BC for detection of BSI-CA at a tertiary care center with patient populations at high risk for
BSI-CA. Methods: Retrospective review was done of all positive IFC and BC between 1-1-2013 and 9-30-
2016. Cultures were reviewed for corresponding results with the alternate method +/- 7 days. Fungal
biomarker results either Fungitell (Associates of Cape Cod, Falmouth, MA, USA) or T2Candida panel
(T2Biosystems, Lexington, MA, USA) were reviewed. Patient characteristics were obtained by review of
medical records. Results: Overall, 121 IFC and 730 BC were positive for yeast. Both IFC and BC were done
in 175 cases that Candida spp. was isolated: 159/175 (92%) were detected by BC and 14/175 (8%) by IFC
alone. Of 121 IFC positive for Candida, 76/121 had corresponding BC: 62/76 (82%) IFC and BC were both
positive, and 14/76 (18%) IFC was positive and BC was negative. In the 14 IFC positive/BC negative cases
C. albicans (6), C glabrata (3) and C. parapsilosis (3) were the common species isolated. In episodes that
were IFC positive/BC negative, 13/14 (93%) IFC had <5 CFU/ml whereas for episodes that were IFC
positive/BC positive, 39% had >5 CFU/ml. Fungal biomarker results were available in 13/14 cases of the
IFC positive/BC negative group and was positive in 12/13 (92%): Fungitell positive 11/13 and T2Candida
positive 1/2 cases. Clinical characetristics of the 14 patients in the IFC positive/BC negative group were:
100% had undifferentiated sepsis; 86% were in the ICU; 21% colonized with candida; 87% had Infectious
Diseases consultation, and all-cause 30-day mortality of 31%. Conclusions: These results suggest that
over 90% of BSI-CA can be detected using routine BC. IFC may complement BC for detection in patients
with low levels of candidemia (<5 CFU/ml). Fungal biomarkers including Fungitell and T2Candida Panel
are highly sensitive for the detection of BSI-CA in the BC negative cases. Utilization of BC together with a
fungal biomarker can eliminate the need for IFC in the detection of BSI-CA.
Session Number: 399
Session Number: 399
Abstract Title:
Growth of Candida Auris and Three Related Species in Aerobic Blood Culture Media and on Selective and
Differential Plated Media
A. Akinlade, R. Scott, L. King, P. Warns, L. Crowther, B. Morrow, V. White, R. Pfeltz; BD Life Sci., Sparks,
MD
Abstract Body:
Background: This seeded study provides specific growth media recovery data for the emerging,
antifungal-tolerant pathogen Candida auris (CA) and three phylogenetically related but less frequently
isolated Candida with which it may be misidentified. Methods: BACTEC™ plastic blood culture bottles of
Plus Aerobic/F (0, 3, 10 mL bagged blood), Peds Plus™/F (0, 1, 5 mL), and Standard/10 Aerobic/F (0, 3, 10
mL) media were inoculated with 10-100 CFU expected per bottle of 23 organisms: 11 CA, eight C.
haemulonii (CH), two C. duobushaemulonii (CD) and two C. pseudohaemulonii (CP). The 621 bottles
(N=3) were incubated in a BACTEC FX™ automated culture instrument and time to detection (TTD) data
was analyzed by ANOVA with Tukey Pairwise Comparisons. The same inocula were plated on nine BBL™
and Difco™ media (N=4; 828 plates): Sabouraud Dextrose Agar Emmons (SDA-E), CHROMagar™ Candida,
and seven selective media containing antifungals (five with cycloheximide, CXD). Plates were incubated
at 35oC and 42oC (N=2), and colonies counted daily for five days; < 10 CFU on either plate per pair was
deemed sporadic recovery. Results: All culture bottles detected within 120-hrs except one Peds Plus
with CH and no blood (121.9 hrs). Differences in overall mean TTDs between media or blood levels were
relatively small, within 3.2 and 5.5 hrs, respectively. Organism was the factor accounting for the most
data variability, with TTD means (range) all differing significantly at 24.7 (15.6-49.6) hrs for CA, 37.0
(31.2-43.6) for CD, 51.9 (29.1-114.8) for CP, and 60.7 (41.7-121.9) for CH (P < 0.05). All strains had
expected CFU at 35oC on CHROMagar, WL Differential Medium (0.0004% CXD), and except for sporadic
recovery of one CH, on SDA-E; six CA had expected CFU on Campy-CSM Agar (0.01% CXD) as did six CA
on BCYE-PAC Agar (0.008% anisomycin). Some CD, CH and CP grew on Dermatophyte Test Medium
(0.05% CXD), Mycosel™ Agar (0.04% CXD), SABHI with CC Agar (0.05% CXD), and Selective 7H11 Agar
(0.001% amphotericin B) at 35oC. Only CA grew at 42oC, with expected CFU from all strains on
CHROMagar and SDA-E, and on WL Differential Medium except for one strain with sporadic recovery;
four and three CA had expected CFU on Campy-CSM and BCYE-PAC, respectively. Conclusions: CA and
related strains reliably detected in BACTEC aerobic blood culture media irrespective of blood volume.
Recovery was optimal on CHROMagar for all conditions, on WL Differential Medium at 35oC and for CA
on SDA-E at 35oC or 42oC. CA thermotolerance and 0.04% CXD sensitivity was confirmed1, but growth
on 0.01% CXD was strain-dependent.
Session Number: 399
Session Number: 399
Abstract Title:
Invasive Infections Due to Trichosporon Spp.: Species Distribution, Genetic Diversity Assessed with
Amplified Fragment Length Polymorphism Analyses, and Antifungal Susceptibility Testing
L-N. Guo, S-Y. Yu, Y-C. Xu; Peking Union Med. Coll. Hosp., Beijing, China
Abstract Body:
Background: The epidemiology of invasive fungal infections is evolving, and many uncommon yeasts
have recently emerged as etiologic agents of bloodstream and deep-seated infections. Trichosporon
spp. has been reported as the second- or third-most-common agent of yeast fungemia, which poses a
major challenge to diagnosis and treatment. Methods: A total of 79 clinical Trichosporon isolates were
collected from 40 different hospitals across China, from 2009 to 2014 (Fig. 1). The diagnostic
performances of VITEK MS and Bruker Biotyper MS system were assessed with sequencing of intergenic
spacer 1 region. Amplified fragment-length polymorphism (AFLP) fingerprinting was applied to
determine their genetic diversities. In addition, the antifungal susceptibility profile of the Trichosporon
isolates was determined. Results: The patients ranged in age from 0 to 86 years (median age 49.4 years),
and males were more affected than females (67.1 vs 32.9%). 20 (25.3%) patients were from ICU, 20
(25.3%) had undergone surgical intervention, 11 (13.9%) had hematologic malignancies. Blood (41.8%)
was the most popular separation site, followed by ascitic fluid (20.3%) and catheter (13.9%).T. asahii was
the predominating species (78.5%), followed by T. dermatis (5.1%), T. japonicum (5.1%), T. inkin (3.8%),
T. dohaense (2.5%), T. asteroids (1.3%), T. faecale (1.3%), T. jirovecii (1.3%) and T. montevideense
(1.3%). The VITEK MS and Bruker Biotyper MS correctly identified 82.3% and 91.1% of 79 Trichosporon
isolates, 98.4% and 100% of T. asahii, respectively. The AFLP dendrogram comprised six main clusters at
the species level, but the profiles did not significantly vary between T. asteroides, T. japonicum and T.
faecale. Additional subgroups within the T. asahii cluster revealed genetic diversitiy, however, no
significant relation was determined between AFLP genotypes and clinical presentations. 27.4% (24h) and
98.4% (48h) of T. asahii isolates exhibited AMB MICs of ≥2 μg/ml, respectively. About 1.6% (24h) and
9.7% (48h) of T. asahii isolates exhibited decreased susceptibility to fluconazole (MICs ≥ 16 μg/ml). One
T. japonicum isolate showed high MIC (128μg/ml) against fluconazole. Voriconazole has the lowest
MIC90 (0.125μg/ml) against Trichosporon isolates. Conclusions: This study outlined the epidemiology,
genetic diversities and antifungal susceptibilities of Trichosporon spp. causing invasive infections in
China.
Session Number: 399
Session Number: 399
Abstract Title:
Identification of Candida Glabrata Complex Species: Use of Vitek Ms Ruo and Clinprotools to Overcome
Limitations of the Vitek Ms Ivd and Bruker Biotyper Databases
X. Hou1, M. Xiao1, S. Chen2, F. Kong2, H. Wang1, X. Fan1, Y-C. Xu1; 1Peking Union Med. Coll. Hosp.,
Beijing, China, 2Univ. of Sydney, Sydney, Australia
Abstract Body:
Background: Candida glabrata species complex includes Candida glabrata sensu stricto and two major
cryptic species, Candida nivariensis and Candida bracarensis. Distinction of these species is relevant for
epidemiological purposes and for antifungal management. Materials/Methods: In this study, two
commercial MALDI-TOF MS systems (Vitek MS system [bioMérieux] and the Bruker MS system [Bruker
Daltoniks]) employing the Vitek MS RUO and Bruker ClinProTools programs, respectively were evaluated
for the identification of 33 isolates of C. glabrata complex (17 C. glabrata, 14 C. nivarensis, 2 C.
bracarensis). Results: C. glabrata sensu stricto was identified correctly by both systems with distinct
principle components compared with the two cryptic species. All C. nivariensis and C. bracarensis could
not be identified to species level by the Vitek MS v2.0 IVD and Bruker Biotyper MS v3.1, but were all
correctly identified by the Vitek MS RUO. The generic algorithm model from ClinProTools software
showed 100% recognition capability and cross validation for the discrimination of C. nivariensis and C.
bracarensis. Spectra peak statistics revealed that five markers (3292.47Da, 4152.64Da, 6245.75Da,
6585.15Da and 7304.68Da) can reliably distinguish between C. nivariensis and C. bracarensis, with area
under the curve (AUC) values of 0.928, 0.9998, 1, 1 and 1, respectively. A small “in-house” Bruker
spectral database was established incorporating spectra of two clinical isolates representing C.
nivariensis and C. bracarensis identified in this study. After complementation with the “in-house”
database, all the remaining 14 C. nivariensis and C. bracarensis isolates were correctly identified to
species level (score >2.00). Conclusions: MALDI-TOF MS enabled rapid and reliable identification of C.
glabrata complex isolates. The use of the Vitek MS RUO system, Bruker ClinProTools software and the
addition of MSP to Bruker MS databases representing the local diversity of isolates assisted with
achieving differentiation of cryptic species within C. glabrata complex.
Session Number: 399
Session Number: 399
Abstract Title:
Verification and Clin. Application of the Maldi-Tof Vitek Ms Saramis Ruo Dermatophyte Database
Enables Faster Resulting of Dermatophytes Cultures in A Pediatric Hospital
C. Burch, H. Wang, A. Leber, S. Antonara; Nationwide Children's Hosp., Columbus, OH
Abstract Body:
Background: In our institution the majority of our fungal cultures are dermatophyte cultures from
outpatients. Speciation of dermatophytes is necessary to initiate the correct treatment and set
prophylactic measures. Laboratory identification depends on phenotypic techniques but is sometimes
problematic. Matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF
MS) presents an alternative way of identification that can result in quick and accurate results. We
wanted to evaluate the use of the Vitek MS SARAMIS RUO dermatophyte database in our patient
population and assess its contribution to reporting faster and more reliable results. Methods: The Vitek
MS Knowledge Base V4.14.0 was evaluated. The database includes 434 reference spectra and 43
SuperSpectra representing 12 Trichophyton species, 7 Microsporum species, 1 Epidermatophyton
floccosum and 1 Arthroderma benhamiae. We followed the manufacturer’s recommendations for
protein extraction. We tested 50 patient isolates and 4 ATCC strains representing 4 Trichophyton
species, 2 Microsporum species and 1 E. floccosum. The isolates were tested after 5 day growth on
Sabouraud agar, mycosel agar and inhibitory mold agar at 30oC. The final identification of the isolates
was compared to phenotypic identification combined with sequencing of the internal transcribed spacer
ITS1. Acceptable identification by MALDI-TOF MS was defined as greater than 70% match to the
reference spectra. Results: All isolates were identified correctly to the genus level. The E. floccosum and
all Microsporum species were 100% correctly identified at the species level and results from all types of
media matched. Among the Tricophyton species, 5/5 (100%) T. rubrum isolates were correctly identified
and 19/20 (95%) T. tonsurans isolates were correctly identified. T. violaceum was not identified from the
5 day old culture due to low biomass. T. soudanese is not included in the database and it was
misidentified as T. rubrum. Sequencing is also unable to differentiate between the two. Modification of
our workflow for reporting dermatophyte cultures can result in faster speciation of isolates between 7-
14 days after receipt of cultures compared to 28 days using phenotypic identification and elimination of
biochemical testing for speciation of Trichophyton species. Conclusions: Using MALDI-TOF MS to identify
dermatophytes results in faster reporting of cultures. T. soudanese, frequently isolated by our patients,
needs to be added to the database to assist with identification.
Session Number: 399
Session Number: 399
Abstract Title:
Validation of A Real-Time Pcr Assay for the Detection of Candida Auris In Urine Matrix and Analysis of
Surveillance Samples
L. Leach, H. Schwab, S. Chaturvedi; Wadsworth Ctr., NYSDOH, Albany, NY
Abstract Body:
Background: Candida auris is an emerging multidrug-resistant yeast, which is causing invasive
healthcare-associated infection with multiple fatalities in the New York. Approximately 50% of the
clinical cases in the NY have been identified from blood culture, and other cases were identified from
various body sites including approximately 25% from urine. The precise mechanism leading to presence
of C. auris in urine is currently not clear but possible colonization might pose serious threat of
transmission in the healthcare-facilities. We have recently developed a real-time PCR assay for the
surveillance samples (J. Clin. Microbiol. doi: 10.1128/JCM.01223-17). In the present study, we have
extended this study to urine matrix for rapid identification. Method: A real-time PCR assay targeting the
ITS2 sequence of C. auris was validated in a urine matrix. DNA was extracted using an in-house
developed freeze/heat/bead beating protocol. The assay was first tested using a spiked urine matrix to
determine the assay’s performance. Following validation of the assay, 27 surveillance urine samples
were screened for C. auris. Results: The limit of detection of the real-time PCR for urine samples was 1 C.
auris CFU/PCR reaction. Real-Time PCR yielded positive results from 7 urine samples with 100% and 95%
clinical sensitivity and specificity when compared to culture results. C. auris DNA was detected in one
urine sample with negative culture results indicating the presence of dead or culture defective C. auris.
Conclusion: We report the validation of a real-time PCR assay for the detection of the emerging fungal
pathogen C. auris in a urine matrix. The results indicated that the assay is a powerful tool for the direct
detection of C. auris from urine. It demonstrated high analytical accuracy and precision. The accurate
and rapid screening of C. auris from urine of patients and colonized individuals would allow for effective
control and prevention of this multidrug-resistant deadly fungal pathogen in healthcare-facilities.
Session Number: 399
Session Number: 399
Abstract Title:
Species-Level Discrimination and Antifungal Susceptibility Profiles of Candida Parapsilosis Complex
Isolates Causing Fungemia
D. A. Wilson, G. W. Procop; Cleveland Clinic, Cleveland, OH
Abstract Body:
Background: Former studies suggested differentiating the members of the C. parapsilosis complex from
one another due to differences in antifungal susceptibility profiles. Differentiation was impractical until
the advent of MALDI-ToF mass spectrometry (MS), requiring DNA sequencing. This retrospective study
investigated the species of the C. parapsilosis complex responsible for fungemia and a review of the
antifungal susceptibility profiles. Methods and Materials: One hundred thirty seven (137), non-duplicate
clinical blood culture isolates identified as C. parapsilosis, collected from 2011 through 2017, were
analyzed on the Bruker Biotyper (Bruker Daltonics, FlexControl software v3.4). MS was performed by the
extended direct colony method (formic acid) or, if needed, by extraction (ethyl alcohol suspension
followed by formic acid/acetonitrile). Manufacturer recommended scores were used to classify results
as species (≥2.000). The reported antifungal susceptibilities (MICs) achieved using the Sensititre™ Yeast
One (ThermoFisher Scientific, Cleveland, OH) were analyzed. Results: 93.4% (128/137) of the yeast were
C. parapsilosis (CP), 2.2% (3/137) Candida orthopsilosis (CO) and 4.4% (6/137) were Candida
metapsilosis (CM). The MIC50 / MIC90 distribution for the three species and antifungal drugs tested is
shown (Table).<table class="AbstractTable" id="{5AD13922-FF50-4247-99C1-77BBCB3C9221}"><caption
rowspan="1" colspan="1"></td><td rowspan="1" colspan="3">Calculated and Estimated MIC50/ MIC90
*</td></tr><tr><td rowspan="1" colspan="1">Drug</td><td rowspan="1" colspan="1">CP
(N=128)</td><td rowspan="1" colspan="1">CO (N=3)</td><td rowspan="1" colspan="1">CM (
N=6)</td></tr><tr><td rowspan="1" colspan="1">Amphotericn B</td><td rowspan="1"
colspan="1">0.5 / 0.5</td><td rowspan="1" colspan="1">0.25/ 0.5</td><td rowspan="1"
colspan="1">0.25/0.25</td></tr><tr><td rowspan="1" colspan="1">Flucytosine</td><td rowspan="1"
colspan="1">0.25 / 0.5</td><td rowspan="1" colspan="1"><=0.06 / 1.0</td><td rowspan="1"
colspan="1"><=0.06/ <=0.06</td></tr><tr><td rowspan="1" colspan="1">Anidulofungin</td><td
rowspan="1" colspan="1">1.0 / 2.0</td><td rowspan="1" colspan="1">0.5 / 2.0</td><td rowspan="1"
colspan="1">0.06 / 0.25</td></tr><tr><td rowspan="1" colspan="1">Caspofungin</td><td rowspan="1"
colspan="1">0.25 / 0.5</td><td rowspan="1" colspan="1">0.25 / 0.5</td><td rowspan="1"
colspan="1">0.12 / 0.12</td></tr><tr><td rowspan="1" colspan="1">Micafungin</td><td rowspan="1"
colspan="1">0.25 / 0.5</td></tr><tr><td rowspan="1" colspan="1">FLluconazole</td><td rowspan="1"
colspan="1">1.0 / 1.0</td></tr><tr><td rowspan="1" colspan="1">Itraconazole</td><td rowspan="1"
colspan="1">0.03 / 0.06</td></tr><tr><td rowspan="1" colspan="1">Posaconazole</td><td
rowspan="1" colspan="1">0.03 / 0.06</td><td rowspan="1" colspan="1">0.06 / 0.06</td><td
rowspan="1" colspan="1">0.015 / 0.03</td></tr><tr><td rowspan="1"
colspan="1">Voriconazole</td><td rowspan="1" colspan="1">0.015 / 0.03</td><td rowspan="1"
colspan="1">0.015 / 0.03</td><td rowspan="1" colspan="1">0.015 / 0.03</td></tr></table> * The
MIC50/ MIC90 values are considered valid for C. parapsilosis since >100 isolates were available for
study, but estimated for C. metapsilosis and C. orthopsilosis, since limited isolates were recovered.
Conclusions: The vast majority of C. parapsilosis complex isolates responsible for fungemia were C.
parapsilosis, rather than C. metapsilosis or C. orthopsilosis. There were too few C. metapsilosis and C.
orthopsolisis isolates to draw definitive conclusions regarding susceptibilities. However, there were not
great differences between these species and C. parapsilosis in this study. Actual MIC data should be
used to guide antifungal therapy for patients with fungemia due to a member of the C. parapsilosis
complex.
Session Number: 399
Session Number: 399
Abstract Title:
Misidentification of Candida Auris Using A Commercial, Manual Biochemical Enzyme-Based Rapid
Identification System
M. Snayd, F. Dias, R. W. Ryan, D. Clout, D. Banach; Univ. of Connecticut Sch. of Med., Farmington, CT
Abstract Body:
Background: As a multi-drug resistant pathogenic yeast, Candida auris is an emerging public health
threat. Despite its capacity for healthcare-associated transmission, rapid, accurate identification remains
challenging. C. auris is misidentified as other Candida species and rare organisms using manual and
automated biochemical-based testing. The RapID Yeast Plus (Remel, USA) is a manual biochemical
enzyme-based system used to identify medically important yeast that relies on chromogenic
biochemical reactions and an electronic coded compendium for organism identification. We sought to
evaluate how this system performed in identifying C. auris. Methods: Ten reference strains of C. auris
were obtained and subcultured to Sabouraud Dextrose Agar in duplicate and incubated. Pure colonies
were isolated from agar and suspended and subjected to 18 biochemical tests through the RapID Yeast
Plus system. Based on the observed chromogenic changes, a numeric microcode was assigned to each
isolate. The microcode was imported into the electronic database for species identification. Speciation
results coincided with a probability level. A probability level listed as implicit, satisfactory, or adequate
was accepted. Each reference isolate was tested in duplicate. If there was disagreement, a 3rd test was
performed for adjudication. Results: Nine of ten reference isolates were misidentified as C. parapsilosis.
Identification of the isolates resulted with satisfactory probability levels ranging from 95.68% - 99.9%.
One was misidentified as C. tropicalis with questionable probability. Glucose utilization, the hydrolysis of
p-nitrophenyl-α-D-glucoside and the hydrolysis of proline-β-naphthylamide was demonstrated by all C.
auris isolates. Conclusions: C. auris may be misidentified as C. parapsilosis using the Remel RapID
system. Glucose utilization, the hydrolysis of p-nitrophenyl-α-D-glucoside, and the hydrolysis of proline-
β-naphthylamide should raise suspicion of a potential C. auris isolate. Laboratories that use the RapID
system should consider molecular testing or matrix-assisted laser desorption/ionization time-of-flight
(MALDI-TOF) mass spectrometry to pursue a definitive identification when C. parapsilosis is identified.
Session Number: 399
Session Number: 399
Abstract Title:
The Impact of Sequence-Based Identification of Infrequently Encountered Yeasts and Non-Sporulating
Molds from Clin. Specimens
S. Jean, C-A. D. Burnham; Washington Univ. in St. Louis Sch. of Med., Saint Louis, MO
Abstract Body:
Background: Fungi that are infrequently encountered in the clinical laboratory may cause opportunistic
infections in immunocompromised individuals. Conventional morphologic identification of fungi can fail
to identify uncommon or non-sporulating molds. Molecular methods, including sequencing, may permit
species-level identification of such fungi but are not available in all laboratory settings and results may
be challenging to interpret, especially when poorly characterized, environmental molds are identified.
Our objective was to determine the clinical impact of molecular identification in this setting. Methods:
We performed a retrospective review of all fungi referred for identification from Barnes-Jewish Hospital
(St. Louis, MO) from June 2013 to July 2017. Following IRB approval, patient demographic and anti-
infective therapy details were obtained from the medical record. Results: During the 4-year period of
review, 93 fungi were sent-out for molecular identification. Seventy-five (75) distinct fungal species
were identified, 53% of which have not been previously documented as pathogens in the literature. The
average time (+/- 2SD) to report for sequencing results was 26 (+/-18) days after the organism was first
observed in culture and 15 (+/-10) days after being sent-out for additional testing. 33 (35%) of the
organisms sent out for identification were preliminarily identified and reported to the genus or family-
level while the remaining isolates (60, 65%) had no preliminary identification reported while awaiting
sequencing results. Isolates with preliminary reports showed 94% “essential agreement” with final
molecular identification and were more likely to be identified as known or putative pathogens (73%).
Isolates without a preliminary identification based on morphology were frequently identified as species
not documented to be a human pathogen (57%, n=34) and antifungal therapy was not frequently
administered for this group (33/55% not treated). Based upon retrospective chart review, molecular
identification guided anti-infective therapy in only 3 (3%) of cases. Conclusions: These data indicate that
molecular identification of infrequently encountered fungi has limited diagnostic yield given the
prolonged turnaround time and lack of impact on anti-infective therapy.
Session Number: 399
Session Number: 399
Abstract Title:
Evaluation of Novel Isothermal Amplification Assay for Direct Detection of Candida Species from Blood
A. Zannini1, K. Chapin2, S. Geffert2; 1Rhode Island Hosp., Providence, RI, 2Rhode Island Hosp. and
Brown Alpert Med. Sch., Providence, RI
Abstract Body:
Background: Invasive fungal infections, including candidiasis result in hundreds of thousands of deaths
yearly world-wide. Current diagnostics lack sensitivity and specificity. An accurate, rapid diagnostic is
essential in improving outcomes. Tangen Biosciences, Inc. has developed a 2 component diagnostic
system that includes a whole blood processing device (LVC™, large volume concentrator) for up to 10 mL
whole blood with filter capture of fungal cells and a detection device (TangenDx™ System) that employs
real-time isothermal LAMP amplification with Stem primers. The system uses the Tangen Candida Blood
stream (BSI) Panel Assay Disk that contains 35 separate channels for discrete reactions including 2
process controls. The disk detects 5 species of Candida, including resistant C. glabrata and C. krusei by
flourescence. Preliminary Tangen data from 140 reactions (28 for each species) indicated sensitivities of
1-3 CFU/ML. This study objective was a performance proof of principle in a clinical lab setting assessing
the processing device, assay system, diagnostic test results, and laboratory work flow using blood
samples spiked with the 5 Candida species. Materials: Individual LVC™ devices were used to process 43,
7 ml whole blood samples with pathogen concentrations of 2-3 CFU/mL. Processed samples were run on
6 TangenDx Systems by 2 technologists. The system is a mobile 7.5” x 3.5” x 2.5” (187mm x 90mm x
64mm), 0.8kg, battery operated unit, with internal data analysis and Bluetooth connectivity. BSI Panel
Assay Disks were used for spiked samples and negative controls. Results: Candida species were detected
correctly 96.1% of the time; each pathogen concentration was 2-3 CFU/ML of whole blood. Sample
processing time was 5 -7 minutes. Time to positive results was 50 minutes, with negative confirmations
taking 70 minutes. 3 of 43 LVC collections required an additional wash, adding 2 minutes. There were no
instrument failures. Conclusions: Initial assessment of the TangenDx system showed high sensitivity for
detection of the major Candida species directly from blood. The small portable device, simple processing
and assay system design along with RT assay disk storage make this a viable and clinically welcomed
diagnostic.
Session Number: 399
Session Number: 399
Abstract Title:
Abstract Body:
scores.
Session Number: 399
Session Number: 399
Abstract Title:
Spectroscopy
Abstract Body:
Session Number: 399
Session Number: 399
Abstract Title:
Pneumocystis Jirovecii Exhalation in the Course of PneumocystisPneumonia Treatment
L. Pougnet1, A. Grall1, M-C. Moal1, R. Pougnet1, Y. Le Govic2, S. Negri1, G. Nevez1, S. Le Gal1;
1Unversity Hosp. Brest, Brest, France, 2Unversity Hosp. Angers, Brest, France
Abstract Body:
Background: We investigated longitudinal Pneumocystis jirovecii air exhalation by a patient developing
Pneumocystis Pneumonia (PCP) and efficiently treated with cotrimoxazole. The patient underwent
kidney transplantation in 2013 and developed PCP in March 2017 while he did not follow PCP
prophylaxis. P. jirovecii was detected in a bronchoalveolar lavage (BAL) sample using microscopy.
Treatment was started using cotrimoxazole (2,880 mg per day). Methods: Five air samples were
collected after treatment initiation during 5 consecutive days in patient's room at one meter from
patient’s head using the Coriolis® μ air sampler (Bertin Technologies, France). P. jirovecii burdens were
determined in the BAL and air samples using a qPCR assay amplifying the mtLSUrRNA gene. Moreover,
P. jirovecii genotyping in the BAL and air samples was performed by examining cytochrome b (CYB) and
mtLSUrRNA genes. Results: The P. jirovecii DNA load was evaluated at 2.97x106 copies/mL of native
sample in the BAL sample (2.97x104 copies/µL of extracted DNA). The P. jirovecii DNA loads were
evaluated at 1.18x107 copies/m3, 2.39x105 copies/m3, 4.48x103 copies/m3 in the first, second and
third samples respectively, and <1.3x103 copies/m3 in both fourth and fifth air samples. A CYB2 allele
was identified in the BAL and the first three air samples, whereas typing at this locus did not give
positive results in the last two air samples. MtLSUrRNA allele 4 was identified in the BAL and in the 5 air
samples. Thus, a perfect match of mtLSUrRNA and CYB genotypes in the BAL and the air samples was
observed consistently with the fact that P. jirovecii detected in air samples was from the patient’s source
and exhaled in his environment. Conclusions: Finally, our study shows that a sharp decrease of P.
jirovecii DNA load was observed between the first and the third air samples. PCP treatment dramatically
decreased P. jirovecii exhalation and supports maintaining preventive measures, which ever they may
be, over at least 5 days after PCP treatment initiation.
Session Number: 399
Session Number: 399
Abstract Title:
Changes in the Epidemiology of Pneumocystis Jirovecii Pneumonia in A Korean Tertiary-Care Hosp. over
A 15-Year-Period
H. Lee, S-H. Choi, J. Chang, S-H. Kim, S. Lee, M-N. Kim, H. Sung; Asan Med. Ctr., Seoul, Korea, Republic of
Abstract Body:
Background: Subsequent to the increasing use of immunosuppressant therapy since the early 1990s,
Pneumocystis jirovecii pneumonia (PcP) has emerged as a life-threatening condition in human
immunodeficiency virus (HIV)-negative patients. We investigated the change of epidemiological and
clinical characteristics, and impact of meteorological factors in patients with PcP. Methods: Data of 424
patients who had been newly diagnosed with PcP were retrospectively analyzed in a 2,700-bed Korean
tertiary care hospital between February 2003 and April 2017. The study included patients with
compatible clinical findings confirmed as PcP with a direct immunofluorescence assay using monoclonal
antibody clone 2G2 (Light Diagnostics™ Pneumocystis carinii DFA Kit; Millipore, Billerica, MA, USA) or an
immunocytochemistry assay using monoclonal antibody clone 3F6 (DAKO Corp., Carpinteria, CA, USA)
using respiratory specimens. Results: The annual average number of cases was increased from 12.2
(initial 5 years) to 42.2 (recent 5 years). The percentage of HIV patients among total PcP cases was
highest in the year of 2004 (66.7%) and rapidly decreased thereafter, reaching 8.4% in recent 5 years. In
most HIV-negative patients, hematologic malignancy (34.8%) and solid organ transplantation (32.9%)
were the major underlying conditions, and immunosuppressive therapies including corticosteroids
(342/362; 94.5%) and chemotherapy history (123/362; 34.0%) were associated with PcP infection (P <
0.001, P < 0.001, respectively). Mean temperature and rainfall were not significantly associated with the
incidence of PcP (R2 = 0.277, P = 0.215, n = 12; R2 = 0.308, P = 0.168, n = 12). Conclusion: Our results
indicate that the incidence of PcP has been increasing in immunocompromised patients for the reasons
other than HIV infection in recent years. These data show that seasonality and humidity do not influence
on fungal spore spreading in susceptible patients.<p><a
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Session Number: 399
Session Number: 399
Abstract Title:
Efficacy of Selective Media for the Isolation of Scedosporium Spp. in Patients with History of Cystic
Fibrosis
W. Seo, A. Mangahis, R. She; Keck Sch. of Med. of the Univ. of Southern California, Los Angeles, CA
Abstract Body:
Background Scedosporium and Lomentospora spp. can colonize previously damaged bronchopulmonary
tracts such as found in patients with a history of cystic fibrosis (CF) or in lung transplant recipients.
However, isolates of Scedosporium spp. may be difficult to recover in mixed cultures with molds like
Aspergillus spp. Studies suggest that the selective SceSel+ media may yield up to two-fold higher
recovery rates of Scedosporium and Lomentospora from CF respiratory cultures. Methods We
evaluated the efficacy of routinely adding SceSel+ (Hardy Diagnostics) to consecutive fungal cultures
routinely performed for adult CF and lung transplant recipients Sept.-Dec. 2017. Conventional media
consisted of Sabaraud dextrose agar (SDA), SDA with chloramphenicol, and Mycosel agar (Hardy). All
cultures were held for 4 wk at 30° C. We separately inoculated mold isolates to SceSel+ media and SDA
for comparison, including 8 S. apiospermum and 5 hyaline molds. Results Of 328 specimens
representing 227 patients, all S. apiospermum (n=4) and S. prolificans (n=2) isolated were recovered by
both conventional and SceSel+ media, but growth of commensal flora (n=6) and A. fumigatus (n=2) was
suppressed partially or completely by SceSel+. Multiple colony morphotypes were noted in 2 of the S.
apiospermum cases on SceSel+ but not SDA media. For the remaining 322 cultures, compared to
conventional media SceSel+ suppressed growth of commensal yeast or bacteria in 83 of 210 (39.5%)
cultures, 2/2 cultures with Cryptococcus neoformans, 17/17 with Aspergillus spp., 2/2 with Penicillium,
2/2 with Coccidioides immitis/posadasii, 2/2 with dematiaceous molds, and 2/2 with other hyaline
molds. SceSel+ media also showed no growth when inoculated with A. fumigatus, A. terreus,
Paecilomyces, Penicillium, or Acremonium spp. isolates. Of 8 S. apiospermum isolates planted on
SceSel+ and SDA, 5 had better growth on SDA, 2 had better growth on SceSel+, and 1 showed equal
growth after 5 days when colony diameters were compared. Conclusions SceSel+ is a highly selective
media for Scedosporium and related molds that was useful in isolating different colony morphotypes
from the same specimen. While we did not appreciate the increased rate of recovery found by other
studies, the overall prevalence of Scedosporium infections at our site among CF and lung transplant
patients remains low overall. Addition of this media to respiratory fungal cultures may not be indicated
in low prevalence areas, but more longitudinal studies are needed to evaluate the needs for a given
geographical area.
Session Number: 400
Session Number: 400
Session Title: CPHM09 - Diagnostic Veterinary Microbiology: Surveillance and Epidemiology of
Pathogens of Veterinary and Human Significance: Towards One Health
Abstract Title:
Application of Whole-Genome Sequencing for Investigation of Epidemiology, Virulence, and
Antimicrobial Resistance of Salmonella Enteritidis
O. Sahin, Z. Wu, A. Krull, T. Frana; Iowa State Univ., Ames, IA
Abstract Body:
Salmonella enterica serovar Enteritidis (SE) is a leading cause of human foodborne gastroenteritis
worldwide, and contaminated poultry egg is recognized as the primary source for zoonotic infections. SE
infection in chickens is typically asymptomatic without producing any clinical signs of disease. However,
during the time when the 2010 nationwide large shell egg outbreak (associated with over 1,600 cases of
human illness) was unfolding, Iowa State University Veterinary Diagnostic Laboratory (ISU VLD) received
several case submissions from laying-hen flocks across five farms in Iowa, including one of the two egg
farms that were implicated as the source of the human outbreak. A common history with these
submissions was elevated mortality in adult laying hens, which had postmortem signs of septicemia and
peritonitis. SE was isolated from the internal organs of all cases, which was found to have a PFGE pattern
indistinguishable from that of the human outbreak isolates. Detailed characterization of isolates
(comprising those from the dead birds, environment of the outbreak farm, and sporadic cases) including
genotyping by standard methods (e.g. PFGE, MLST, MLVA), virulence gene screen by PCR, and
antimicrobial susceptibility testing (AST) were then performed. Here, whole-genome sequencing (WGS)
of 42 SE isolates was conducted to evaluate its utility for various epidemiological analyses. Using WGS
single-nucleotide polymorphism (SNP) analysis, almost all isolates from the egg farm implicated in the
human outbreak (both from internal organs and the farm environment) were clustered closely together
along with the CDC human and shell egg isolates associated with the same human outbreak. Although
the bird septicemic isolates from other farms were placed together into a separate cluster, they
noticeably diverged from the outbreak isolates. Sporadic isolates from unrelated times and sources
formed distant clades to both the outbreak and septicemia isolates. In silico analyses found that WGS
results displayed 100% concordance with serotyping, AST, and MLST data. These findings show that
WGS is a high resolution typing platform for detection and traceback investigation of SE outbreaks, as
well as a viable tool for fast and accurate prediction of antimicrobial resistance profiles and serotypes of
SE.
Session Number: 400
Session Number: 400
Abstract Title:
Convergent Microbial Composition from Two Sympatric Dolphin Species: A Case Study of Bottlenose
Dolphins (Tursiops Truncatus) and Indo-Pacific Humpback Dolphins (Souca Chinensis) Living in Captivity
X. Wan1, R. Tian2, M. Kempher2, J. Zheng1, D. Wang1; 1Chinese Academy of Sci., Wuhan, China, 2Univ.
of Oklahoma, Norman, OK
Abstract Body:
Background: The mammal associated microbiome plays an important role in host defense against
pathogens and disease. Fundamental knowledge of a host microbiome is needed before understanding
its role in health and disease resistance. However, cetaceans are an ecologically and evolutionarily
unique group of aquatic mammals and their microbiome remains relatively unexplored. Methods: Here,
we analyzed the bacterial communities from two sympatric dolphin species, bottlenose dolphins
(Tursiops truncatus) and Indo-pacific humpback dolphins (Sousa chinensis) living in captivity, as well as
wild Indo-pacific humpback dolphin, using high-throughput 16S rRNA gene sequencing. Samples were
taken from two body sites known to be important lines of defense for pathogen infections (intestinal
and respiratory tract). Results: Comparisons of sympatric and allopatric dolphin species revealed that
two cohabitating dolphin species shared a more similar microbiome (both intestinal and respiratory
microbiome) than members of the same species across two habitats. Moreover, body sites (intestinal
and respiratory tract), intestinal region (i.e., foregut, midgut and rectum) and intestinal location (mucus
and content) all contribute to variation in the dolphin microbiome. We also detected the presence of
potential pathogens in sampled dolphins, such as Plesiomonas, Aeromonas and Streptococcus.
Conclusions: Our finding helps elucidate the factors shaping dolphin microbiome, which highlights the
stronger influence of habitat on microbiome composition than the host species. This finding expands our
understanding of the factors shaping microbial communities in threatened cetaceans and provides
baseline information for predicting disease-related alterations in host microbial communities. In future,
environmental (habitat) monitoring should be considered as an important part in cetacean health
management in both the wild and captivity.
Session Number: 400
Session Number: 400
Abstract Title:
Occurrence of Salmonella Spp. in Urban Wildlife of Central Mexico
A. M. Olvera-Ramírez, G. Arteaga-Salazar, C. A. López-González, L. Salas-Rosas, G. M. Nava-Morales, G.
Aguilar-Tipacamú; Univ. Autónoma de Querétaro., Querétaro, Mexico
Abstract Body:
Salmonella spp. is an agent implicated in human foodborne infections and resides in a variety of
different hosts. The urbanization process increases the contact between wildlife and humans, and
Salmonella transmission to humans may occur by direct contact with animals (domestic or wild). The
aim of this study was to investigate the prevalence of Salmonella spp. in urban wildlife in central Mexico.
Our study models were Virginia opossums (Didelphis virginiana) and rock squirrels (Otospermophilus
variegatus) in Queretaro, Mexico. Sampling period for opossums was August to December of 2016, and
February to April of 2017 for rock squirrels. We used Tomahawk traps (32”x10”x12”) to capture
opossums, and sardines as an attractant. The traps were active at night, checked and inactivated during
the first hours of the morning. In the case of rock squirrels, Sherman cages (3”x3.5”x9”) baited with a
mixture of oats, peanuts, sunflower seeds and peanut butter were used to capture them and remained
active during morning hours. After physical restrain, animals were anesthetized with Tiletamine
Zolazepam (Zoletil® 100) by intramuscular injection (15 mg/kg for opossums and 6 mg/kg for rock
squirrels). We monitored physiological constants (body temperature, heart rate and respiratory rate)
and measurements including weight and sex. We obtained feces, anal and salivary swabs from each
animal. After recovery, animals were released at the capture site. Fecal samples were kept in dry ice,
swabs were refrigerated, and taken to the laboratory of Veterinary Microbiology (UAQ). Salivary and
anal swabs were subject to pre-enrichment. Extraction of DNA was done either by a heat method or
Qiamp stool kit (Qiagen). Detection of Salmonella spp. was done by PCR, using primers to identify InvA
gen. We captured a total of forty opossums and one hundred squirrels. The prevalence of Salmonella
spp. in opossums was 57.5% and 94.5% in rock squirrels. We found no significant relationship between
the excretion of Salmonella spp. to weight, age, or sex in opossums or rock squirrels (P>0.05). We can
conclude that prevalence of Salmonella spp. is higher than 50% in the models used in urban wildlife.
Session Number: 400
Session Number: 400
Abstract Title:
Retrospective Surveillance of Escherichia Albertii in the E. coli Isolates from Pet Animals and Poultry in
the United States
A. Hinenoya1, X. Zeng1, B. Gillespie1, O. Sahin2, C. Logue3, S. Yamasaki4, J. Lin1; 1Univ. of Tennessee,
Knoxville, TN, 2Iowa State Univ., Ames, IA, 3Univ. of Georgia, Athens, GA, 4Osaka Prefecture Univ.,
Osaka, Japan
Abstract Body:
Background: Escherichia albertii is considered to be an emerging human enteropathogen and avian
pathogen with epidemic mortality. Some E. albertii strains produce Shiga toxin 2 with the potential to
cause hemorrhagic colitis and a life-threatening complication, hemolytic uremic syndrome in humans.
However, due to the close relatedness with other members of the Enterobacteriaceae, E. albertii is
often misidentified as Escherichia coli or Shigella boydii, resulting in an underestimation of E. albertii-
related infections and its impact as an emerging foodborne pathogen. In this study, to understand the
prevalence of E. albertii in the US animal system, we performed a retrospective screening for E. albertii
in E. coli strains isolated from pet animals and poultry in the US and elsewhere. Methods: A total of 200
E. coli clinical isolates obtained from feces, intestinal and extra-intestinal parts of pet animals (103
canine, 27 feline, 38 equine, 30 mustelid and 2 lagomorpha) from diverse areas (2015 to 2017), and 522
E. coli strains isolated from poultry production systems (2004 to 2015) in US and elsewhere consisting of
strains recovered from healthy and diseased birds were subjected to PCR analysis in this study. Template
DNA of each isolate was prepared by the boiling method and examined by two pairs of E. albertii-
specific PCR primers that specifically target E. albertii cytolethal distending toxin genes, and part of yejH
and yejK gene, respectively. In addition, an established multiplex PCR was performed for all isolates to
specifically differentiate E. albertii, E. coli and E. fergusonii. Results: All the 722 E. coli isolates were
negative for the PCR using the both E. albertii-specific PCR primer pairs. The multiplex PCR confirmed
that all the isolates were E. coli except for one turkey isolate that was determined to be E. fergusonii.
The genomic DNA from E. albertii strain LMG20976T was used as a positive control, which generated
three PCR products with sizes of 449, 846 and 393 bp by using the two pairs of E. albertii-specific PCR
primers and the multiplex PCR, respectively. Conclusions: E. albertii was not detected in the US pet and
poultry E. coli isolates examined in this study. A more extensive retrospective and prospective
surveillance of E. albertii in the E. coli from food animals and humans in the US is highly warranted.
Session Number: 400
Session Number: 400
Abstract Title:
Molecular Detection and Epidemiology of Anaplasma and Ehrlichia Species in Korean Native Goats
H. Seo; Animal and Plant Quarantine Agency, Gimcheon, Korea, Republic of
Abstract Body:
Background: Tick-borne disease has given a fatal damage to both human and animals and caused large
economic losses to livestock industry worldwide. Many epidemiological surveys of tick-borne disease
reported in many countries. In the Republic of Korea, serological and molecular surveillance were
carried out for the dog, cattle, horse and human. However there are no available data on the molecular
surveillance of Anaplasma and Ehrlichia spp. in Korean native goats. Therefore, the current situation was
investigated in this study through molecular survey of Rickettsia disease in goat collected in Ulsan from
2016 by polymerase chain reaction (PCR) and sequencing analysis of 16S rRNA gene. Method: A total of
452 goat blood samples were collected from 20 farms. Whole bloods DNA were extracted from the
samples using Maxwell® 16 Whole blood DNA Kit according to the manufacturer’s instructions. PCR
reaction was carried out by previously described methods. Nucleotide sequence homology searches
were analyzed by the National Center for Biotechnology Information BLAST network service and aligned
using the MegAlign software package (Windows version 7.1;DNA-STAR,USA). Results: A total of
50/452(11.06%) were positive by polymerase chain reaction for Anaplasma and Ehrilcha spp. Positive
samples carried out sequence analysis for the 16S rRNA gene. A total of 1/452(0.22%) were showed
100% degrees of similarity to Ehrlichia chaffeensis. A total of 10/452(2.21%) were showed 100% degrees
of similarity to Anaplasma spp. And a total of 39/452(8.63%) were showed 100% degrees of similarity to
A. bovis. Conclusions: In this study, a molecular surveillance was conducted to detect E.chaffeensis,
Anaplasma spp. and A. bovis. The positive rate of Anaplasma and Ehrilcha antigen in Ulsan goats was
shown with 11.06% showing the possibility of goat infection in this region. These findings suggest that
Korean native goats were easily exposed to Anaplasma and Ehrilcha spp.
Session Number: 400
Session Number: 400
Abstract Title:
Molecular Epidemiology of Emergent Pathogens Associated with Canine Infectious Respiratory Diseases
in the United States
G. Maboni1, A. Lorton2, R. Berghaus2, P. Bartlett2, I. Fernandez2, S. Sanchez2; 1Univ. of Georgia,
Athens, GA, 2Univ. of Georgia, ATHENS, GA
Abstract Body:
Canine infectious respiratory disease (CIRD) is an endemic syndrome with multiple viral or bacterial
pathogens being involved sequentially or synergistically to cause disease1. The clinical signs caused by
the different agents are similar, which makes differential diagnosis challenging2. There is limited
information about the prevalence of pathogens associated with CIRD in the United States. To attain new
insights into the disease epidemiology, we conducted surveillance using molecular methods to target
the main viral and bacterial agents associated with CIRD in respiratory samples. Additionally, we aimed
to develop a novel probe-based multiplex qPCR to simultaneously detect and differentiate two species
of Mycoplasma (M. canis and M. cynos). Nasal, oropharyngeal, tracheal swabs, and lung tissues from
clinically ill dogs (n=562) were processed at Athens Veterinary Diagnostic Laboratory (University of
Georgia, USA) between 2011 and 2017. Canine Adenovirus 2 (CAdV-2), Canine Distemper Virus (CDV),
Canine Parainfluenza Virus (CPIV), Bordetella bronchiseptica, Coronavirus, Influenza, Streptococcus equi
subsp. zooepidemicus, M. canis and M. cynos were detected by standard or Real-Time PCR. For the
development of the multiplex qPCR for M. cynos and M. canis, primers and probes were designed
targeting species-specific gene regions identified in the 16S/23S rRNA intergenic spacer region. Results
revealed that CPIV (29%), M. canis (23.6%) and M. cynos (24.5%) were the most commonly detected
pathogens followed by Influenza (H3N2) (11.2%), B. bronchiseptica (9%), Coronavirus (4.6%), CAV
(2.5%), CDV (2%) and S. equi subsp. zooepidemicus (0%). Nasal-pharyngeal and oropharyngeal swabs
had the highest percentage of positive results. Co-infections occurred in 46 specimens, which were
positive for 2 to 5 different CIRD agents. The probe-based multiplex qPCR was successfully developed
and specifically detected M. cynos or/and M. canis. In summary, while confirming that CPIV is one of the
main pathogens associated with CIRD, this study highlights the role of newly emerging bacteria, such as
M. canis and M. cynos, as well as the importance of co-infections. These results may aid veterinarians to
adopt strategies to improve empiric antibiotic selection to treat CIRD. Further analysis will elucidate the
role of co-infections in clinically ill and asymptomatic dogs. Our novel multiplex qPCR for M. canis and
M. cynos provides an efficient diagnosis alternative that will allow for accurate disease therapy and
control.
Session Number: 400
Session Number: 400
Abstract Title:
Serological Surveillance of Equine Piroplasmosis in Republic of Korea, 2017
H. Seo; Animal and Plant Quarantine Agency, Gimcheon, Korea, Republic of
Abstract Body:
Background: Equine piroplasmosis is caused by Babesia caballi and Theileria equi that infects equid
species, such as horses, mules, donkeys and zebras. These two protozoa are transmitted by tick of
genera Dermacentor, Hyalomma, and Rhipicephalus. Horse population has been increasing with racing
industry in the Republic of Korea (ROK). Equine piroplasmosis is one of the most important tick-borne
diseases in the racing industry worldwide. However, there is no available information on the disease in
ROK. Therefore, we conducted a serological survey of equine piroplasmosis in ROK from May to July in
2017. Method: We survey the antibody of equine piroplasmosis in sera collected from horse stable in 5
metropolitan cities and 8 provinces. Serum samples were tested anti-B. caballi, and anti-T. equi
antibodies, using competitive ELISA kits (VMRD, Pullman, USA). Results: A total of 926 serum samples
were tested for antibody detection of B. caballi, and T. equi using cELISA kits for each pathogen. There is
no B. caballi and/or T. equi antibody positive horse of 926 horses in 2017. Conclusions: In this study,
serological surveillance was conducted to detect the antibody to two pathgens of equine piroplasmosis.
B. caballi and T. equi were not detected in the tested samples this year. However, continuing climate
change, increasing the number and distribution of tick vectors, and increasing imported equid species
from equine piroplasmosis-occurred countries will enhance the risk of introduction of equine
piroplasmosis into ROK. Considering one health and environmental change, further studies are required
continuous monitoring of tick-borne equine piroplasmosis in ROK.
Session Number: 400
Session Number: 400
Abstract Title:
Prevalence and Molecular Characterization of Multiple Enteric Viruses in Dogs with Diarrhea from
Bangkok, Thailand
S. Saengchoowong, T. Jinato, S. Rattanaburi, S. Payungporn; Faculty of Med., Chulalongkorn Univeristy,
Bangkok, Thailand
Abstract Body:
Background: Canine parvovirus (CPV), canine distemper virus (CDV), and canine enteric coronavirus
(CCoV) are major causes of infectious diarrhea and mortality in dogs worldwide. Besides, newly
described canine kobuvirus (CKoV) and bocavirus (CBoV) have been to date detected in dogs with
gastroenteritis in many countries. However, there was no prior information regarding the occurrence of
these emerging viruses in Thailand. Therefore, the objective of this investigation was to study the
prevalence and molecular characterization of CPV, CDV, CCoV, CKoV, and CBoV collected in dogs with
diarrhea in Bangkok. Methods: In total, sixty rectal swab samples were obtained in dogs with diarrhea
from three animal hospitals located in Bangkok, Thailand. Viral nucleic acids were extracted using
GenUP™ Virus DNA/RNA Kit. After that, DNA of CPV and CBoV and cDNA of CDV, CCoV, and CKoV was
detected by conventional and semi-nested RT-PCR, respectively. After sequencing, all sequence identity
analyses were performed with the nucleotide and amino acid sequences aligned with the ClustalW
method using the BioEdit Sequence Alignment Editor. Neighbor-joining phylogenetic trees were built in
MEGA 7 with 1,000 bootstrap replicates. Results: Among sixty specimens, single infections were
predominant in dogs with diarrhea (40%), followed by double (18.33%), triple (11.67%), and quadruple
(1.67%) infections. However, 17 out of 60 (28.33%) was not detected any of the five examined viruses. In
single infections, CCoV was the predominantly found virus, accounted for 26.67%. The second most
common virus was CDV (8.33%), followed by single infections of CPV (3.33%) and CBoV (1.67%).
Although single CKoV infection was not examined, it was detected in multiple infections. For double
infections, the most frequently found viruses were CCoV co-infected with CDV at 10%, followed by
CCoV+CPV (5%), CPV+CDV (1.67%) and CCoV+CBoV (1.67%). For triple infections, CCoV and CPV were
frequently detected with CKoV and CDV at 5% and 3.3%, respectively. Moreover, CCoV+CKoV+CDV
infections were also found at 3.3%. In addition, one sample was positive for CPV+CDV+CKoV+CBoV
infection. Conclusions: This study discloses prevalence and molecular characterization of five viruses in
dogs with diarrhea in Bangkok, Thailand. Although single infections were frequently detected the most,
multiple infections should be considered for viral diarrhea. In addition, CCoV was the most common
found in single infections or co-infected with other enteric viruses.
Session Number: 400
Session Number: 400
Abstract Title:
Occurrence of Escherichia coli O157 of Publ. Hlth. Significance from Slaughtered Animals in Pakistan
A. Ahsan1, H. Irshad1, A. Ullah1, A. Bin Zahur2; 1Animal Sci. Inst., Islamabad,, Pakistan, 2Natl. Vet. Lab.,
Islamabad,, Pakistan
Abstract Body:
Aims: Shiga toxin-producing Escherichia coli O157 is considered an important food-borne pathogen of
zoonotic importance causing diarrhoea, haemorrhagic colitis (HC) and haemolytic uraemic syndrome
(HUS) in humans. Ruminants are considered important reservoir for E. coli O157. The aim of the study is
to find the occurrence of E. coli O157 in slaughtered animals in Pakistan. Methods and Results: In total
400 recto-anal mucosal swabs (RAMS) were collected from cattle (n=104), buffaloes (n=96), sheep
(n=75) and goats (n=125) slaughtered in two abattoirs of Islamabad/Rawalpindi, Pakistan. RAMS were
enriched in buffered peptone water at 37 °C for 24 hours and analysed for presence of E. coli O157 using
PCR. The samples positive for E. coli O157 were subjected to isolation using Sorbitol MacConkey agar
(SMAC) to differentiate between sorbitol fermenting and non-sorbitol fermenting isolates. The overall
prevalence of E. coli O157 was 0.5% (2/400). The prevalence of E. coli O157 in sheep and cattle was
1.33% and 0.96% respectively. All the samples from buffaloes and goats were found negative for E. coli
O157. Processing of two E. coli O157 positive samples resulted in one E. coli O157 isolate. This isolate
was analyzed for presence of virulence genes (stx1, stx2, eae and ehxA) using multiplex PCR. The results
of multiplex PCR indicated the presence of stx1 and stx2 genes in E. coli O157 isolate obtained from
sheep. Conclusions: The study indicated very low prevalence of E. coli O157 in slaughtered animals
however, it was a small scale study limited to small geographical area and further large scale studies are
required to investigate the prevalence of E. coli O157 in slaughtered animals in Pakistan.
Session Number: 400
Session Number: 400
Abstract Title:
Occurrence and Pathogenicity of Escherichia coli Isolates from Gastrointestinal Tract of Fresh Water
Fishes in Abuja, Nigeria
S. Mailafia, G. R. Okoh; Univ. of Abuja, Nigeria, Gwagwalada, Nigeria
Abstract Body:
Escherichia coli (E.coli) are bacterial micro flora of fresh water fishes. They form part of its complex
ecosystem and could be responsible for a variety of diseases in fishes, man and animals. A total of 220
samples were randomly collected from the Gastro Intestinal Tract (GIT) of fishes belonging to two
species of Clarias gariepinus (110) and Heterobranchus bidorsalis (110). The GIT contents were analyzed
by culture, microscopy, and biochemical testing. MicrobactTM 24E System identification kit (Oxoid,
London, UK) was used for the confirmation of the Escherichia coli (E.coli) isolates, which yielded an
overall isolation rate of 80(36.36%). On individual fish prevalence, Clarias gariepinus had higher
prevalence of 50(45.45%) while Heterobanchus bidorsalis had a lower prevalence rate of 30(27.27%).
Molecular typing using Polymerase Chain Reaction (PCR) confirmed that the isolates were E. coli but
none of them belonged to E. coli O157 serotype. Statistical analysis using X2 showed no significant
difference in prevalence of E. coli between the two species of fishes P> 0.05, df=2. Pathogenicity studies
was determined for one isolate using Lethal Dose Assay (LD50) and the titer was 10-3 ul/ml. Virulence
factors were determined for all isolates and the results revealed that 25.0% of the isolates produced
verotoxins while heamolysin production was indicated in about 14.7% isolates and cell surface
hydrophobicity was found in 50.0%. Serum resistance was found in 33.3% of the isolates and also,
gelatinase production was 33.3%. Our research clearly showed that E.coli isolates from Nigerian fishes
have very high pathogenic potential and that E.coli O157 was not responsible for the array of pathogenic
factors dissipated in our study. It is therefore suggested that necessary public health education and
awareness campaign should be intensified on the need for proper handling and preparation of fish
before consumption especially in our rural communities. More so, the Federal Government of Nigeria
needs to develop research centers for aggressive formulation of strategies useful in monitoring and
control of pathogenic E. coli from the human population.
Session Number: 400
Session Number: 400
Abstract Title:
Evaluation of A Commercial Real-Time Pcr Kit for the Detection of Mycobacterium Avium Subspecies
Paratuberculosis in Milk
A. Alajmi; Publ. Authority of Agriculture Affairs & Fish Resources (PAAF),, Kuwait, Kuwait
Abstract Body:
Mycobacterium avium subspecies paratuberculosis (MAP), the etiological agent of paratuberculosis, is
economically important to dairy operations because it affects ruminants such as cattle as the causative
agent of Johne's disease and it is also perhaps the causative agent of Crohn's disease. The many
different commercially produced tests for detecting MAP in different matrices have varied advantages,
disadvantages, and applications. In this work, the integrated VetMAX™ MAP Real-Time PCR kit was
evaluated using artificially spiked milk, reconstituted infant milk, and field milk samples, including a
unique transposon sequence; ISMAP02 was targeted to provide sensitive-specific results. The analytical
sensitivity of the assay was estimated using MAP type strain ATCC 19698 (DSM 44133) at 141.2 fg µL-1
with a detection probability of 100% based on 10-6 serial dilutions of MAP DNA and at 14 fg µL-1 a
detection probability of 66.6% based on a 10-7 dilution. The assay specificity was validated by testing
fifteen isolates of MAP, thirteen isolates of non-MAP Mycobacterium species and eight isolates of other
related bacterial non-Mycobacterium species. Six spiked experiments were performed using multiple 50
mL-1samples of both raw milk and reconstituted infant milk (BEBA®) that were spiked with tenfold serial
dilutions containing 100 to 105 MAP cells mL-1. However, three raw milk samples from a local farm and
three infant milk formula samples were tested independently. The detection probability in raw milk for
the samples containing 1.4 × 101 MAP cell 50 mL-1 were 16.6% and the detection probability for
reconstituted infant milk samples containing 1.7 × 101 MAP cell 50 mL-1 was 91.6%. The validity of using
the MAP real-time PCR kit to detect MAP in milk was supported by the study results.
Session Number: 400
Session Number: 400
Abstract Title:
Optimization & Application of Loop-Mediated Isothermal Amplification (Rt-Lamp) for Rapid Diagnosis of
Local Serotype of Foot-And-Mouth Disease Virus in Beef Cattle
K. Hanif1, A. Raza2, I. Raseed2, Z. Rafiq3; 1Animal Hlth.& Food Safety Lab., Fauji Meat Limited, Thatta,
Pakistan, 2Univ. of Agriculture, Faisalabad, Pakistan, 3Everfresh Farms, Sargodha, Pakistan
Abstract Body:
Background: FMD is highly infectious & contagious viral disease of cloven footed animals. This disease is
considered as a transboundary disease & characterized as a list of Type A diseases by OIE. The disease
causes severe economic loss with its impact on national & international trade of livestock & their
products. In Pakistan, various serological & molecular techniques are available for diagnosis of FMDV
but none of the method can be applied in field condition for rapid & less expensive detection of FMDV.
The current study was designed to optimize the novel RT-LAMP technique for rapid & accurate detection
of FMDV & its local serotypes. The outcomes of this study was enabled the field veterinarians &
technicians for early diagnosis of FMD in field conditions. Methods: A total 42 suspected field samples
(epithelial fluid) were collected from beef cattle farms located at District Hyderabad, Pakistan. Molecular
characterized three FMDV serotypes (A, O & Asia 1) were also collected from QOL Laboratory, Lahore as
reference serotypes. Suspected field samples & reference FMDV serotypes were subjected to RNA
extraction. For comparative study, RT-LAMP & RT-PCR were applied on RNA samples with certain
modifications in RT-LAMP targeting the 3D gene & VP1 for FMDV & its serotypes, respectively. Effect of
variation in RNA concentration (log 10 - log10-10), incubations time (45 min, 60 min & 75 min) &
temperatures (55 - 65 °C) were also studied. Results: The RT-LAMP amplified the target 3D gene using
specific primers at 64 °C for 60 min and 80 °C for 10 min. RT-LAMP also amplified the target VP1 gene by
using serotype specific primers at 62 °C for 60 min and 80 °C for 10 min. Amplified RT-LAMP product was
visualized through the change in color & further confirmed through agarose gel electrophoresis. RT-
LAMP detected upto 10-4 dilution for serotype O, A & Asia 1. A total of 25 samples out of 42 were found
positive by RT-LAMP & RT-PCR and identified serotypes were A (n=12), O (n=09) & Asia-1 (n=04).
Conclusions: The proposed study showed that RT-LAMP sensitivity appears 100% which is comparable to
RT-PCR. Furthermore, the specificity of RT-LAMP is 100% with no cross reactivity. These outcomes make
RT-LAMP a simple, deployable & cost effective technique for timely detection of FMDV in field
conditions. RT-LAMP plays an effective role in control & eradication programs of FMD in Pakistan &
uplifting the economy of the country.
Session Number: 400
Session Number: 400
Abstract Title:
Isolation of A Novel Campylobacter Species from Bovine Genital Sample
O. Okwumabua; Midwestern Univ., Glendale, AZ
Abstract Body:
Campylobacter fetus subsp. venerealis is a cause of bovine venereal campylobacteriosis that has
socioeconomic, public health and international trade of animals and animal products implications.
Semen export guidelines require that bulls be Campylobacter fetus subsp. venerealis negative as such
accurate diagnostic results are crucial. A slow growing, curved, Gram-negative, microaerophilic
bacterium was isolated from bovine vaginal sample submitted for Campylobacter fetus subsp. venerealis
culture. Characterization of the isolate showed that like C. fetus subsp. venerealis the bacterium was
oxidase positive, cephalothin sensitive and it failed to grow in 1% glycine. Unlike C. fetus subsp.
venerealis it was negative by the polymerase chain reaction (PCR) for C. fetus but positive for C. fetus
subsp. Venerealis specific PCR. The matrix-assisted laser desorption ionization time of flight mass
spectrometry (MALDI-TOF MS) and cellular fatty acid methyl ester (FAME) analysis yielded no
identification. The isolate could not be sub-typed by pulsed field gel electrophoresis (PFGE) or the
arbitrary fragment length polymorphism (AFLP). The 16S rRNA gene sequence analysis indicated that the
organism formed a hitherto unknown sub-line within the genus Campylobacter. The whole genome
sequence analysis revealed that the organism has a 1,338 bp region that shared 100% identity with C.
fetus subsp. venerealis and contained the C. fetus subsp. Venerealis specific PCR primers sequences. The
16S and whole genome sequences displayed some affinity with Campylobacter mucosalis and
Campylobacter concisus. We conclude that the isolate represents a novel species of the genus
Campylobacter and that the sole use of PCR-based organism identification in clinical diagnostic
microbiology laboratories has negative implications if not interpreted carefully.
Session Number: 400
Session Number: 400
Abstract Title:
Investigation of A Mesophilic A. Salmonicida Strain Isolated from An Unsuspected Host: the Migratory
Bird Pied Avocet
A. Bernatchez1, A. Vincent1, J. Frey2, S. Charette1; 1Université Laval, Québec, QC, Canada, 2Univ. of
Bern, Bern, Switzerland
Abstract Body:
Aeromonas salmonicida is a Gram-negative bacterium, which is ubiquitous in fresh water. It is the
causative agent of furunculosis, a systemic disease that affects salmonids in the wild and mainly in
aquaculture. Although the species was considered psychrophilic for many years, a new mesophilic
subspecies, Aeromonas salmonicida subsp. pectinolytica, has been discovered in a polluted river in
2000. Additional mesophilic subspecies have then been sampled from various sources, such as Indian
market fishes, confirming that the salmonicida species is not exclusively psychrophilic. Phylogenomic
analysis of the different subspecies showed an important dichotomy and genetic variability between the
mesophilic and psychrophilic strains. Up to now, the hosts known so far for this bacterium are different
fish species. However, a new strain, JF2480, has recently been sampled from ill pied avocets, a migratory
bird that feeds in shallow lake and mud ponds. A phylogenomic analysis that contains several hundreds
of gene sequences suggests it is taxonomically distant from other known Aeromonas salmonicida
subspecies. The genome sequence that has been obtained from Illumina MiSeq confirms that the strain
possesses the key virulence genes that are present in the typical Aeromonas salmonicida psychrophilic
subspecies, with the exception of the gene encoding the type three secretion system (TTSS), a protein
complex used by virulent strains to inject toxins into the host cells, which is incomplete. Bacterial
virulence assays conducted on the surrogate host Dictyostelium discoideum amoeba confirmed that the
strain is virulent despite the lack of TTSS. Bacterial growth curves showed optimal growth at 37°C but
also capacity to grow at higher temperatures. Additionally, extensive phenotypic characterization tests
have been conducted using API®bioMerieux, demonstrating that A.sal JF2480 has a more active
metabolism at 37°C than 25°C. The discovery of this strain further demonstrates the extent of the
specie’s phylogenomic tree. This study also suggests that Aeromonas salmonicida can infect a wider
array of hosts than previously suspected and that we need to rethink the way we perceive Aeromonas
salmonicida’s natural environment.
Session Number: 400
Session Number: 400
Abstract Title:
Investigation on Bonobo Die-Off of Unknown Origin At the Sanctuary “Lola Ya Bonobo” At Kinshasa, Drc,
2015-2016
C. Kumakamba; Metabiota RDC, Kinshasa, Congo, Democratic Republic of the
Abstract Body:
Emergence of new or unknown pathogens in animals may present a threat to human health. Are
specially at risk: animal keepers and veterinarians, because of very close and regular contact with
animals. The PREDICT-2 project was requested by the team at "Lola ya Bonobo" to help identify the
pathogens causing the death of 4 females bonobos between April 2015 and May 2016. The animals
presented with neurologic symptoms without fever, and respiratory distress, with a rapid evolution to
death within four days. Biological specimens were collected by the Sanctuary staff from sick animals as
well as during necropsy. Suspicion of encephalitis (viral and bacterial encephalitis, Encephalomyocarditis
Virus – EMCV, toxoplasmosis, and oesophagostomum) was made and biochemistry, bacteriology,
parasitological, serology (IgM/IgG anti-toxoplasma and anti-trypanosome antibodies) and hem culture
analyses were performed at INRB. Pathological slides were prepared from brain tissues and sent to the
Mountain Gorilla Veterinary Project team in Goma for analysis. Oral swabs and tissue samples (lung,
liver, spleen, kidney) were tested for the following viral families at the PREDICT lab: Filovirus, Flavivirus,
Bunyavirus, Hantavirus, Paramyxovirus, Influenza virus, Coronavirus and Enterovirus including EMCV
virus and Herpes virus. All results were negative for all viral families tested except one. The positive
amplification for Herpes virus was obtained for DNA extracted from CSF (Cerebral-Spinal Fluid) sample
for one of two animals tested. This herpes virus strain is closely related to Pan paniscus
lymphocryptovirus, in the Gammaherpesvirinae subfamily found in bonobos and previously described as
a novel simian homologue of human Epstein-Barr virus. No human cases of disease in relation with these
animal events were reported. The die-off appeared to be contained to the bonobo sanctuary, were the
staff implemented hygienic measures in dormitories, cages, enclosures and feeding areas.
Session Number: 400
Session Number: 400
Abstract Title:
Esbls and Carbapenemase-Producing Escherichia coli in Companion Animals with Urinary Tract Infection
in Italy in 2017
A. Grassi1, L. Maniscalco1, L. Facchetti1, V. Baldo2, G. Alborali2, L. Conter3, S. Allibardi3; 1I-Vet
laboratory, Brescia, Italy, 2Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna,
Brescia, Italy, 3Copan, Brescia, Italy
Abstract Body:
Background: Infections with extended-spectrum β-lactamase (ESBL)-producing gram-negative bacteria
and carbapenem-resistant Enterobacteriaceae (CRE) have been reported worldwide. The emergence of
CRE has followed closely behind the rapid global dissemination of extra-intestinal pathogenic E. coli
(ExPEC), which are highly virulent and can exhibit resistance to important antimicrobials. Little is known
about the occurrence of ESBL-producing and CRE bacteria in companion animals. Therefore, we
characterized ESBL producing bacteria and the presence of CRE genes (KPC, NDM, VIM, IMP, and OXA-
48-like) in uropathogenic E. coli in companion animals by phylogroups analysis, antimicrobic
susceptibility and molecular diagnostic testing. Methods: 368 urinary samples of companion animals
(224 dogs, 144 cats) with suspected UTI were collected by cystocentesis between May and October
2017. Samples were plated on blood agar and MacConkey agar and incubated overnight at 37°,
presumptive identification of colonies were performed by biochemical tests and confirmed by MALDI-
TOF. The isolated E. coli were tested for antimicrobial susceptibility using Kirby-Bauer disc diffusion
method and bacterial genomic DNA of each isolate was extracted for identification of ESBL genes TEM,
SHV, CTX-M1, multi-locus sequence typing (MLST), mcr-1 gene (end point PCR), and analysis of E. coli
phylogroups. In addition, identification of CRE resistance genes (IMP-1, NDM, KPC, VIM, OXA-48) was
performed using Xpert CARBA-R assay (Cepheid) after eSwab (COPAN Italia) sampling from 23 isolated E.
coli. Results: 142 out of 368 samples (99 dogs, 43 cats) were positive for bacterial growth. Escherichia
coli were identified in 55/142 and 5/142 samples, respectively. E. coli isolated strains were phylogroups:
A (6/55), B1 (6/55), B2 (39/55), D (3/55) and F (2/55). No CARBA-R resistance genes were identified in
the selected samples. Tests for antimicrobial susceptibility revealed that Amikacin, Imipenem,
Gentamicin, Amoxicillin+Clavulanic Acid and Chloramphenicol can be useful in 85% of cases while only
55% of the isolated strains were sensible for fluoroquinolones and Trimethoprim-Sulfamethoxazole.
Conclusion: this study revealed that E.coli represents the main isolated bacteria from canine and feline
urinary samples confirming its role in UTIs in both human and animals. In this study antibiotic resistance
was found in more than 34% of cases and associated to ESBL and also mcr-1 positivity, for this reason
empiric treatment should be avoided.
Session Number: 400
Session Number: 400
Abstract Title:
MecA Genes Detection in Dogs in Kenya
E. S. Mitema, C. W. Njoroge; Publ. Hlth., Pharmacology and Toxicology, Nairobi, Kenya
Abstract Body:
Background: Staphylococcus spp are recognized skin commensals and opportunistic pathogens.
Treatment of infections due to Staphylococcus spp is becoming challenging due to the emergence of
antimicrobial resistance in both human and veterinary medicine. Methicillin resistant Staphylococcus
aureus (MRSA) and methicillin-resistant Staphylococcus pseudintermedius (MRSP) in dogs represent a
major challenge for small animal clinicians due to their characteristic as nosocomial pathogens and their
multidrug resistance phenotype. No data is available on MRSA in dogs in Kenya. The present study was
undertaken to investigate any possible presence of MRSA/MRSP in healthy and dogs with wounds in our
university small animal hospital using phenotypic and genotypic assays. The study also undertook Basic
Local Alignment Search Tool (BLAST) analysis of sequenced polymerase chain reaction (PCR) amplicons
of the resistance determinants. Methods: A total of 191 samples were obtained from the nose, buccal
mucosa, perianal area, wounds and ears of healthy and dogs with wounds on the skin. Pooled samples
of each dog were cultured on Mannitol salt agar (MSA), and thereafter catalase and tube coagulase tests
were performed. Antimicrobial susceptibility testing for positive isolates was done using disc diffusion
with oxacillin as surrogate for methicillin. Clinical and laboratory standards institute (CLSI) guidelines
were employed for susceptibility studies. Conventional PCR detection of nuc and mecA genes was done
to confirm MRSA using specific 16S rRNA gene of Staphylococcus Genus and primers. Resistant
amplicons were sequenced, analyzed and thereafter BLAST analysis undertaken. Sequenced resistance
genes wwere submitted to NCBI GenBank for assignment of accession numbers Results: Presumptive
Staphylococcus spp were isolated from 34% (65/191) of the samples. Coagulase positive Staphylococcus
spp (COPS) accounted for 43% (28/65) of the Staphylococcus spp isolated. Phenotypic resistance to
oxacillin was detected in 53.6% (15/28) of COPS. The PCR assay detected mecA gene as a 286 bp gene
fragment amplicon in only 2 of the 15 (7%) oxacillin resistant phenotypes. Conclusions: This study
reports the first case of MRSA in dogs in Kenya. The mecA genes detected in this study were associated
with mobile genetic elements (SCCmec) and were similar to human like isolates hence can be a public
health risk.
Session Number: 400
Session Number: 400
Abstract Title:
Reemergence of Novel Reassortant Avian Influenza H14n3 in Asia
N. Siddique; Natl. Agricultural Res. Ctr., Islamabad, Pakistan
Abstract Body:
Background: Since the isolation of original four isolates of H14 Avian Influenza Virus in central Asia
during 1982, these rare viruses had never been reported in any Asian country. Exceptionally, the four
H14 isolates recovered from Wisconsin and California during 2010-11. Pakistan homes a wide variety of
migratory birds flocking up from Siberia, Russia and Europe during winters through Indus flyway. In 2014
during Avian Influenza virus surveillance three H14N3 isolates recovered from Duck, Geese and Pigeon
from Live bird market in Pakistan. Methods: The clinical specimens (cloacal and tracheal swabs) were
collected and subjected to virological evaluation through embryonated SPF chicken egg inoculation.
Subtype identification was determined by HA, HI techniques along with RT-PCR and QRT-PCR procedures
using sequence specific primers and probes. The isolated H14N3 AIVs were subjected to whole genome
sequencing. The purified PCR products were directly used for cycle sequencing reactions in a genetic
analyzer. Phylogenetic analysis was conducted using MEGA -4. The gene sequences were submitted to
GenBank. Results: The whole genome sequencing revealed introduction of a unique reassortant
Eurasian- American avian strain into Pakistan. Phylogenetically HA gene shares 92% nucleotide sequence
identity with H14 from Wisconsin and California. The NA genes showed 97.7% homology with European
H11N3. The M, PB1, PB2 and PA genes were Asian in origin whereas NS and NP genes showed maximum
sequence identity with the Pakistani H3N1 and H4N6. Sequence analysis revealed a LP amino acid motif
(PDKQAK ) at HA cleavage site, avian-like receptor specificity (Q243, G241) and 7 N- linked glycosylation
sites while 2 glycosylation sites were lost due to S23I and N278G mutations.The amino acids known to
be associated with sensitivities to antiviral drugs were found conserved except the unique mutation
Ile27Val on matrix protein. Seroprevalence was negligible in various birds species Conclusions: These
observations suggested that these AIVs may persist in environmental stasis for long periods of time,
undetected by surveillance, until a time when agent, host, or environmental factors allow for
reemergence in susceptible hosts within a region. Some IAV strains are preferentially maintained within
individual migratory flyways and may be endemic to certain regions of the world The various point
mutations in these novel reassortant H14N3 and close relationship with American, European and Asian
AIVs also reflect the genetic diversity and intercontinental spread.
Session Number: 400
Session Number: 400
Abstract Title:
Study of Anthrax Focy in Georgia Along Azerbaijan-Georgian Boardes
M. Nikolaishvili, Lela Kerdzevadze,Marina Zakareishvili, Irma Beradze, Marina Donduashvili, Nino
Vepkhvadze, Maka Kokh; Lab. of the Ministry of Agriculture, Tbilisi, Georgia
Abstract Body:
Introduction: Anthrax is endemic in the South Caucasus region. There is a lack of understanding of the
regional epidemiology of the causative pathogen, Bacillus anthracis, and the trans-boundary factors
related to its persistence. There are several known B. anthracis foci in the Georgia-Azerbaijan border
region, although the exact location of the foci was unknown; The purpose of this study was to describe
anthrax foci along the Georgia-Azerbaijan border and to describe control measures in identified areas as
well as to increase the local and regional understanding of anthrax ecology, ecological risk factors, and
the genetic relationships and distribution among Georgian and Azerbaijani B. anthracis strains, a
regional study of the ecology of anthrax foci was conducted in Georgia and Azerbaijan. Methods:
According to the study scope, 1000 samples out of 344 foci were collected from Kakheti (Sighnaghi,
Sagarejo, Lagodekhi, Dedoplistskaro, Gurjaani) and Kvemo Kartli (Marneuli, Gardabani, Rustavi) regions
of Georgia and tested at the Laboratory of the Ministry of Agriculture using standard bacteriological and
molecular biology methods. Sites were selected based on historical data, included known B. anthracis
foci, animal burial sites, livestock grazing and holding areas around the selected villages along the
Georgia-Azerbaijan borders. Samples were collected from different sites. From 1 to 15 samples from one
foci. Soil samples were processed, heat-shocked, and cultured. Suspect colonies were tested using Gram
stain; gamma phage lysis; motility test; detection of the poly‐D‐glutamic acid capsule by DFA; and
detection of cell‐wall-associated polysaccharide by DFA. Cultures isolated through bacteriology tests
were confirmed by PCR assay. Results: Bacteriological tests revealed 15 positive samples from Kakheti
and 11 from Kvemo Kartli from which, cultures were isolated and confirmed by PCR. Conclusions: All the
positive samples were sent to NCDC Georgia for the molecular characterization of the pathogen. As a
result, some new anthrax foci were detected to the historical known ones and new maps will be done by
LMA/NFA based on the new data using GIS technologies. This study will assist in the formulation of
targeted public health interventions aimed at increasing knowledge of the disease within specific
demographics. Public health interventions and veterinarians will focus on livestock surveillance and
control in identified areas.
Session Number: 400
Session Number: 400
Abstract Title:
Glyphosate Inhibits Keratinolytic Activity of Bacillus Spp. Isolated from Wild Songbirds
S. E. George1, E. G. Urbanski1, M. M. Vroom2, L. Tuhela1; 1Ohio Wesleyan Univ., Delaware, OH, 2Univ.
of Florida, Merritt Island, FL
Abstract Body:
Glyphosate is a frequently-used herbicide both domestically and in the agricultural industry worldwide.
While the effect of glyphosate ingestion on bird health has been studied, less is known about the
potential impact of glyphosate on bacteria found on avian plumage. This study investigated the effect of
glyphosate on the degradation of bird feathers by keratinolytic plumage bacteria. To determine the
effect of glyphosate on bacterial feather degradation, two strains of Bacillus spp. isolated from the
plumage of wild songbirds captured in mist nets were tested. A disk-diffusion assay was used to
determine that 8.44 g/L of glyphosate inhibited the growth of these two isolates. Feather degradation
assays were subsequently performed on each of the Bacillus isolates to determine whether the
keratinolytic activity of the bacteria was affected. For each isolate, four tubes were prepared that
contained a basal salts medium and a white goose feather as the sole carbon and nitrogen source. To
two of those tubes, 8.44 g/L of glyphosate was added, then one tube with glyphosate and one tube
without glyphosate were inoculated with a Bacillus isolate. The other tubes with and without glyphosate
were left uninoculated as controls. All experiments were done in replicates of 10, and all tubes were
incubated at 37⁰C at 125 rpm for seven days. Aliquots from each tube were removed once every 24
hours. Feather degradation was determined by measuring the absorbance of the aliquots at 230 nm. At
this wavelength, an increase in the absorbance indicated an increase in oligopeptides resulting from the
degradation of keratin in the feathers. The results from uninoculated tubes indicated that glyphosate
alone did not damage feathers. However, glyphosate inhibited bacterial feather degradation from 86%
to 98% depending upon the isolate. These data suggest that exposure to glyphosate disrupts feather
degradation by Bacillus spp.
Session Number: 400
Session Number: 400
Abstract Title:
Penetrating Trauma and Bronchopneumonia in A Ram
O. Orakpoghenor, T. P. Markus; Ahmadu Bello Univ., Zaria, Nigeria
Abstract Body:
Background: This case report documents the gross and histopathological findings in the lungs of a ram
that died after a period of prolonged sternal recumbency and dyspnoea. The carcass of a nine months
old Yankasa ram was presented to the necropsy unit after three weeks of dysnoea, prolonged sternal
recumbency and treatment with tylosine, penicillin-streptomycin and griseofulvin prior to death.
Methods: The ram was observed grossly and histopathologically, and lung tissue was taken to the
microbiology laboratory. Results: An area of necrotic dermatitis on the sternum and penetrating wound
area with haemorrhages on the left lateral thoracic wall between 8th-10th intercostal spaces were
observed. On gross examination, hydrothorax, hydropericardium and hepatization of the lungs were
observed. On histopathology, fluid was accumulated in the alveoli, bronchioles and bronchi coupled
with thickening of the interalveolar walls. On microbial culture and isolation, Staphylococcus aureus was
isolated and identified. Conclusion: Based on these findings, a diagnosis of penetrating trauma and
bronchopneumonia were made. This is the first documented report of bronchopneumonia in Zaria,
Nigeria.
Session Number: 400
Session Number: 400
Abstract Title:
Isolation and Identification of New Salmonella From Calf
S. Zhang, C. Wang, S. Su, Q. Wan, H. Li, X. Xu, B. Pan; China Agricultural Univ., Beijing, China
Abstract Body:
Calf diarrhea is a common disease in calf rearing. In this study, a calf diarrhea incidence in a large-scale
dairy farm in Shanxi Province was investigated and diagnosed, and we have identified that it was
Salmonella diarrhea, through experimental diagnosis. According to the antibiotic susceptibility
tests,Salmonella was highly sensitive with amikacin, ceftriaxone, which were selected for clinical
treatment. At the same time, prevention and control measures of the disease were put forward, and
better control effect was achieved. This study initially provides a reference for the diagnosis and
treatment of salmonella of calf diarrhea. Key words: Calf diarrhea; Salmonella; Experiment;
Identification
Session Number: 400
Session Number: 400
Abstract Title:
Protective Effect of A Bioprocessed Polysaccharide from Lentinus Edodes Mycelia Cultures with Turmeric
against Salmonella Gallinarum
J. B. Lee, S. M. Wi, S. K. Kim, J. W. Yoon; Coll. of Vet. Med. & Inst. of Vet. Sci., Kangwon Natl. Univ.,
Chuncheon, Korea, Republic of
Abstract Body:
Our previous studies demonstrated that a bioprocessed polysaccharide (BPP) isolated from Lentinus
edodes mushroom mycelia cultures supplemented with black rice bran can protect mice against
Salmonella lipopolysaccharide-induced endotoxemia and reduce the mortality from S. Typhimurium
infection through upregulated Th1 immunity. Here we report that a BPP from L. edodes mycelia cultures
supplemented with turmeric (referred to as BPP-turmeric) alters chicken macrophage responses against
avian-adapted S. Gallinarum and protects chicks against a lethal challenge from S. Gallinarum. In vitro
analyses revealed that the water extract of BPP-turmeric (i) changed the protein expression/secretion
profile of S. Gallinarum, although it was not bactericidal, (ii) reduced the phagocytic activity of the
chicken-derived macrophage cell line HD-11 when infected with S. Gallinarum, and (iii) significantly
activated the transcription expression of interleukin (IL)-1β, IL-10, tumor necrosis factor-α (TNF-α), and
inducible nitric oxide synthase (iNOS), whereas repressed that of IL-4, IL-6, interferon (INF)-β, and INF-γ.
We also found that BPP-turmeric (0.1 g/kg feed) as a feed additive provided significant protection to 1-
day-old chicks infected with a lethal dose of S. Gallinarum. Collectively, these results imply that BPP-
turmeric contains biologically active component(s) that protect chicks against S. Gallinarum infection,
possibly by regulating macrophage immune responses. Further studies are needed to evaluate the
potential efficacy of BPP-turmeric as a livestock feed additive for the pre-harvest control of fowl typhoid
or food-borne salmonellosis.
Session Number: 400
Session Number: 400
Abstract Title:
The Predominance of Campylobacter Coli St1068 in Cattle in the United States
Z. Wu, Y. Tang, S. Muirhead, O. Sahin, N. Pavlovic, Q. Zhang; Iowa State Univ., Ames, IA
Abstract Body:
Campylobacter is a major foodborne pathogen and a leading cause of human gastroenteritis in the U.S.
and other countries. Campylobacter is commonly present in food-producing animals, and ruminants are
important reservoirs. To investigate the population structure of C. coli in cattle, we collected 3,184 fecal
samples from 35 feedlots in five different states, and isolated Campylobacter from the samples. In total,
356 C. coli isolates were obtained, and then 116 C. coli isolates were randomly chosen for genotyping
using pulsed-field gel electrophoresis (PFGE), multi-locus sequence typing (MLST), and whole genome
sequencing (WGS). Notably, the majority (75%) of the C. coli isolates belonged to a single sequence type
(ST-1068), and the isolates were highly resistant to tetracycline (73.3%), ciprofloxacin (83.6%), and
nalidixic acid (82.8%). To further determine the distribution of ST1068 in cattle, 1902 genomes of C. coli
were retrieved from NCBI database. The isolates were derived from pig (n=560), chicken (n=729), and
cattle (n=613) from 2015 to 2017 across the country. Interestingly, 71.1% of the cattle C. coli isolates in
the NCBI database belonged to ST1068, consistent with the result obtained from our surveillance study.
However, ST1068 only accounted for 0.6% and 5.4% of the isolates from chicken and pig, respectively.
Contrary to the C. coli isolates in cattle, there was not a predominant sequence type in the chicken and
pig isolates. Whole genome-based phylogenetic analysis showed that all the ST1068 isolates were
closely related, indicating an evolutionarily well-adapted genotype occupies the cattle niche in the
United States. These results reveal the wide dissemination and high-level antibiotic resistance of a
predominant C. coli clone in cattle in the U.S.
Session Number: 400
Session Number: 400
Abstract Title:
Transfer of Class 1 Integron-Mediated Antibiotic Resistant Genes from Salmonella of Fly Origin to
Susceptible E. coli and Salmonella Strains
J. Chen1, Y. Xu2; 1Univ. of Georgia, Griffin, GA, 2Ohio State Univ., Columbus, OH
Abstract Body:
Background: Foodborne bacterial pathogens, especially those that have developed resistance to
antibiotics are a serious threat to human health. Mobile DNA elements such as integrons are a critical
source of antibiotic-resistant genes. The purpose of this study was to determine the prevalence of
integrons in Salmonella strains isolated from flies captured on cattle farms, characterize the integron
structures, and determine the transferability of identified integrons.Methods: Presence of integrons and
integron gene cassettes were screened, using PCR, in 606 Salmonella isolated from flies captured on
cattle farms. DNA sequences of the identified gene cassettes were determined and compared against
those deposited in the NCBI database. Integron-positive Salmonella strains were subsequently
conjugated with an integron-negative Salmonella strain of fly origin and E. coli C600 on tryptic soy agar
(TSA) and selected farm samples including calf milk powder, 3 bovine feeds, drinking water, tail hair,
bedding sand, and bovine feces.Results: Two out of 606 (0.3%) isolated Salmonella, 438 and 442,
harbored class 1 integrons. Salmonella 438 carried a gene cassette with aadA7 (ca. 1.1 kb) and
Salmonella 442 had a cassette with drfA12-orfF-aadA2 (ca. 2.0 kb). The two integrons were transferrable
through conjugation on TSA to the Salmonella strain of fly origin and E. coli C600 at efficiencies ranging
from 1.47 × 10-6 to 4.25 × 10-5. However, only Salmonella 442 was able to transfer its integron to the
recipient cells on 3 out of the 8 farm samples, namely cattle hair, bedding sand, and drinking water, with
conjugation efficiencies ranging from 4.26 × 10-10 to 1.36 × 10-8. Antibiotic-resistant genes not carried
by integrons were co-transferred with integron-mediated antibiotic resistance genes.Conclusions: These
data revealed that Salmonella isolated from flies on cattle farms carried integrons which could
disseminate antibiotic resistance genes through horizontal gene transfer in the farm environments.
Session Number: 400
Session Number: 400
Abstract Title:
Performance of Three Commercially Available Automated Biochemical Testing Platforms for
Identification of Staphylococcus Delphini
T. Tekle1, S. Compton2, E. Burd3, B. Zimmer4, K. Carroll5, D. Bemis2, L. Westblade6; 1Johns Hopkins
Hosp., Baltimore, MD, 2Univ. of Tennessee Coll. of Vet. Med., Knoxville, TN, 3Emory Univ. Sch. of Med.,
Atlanta, GA, 4Beckman Coulter, Inc., West Sacramen
Abstract Body:
Background: The Staphylococcus intermedius group (SIG) is a collection of coagulase-positive
staphylococci composed of three species: S. delphini, S. intermedius sensu stricto and S.
pseudintermedius. S. delphini is further divided into two distinct phylogenetic clades referred to as
Group A and B. The SIG are notable animal pathogens and S. pseudintermedius has emerged as a rare
agent of human infection. The SIG may, due to their coagulase activity, be misidentified as
Staphylococcus aureus which is known to influence accurate interpretation of antimicrobial
susceptibility testing (AST) results for S. pseudintermedius, and may be of importance for S. delphini.
Therefore, the purpose of this study was to determine the performance of automated biochemical
platforms for the identification of S. delphini in the clinical microbiology laboratory. Methods: A
collection of 21 genetically confirmed S. delphini isolates (9 Group A and 12 Group B isolates) was
analyzed using three automated biochemical platforms: MicroScan (Beckman Coulter), Phoenix (Becton,
Dickinson and Company), and Vitek 2 (bioMérieux) per each manufacturer’s guidelines. Results: No
platform identified S. delphini to the species level due to its omission from the database of all three
systems. When considering high confidence/probability identification results for a member of the SIG or
S. aureus, MicroScan identified 52.4% (11/21) and 4.8% (1/21) of isolates as S. intermedius and S.
aureus, respectively; Phoenix identified 81% (17/21) and 9.5% (2/21) of isolates as S. intermedius and S.
aureus, respectively; and Vitek 2 identified 57.1% (12/21) and 28.6% (6/21) of isolates as S.
pseudintermedius and S. aureus, respectively. The remaining isolates were either low probability
identification results, identified as another species, or unidentified. Conclusions: This study suggests
accurate identification of S. delphini, which could be important for epidemiological or AST interpretation
purposes, is dependent upon molecular-based methods rather than biochemical methods. However,
these biochemical data could be appropriated to develop the databases of the various identification
platforms and ultimately enhance their performance for identifying SIG members to the species level.
Session Number: 401
Session Number: 401
Session Title: CPHM10 - Diagnostic Virology: The Global View of Virology
Abstract Title:
Seroprevalence and Correlates of Hepatitis B and C Viruses among Hiv Infected Children Attending An
Antiretroviral Therapy Clin. in Keffi, Nigeria
V. B. Oti, G. Pennap; Nasarawa State Univ., Keffi, Nigeria
Abstract Body:
Hepatitis B virus (HBV), Hepatitis C virus (HCV) and Human immunodeficiency virus (HIV) are common
blood-borne viral infections in Nigeria. The knowledge of their shared transmission routes has raised
alarm about the possibility and impact of coinfection with the viruses in pediatric patients. The study
determined the seroprevalence and correlates of these viruses among HIV infected pediatric patients
attending an antiretroviral therapy clinic in Keffi, Nigeria. Two hundred participants were screened for
HBsAg and anti-HCV antibodies using rapid test kits (ACON Laboratories Inc, USA). Informed written
consent was obtained from their parents/guardians and information on their socio-demographics and
exposure to some possible predictors were also obtained. A general prevalence of Hepatitis B and C
virus infections in the study population was 24.5%. The prevalence of HBV was 11.0% while HCV was
13.5% and no child was coinfected with all the 3 viruses. The viral infections were statistically associated
with having scarification marks (p < 0.05). However, gender, age, history of blood transfusion and HBV
vaccination were not statistically significant possible risk factors for HBV and HCV infections (p > 0.05),
although there were arithmetic difference among the risk factors. The HIV/HBV and HIV/HCV coinfection
prevalence of 11.0% and 13.5% respectively is an emerging problem that cannot be downplayed.
Routine screening of all HIV infected children for HBV and HCV and appropriate placement of patients
on antiretroviral treatment with coinfection according to the National guideline is suggested.
Session Number: 401
Session Number: 401
Abstract Title:
Seroprevalence of Torc Infections Amongst Pregnant Women in West French Guiana
J-F. Carod1, S. Beneteau1, G. Carles1, B. Seve1, P. Brousse2, R. Boukhari1, M-F. Manca1, A. Jolivet1;
1West French Guiana Hosp. Ctr., Saint-Laurent-du-Maroni, French Guiana, 2Cayenne Hosp. Ctr.,
Cayenne, French Guiana
Abstract Body:
Background: French Guiana is a territory of France in Latin America. It is remarkable for the diversity of
its community and its population growth that resemble developing countries. The follow-up of pregnant
women is one of the major activities of the regional hospital of Saint-Laurent-du-Maroni and strictly
follows the French Guidelines. The aim of this study was to evaluate the seroprevalence of TORC
infections among pregnant women in West French Guiana. Methods: During 2015-2017, a
seroepidemiological retrospective study was carried out in West French Guiana. The study included all
pregnant women between 13-46 years old that consulted or were admitted to the West French Guiana
Hospital Center at Saint-Laurent-du-Maroni, French Guiana. Serological evaluation for TORC s was
carried out by IgG Enzyme Linked Immunosorbant Assay (Architect, Abbott, USA). Acute infections were
documented with positive IgM antibodies with low IgG avidity. Data was analyzed using The R Project
for Statistical Computing (R-3.4.3).<br />Result:<br /><table class="AbstractTable" id="{0F6913AE-5557-
41D4-B829-D7BB8E9226C2}"><caption class="AbstractTableCaption"></caption><tr><td rowspan="1"
rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1"></td><td rowspan="1"
colspan="1">T. gondii</td><td rowspan="1" colspan="1">Rubella</td><td rowspan="1"
colspan="1">Cytomegalovirus</td></tr><tr><td rowspan="1" colspan="1">Number of requests</td><td
colspan="1">3289</td></tr><tr><td rowspan="1" colspan="1">Median age</td><td rowspan="1"
colspan="1">25</td></tr></table><br />The IgG sero-positivity to T. gondii, Rubella and CMV in this
population was 19,5%; 87% and 98,7% respectively. Acute toxoplasmosis, Rubella and CMV infection
were documented in 0.4%, 0.02% and 0.2% of women respectively. Seroprevalence from 2015-2017 was
stable except for Rubella which had a higher seroprevalence rate in 2017 (p<0.005). Seropositivity to T.
gondii, Rubella and CMV, varied significantly between age groups (p<0.05). Native Indian women
showed a significantly lower seroprevalence rate for T. gondii. Conclusion: Toxoplasmosis
seroprevalence among pregnant women in Western French Guiana remains below the seroprevalence in
France (36,7%) and Brasil (50-80%). Vaccination enabled French Guiana to reach high immunization
levels against Rubella. CMV exposure is highly prevalent among the population studied and infection
occurs in childhood. The same pattern is observed in South-America but not in Western-Europe
including France were rates are significantly lower (37.7%). Unfortunately, no current data is available
for Surinam where most of our patients originate.
Session Number: 401
Session Number: 401
Abstract Title:
Seoprevalence and Risk Factors of Human Immune Deficiency Virus (Hiv) and Hepatitis C Virus Infections
among Pregnant Women Attending Antenatal Care Clin. in Western Ethiopia
E. Ejeta1, R. Dabsu2; 1Jimma Univ., Jimma, Ethiopia, 2Wollega Univ., Nekemte, Ethiopia
Abstract Body:
Background: Human Immune Deficiency Virus and Hepatitis C virus (HCV) infections are global public
health challenge. Both HIV and HCV share common modes of transmission and also have serious effects
both on pregnant women and infants. However, there is limited information on sero-prevalence of HIV
and HCV infection among pregnant women in West part of Ethiopia. Hence, this study was conducted to
assess sero-prevalence and predictor factors of HIV and HCV infection among pregnant women
attending antenatal care in West Ethiopia. Methods: Institutional based cross-sectional study was
conducted from July to September, 2014 among 421 pregnant women’s attending antenatal care
services in purposively selected health facilities, East Wollega Zone, Ethiopia. The HCV and HIV sero-
markers were tested from aseptically collected serum samples. Hepatitis C virus was detected using an
enzyme linked immunosorbent assay (ELISA). HIV infection was also detected using the national HIV test
algorithms. The pretested-structured questionnaire were used to collect socio-demographic data, and
predictor factors of HIV and HCV infection. The collected data were analysis using SPSS version 20.
Results: The overall sero-prevalence for HCV and HIV among the study population was about 8.1%
(34/412; 95%CI: 5.7-10.7) and 1.0% (4/421; 95%CI: 0.2-2.0), respectively. The HCV-HIV co-infected
prevalence was 0.23% (1/421). Among HIV infected women, the prevalence of HCV infection was 25%.
The risk of HCV infection was significant low for urban residents (AOR=0.38, 95% CI: 0.16-0.90)
compared to their rural counterparts. Significantly low risk of HCV infection was also observed among
illiterate (AOR= 0.24, 95%CI: 0.06-0.85) population comparing to those attending higher level of
education. For HIV infection, the history of blood transfusion was significant increase the risk (AOR =
19.52, 95%CI: 1.80-150.6). Conclusions: The study showed that HCV and HIV infections are important
public health problem in the study area. All pregnant women need to be screened for both HCV and HIV
infection during antenatal care. Thus, HCV testing and diagnosis need to be included in the antennal
care services, and awareness creation is need on the prevention and mode of transmissions HCV and
HIV in general.
Session Number: 401
Session Number: 401
Abstract Title:
Occult Hepatits B Virus Infection among Blood Donors At Two Teaching Hosp. in Nigeria: Implications for
Blood Transfusion Occult Hepatits B Virus Infection among Blood Donors At Two Teaching Hosp. in
Nigeria: Implications for Blood Transfusion Occult Hepa
N. R. Agbakoba, I. A. Osuji, M. O. Ifeanyichukwu; Nnamdi Azikiwe Univ., Nnewi Campus, Nnewi, Nigeria
Abstract Body:
Background: Occult hepatitis B virus infection (OBI), characterized by detection of HBV DNA in the serum
or tissues of subjects who tested negative for HBsAg has become a challenge to blood safety. The aim of
this study is to determine the prevalence rate, viral markers, viral load and genotypes of occult HBV
infection among blood donors in Nnamdi Azikiwe University Teaching Hospital, Nnewi (NAUTH) and
University of Abuja Teaching Hospital (UATH) - both in Nigeria. Methods: Sera from 212 healthy blood
donors (108 and 104 from UATH and NAUTH respectively) were re-tested for HBsAg, HIV, Syphilis
antibodies and anti-HCV using 4th generation Enzyme Linked Immunosorbent Assay (ELISA). One
hundred (100) samples (50 samples from each study site) that were seronegative for HBsAg, HIV, HCV
and Syphilis were examined for the presence of HBV-DNA by conventional Polymerase Chain Reaction
(PCR). HBV DNA (Viral load) was determined on some positive samples by Real Time PCR. Gene
sequencing was done to determine HBV genotypes. Results: Of the 100 seronegative blood donors,
14(14%) were confirmed as OBI. Of the 14 OBI samples, 12 (85.7%) were seropositive. and among the
seropositives, 7 (58.3%) were positive for anti-HBs, 3 (25%) for anti-HBc, and 2(16.7%) for both HBsAb
and HBcAb (P<0.0001). Three (3) out of the 14 OBI blood donors (21.4%) were positive for anti-HBc IgM
(p<0.05). None of the OBI samples was positive for HBeAg and anti-HBe markers. The mean viral load of
OBI blood donors was <100 IU/mL. DNA sequencing and Phylogenetic analysis showed that all OBI
isolates belong to Genotype E and most of the isolates have gene sequences similar to HBV isolates from
Sudan. Conclusions: There is a high prevalence of OBI among blood donors in these two Teaching
Hospitals. This study therefore recommends that blood donors in these Hospitals in particular and
Nigeria in general be screened for OBI by Nucleic Acid Testing (NAT). In addition, screening for HBV
markers (anti-HBc and anti-HBs) is also recommended. Implementation of these findings in blood
transfusion services will minimize transfusion transmissible HBV infection risk in Nigeria.
Session Number: 401
Session Number: 401
Abstract Title:
Molecular Detection of Hepatitis E Virus in Wild Caught Rodents in Lagos, Nigeria
S. A. Osanyinlusi1, O. B. Salu2, A. B. James2, R. M. Orenolu2, R. A. Anyanwu2, S. A. Omilabu2; 1Federal
Univ. Oye-Ekiti, Ekiti State, Nigeria, Oye Ekiti, Ekiti State, Nigeria, 2Coll. of Med., Univ. of Lagos, Idi-
Araba, Lagos, Nigeria
Abstract Body:
Background: Hepatitis E virus (HEV), an important cause of acute viral hepatitis globally has been
detected from the brown rats (Rattus norvegicus). However, HEV is yet to be reported among wild
rodents in Nigeria. To investigate the carriage of HEV by rodents found within human dwellings within
Ikotun, Alimosho Local Government Area of Lagos State, Nigeria. Materials and Methods: A cross-
sectional study comprising Rodents, brown rats (Rattus norvegicus) and Musk shrew (Crocidura
dolichura) captured within urban human habitation. Rodents were sacrificed by cervical dislocation at
the Virology Laboratory, College of Medicine of the University of Lagos. Viral RNA was extracted from
blood, liver, kidney, heart and lung tissues. Purified RNA amplification was by nested reverse
transcription polymerase chain reaction (RT-PCR) and amplicons detected by agarose gel
electrophoresis. Results: HEV RNA were detected from the following organs of R. norvegicus; blood
(50%), kidney (33%), lungs (42%), liver (33%), and heart (42%) with an overall prevalence rate of 40% (24
of 60). One sample of blood from C. dolichura was positive for HEV RNA. No significant difference in the
detection of HEV among the distinct tissue analyzed (P>0.05; α=5%). Conclusion: The high prevalence
(40%) HEV RNA detected in R. norvegicus, makes rodents an obvious target for further investigations of
their role in HEV transmission. Keywords: Hepatitis E Virus, Rodents, Reverse Transcriptase-Polymerase
Chain Reaction, Zoonosis, Human habitation.
Session Number: 401
Session Number: 401
Abstract Title:
Tracking and Epidemiological Characterization of Acute Encephalitissyndrome in Uttar Pradesh, India
S. Shukla, S. Prakash, R. Garg, R. Kumar, A. Jain; King Georges Med. Univ., Lucknow, India
Abstract Body:
Background: Uttar Pradesh (UP), a northern state of India is endemic zone of acute Encephalitis
syndrome (AES) presenting as seasonal disease, of which aetiology remains undetermined in around
50% of cases. Recently, Scrub typhus has been discussed as an important aetiology of AES. Hence, this
study was conducted to establish scrub typhus as aetiology of AES in UP. Materials/methods: AES
suspects attending virology laboratory, department of Microbiology, KGMU, U.P, India, during 1st Jan
2017 to 31st October 2017, consenting to participate and having sufficient volume of both serum and
CSF samples were enrolled. By using serial algorithm of AES testing, serum and CSF samples were tested
by ELISA and/or RT PCR for Japanese Encephalitis, Dengue, West Nile Virus, Chikungunya, Enterovirus,
Herpes Simplex virus, bacterial (N. meningitides, S. pneumoniae and H. Influenza), Scrub typhus and
Leptospira. To investigate acute CNS involvement in cases of scrub typhus, we further tested scrub
typhus DNA in CSF by RT PCR. Clinical and epidemiological characterization of these patients was done
through predesigned questionnaires. Genotype analysis of scrub strains was done by sequencing.
Results: Total 602cases were enrolled and tested for AES pathogens from different districts of UP. Out
which 179 (29.7%) patients were positive for any of the above listed pathogens. Out of total 602 , 315
(52.3%)patient s were positive for scrub typhus(309 IgM + and 6 DNA PCR + and 38 both+). Majority of
above AES tested pathogens were coinfected (103(57%)) with scrub typhus. Six cases were only
detected by RT PCR on CSF sample. Epidemiological study suggested a post monsoon hike in scrub
typhus cases. Majority of cases was from eastern UP. Molecular sequencing showed scrub typhus
circulating in our region was related to Vietnam, Cambodia and Thailand origin. Conclusions: Scrub
typhus is emerging as one of the major aetiology of AES in northern state of India. We emphasize scrub
antibody and RT PCR testing to be included in AES algorithm testing. This will aid clinician to initiate early
antimicrobial therapy against Scrub typhus encephalitis and reduce morbidity and mortalities of AES.
Session Number: 401
Session Number: 401
Abstract Title:
Is the Roof Cracking? Systematic Review and Meta-Analysis of Hiv, Hbv and Hcv Prevalence in Sudan
M. Suluiman1, M. Atif2, Y. Mustafa2; 1Elrazi Univ., Khartoum, Sudan, 2Univ. of Khartoum, Khartoum,
Sudan
Abstract Body:
Viral hepatitis constitutes a global health problem; previous studies have affirmed a considerable
morbidity and mortality from both acute infections and chronic complications. On the other hand,
Human Immunodeficiency Virus (HIV) infection is also of known burden. Determining prevalence
estimates of these viruses is crucial for establishing appropriate country specific strategies regarding
prevention, diagnosis, and containment. This systematic review was aimed to provide pooled
seroprevalence estimates of the three viruses in Sudan. Structured review of the literature was
conducted to obtain relevant studies published in both national and international databases. After
assessment of quality and bias in all proposed studies, 57 prevalence studies were included. Meta-
analysis was conducted for all studies as well as subgroup analysis was also approached. The total
sample size of participants in included studies providing HIV antibodies prevalence was 15,479. Based on
information retrieved from these studies, HIV prevalence ranged from 0 to 18.3% among different study
populations. However, pooled prevalence estimate for HIV antibodies was 1%. Kassala, Eastern Sudan
was the most endemic State (4.18%). The HBV reported seroprevalence rates ranged from 5.1 up to
26.81% among different populations and the overall pooled prevalence was 12.07%. For HCV antibodies;
2.74% was determined to be the pooled prevalence. Khartoum State was the most endemic State of
both HBV and HCV with seroprevalence of 12.69% and 6.78%, respectively. Based on the data reviewed
and synthesized, there is no evidence for an HIV endemic in the general population of Sudan. However,
both HBV and HCV seroprevalence rates are indicating otherwise. Reducing the overall burden of HIV,
HBV and HCV infections will require new measures and national strategies and the recognition of the
infections as one of the country’s priority issues
Session Number: 401
Session Number: 401
Abstract Title:
Molecular Epidemiology of Rsv B Strains Circulating in Children Less Than 5 Years in Pakistan
F. Aziz1, S. Urooj Kazmi2, S. Ali3, S. Asad Ali1, S. Hani Abidi1; 1Aga Khan Univ. Hosp., Karachi, Pakistan,
2Dadabhoy Inst. of Higher Ed., Karachi, Pakistan, 3Nazarbayev Sch. of Med. Nazarbayev Univ., Astana,
Kazakhstan
Abstract Body:
Background: Viruses are capable of causing acute respiratory infections and are most common cause of
morbidity and mortality in children worldwide. Globally, it is estimated that 33·1 million episodes of
human respiratory syncytial virus (RSV) resulted in 3·2 million hospital admissions and 59, 600 hospital
deaths in children younger than 5 years. The disease burden of RSV in low-income countries is poorly
studied, but available data indicates that the virus is responsible for a high proportion of childhood
acute respiratory tract infections in these parts of the world. The aim of the study was to elucidate the
circulating genotypes of RSV strains in children < 5 years admitted to a tertiary care hospital in Karachi,
Pakistan. Methods: A total of 1,121 children admitted with respiratory illness were recruited in this
study. Nasopharyngeal swabs were collected and real- time-PCR was performed. To genetically
characterize the RSV, targeting Matrix gene of RSV from real- time PCR positive samples were amplified
using nested rt-PCR and sequenced. The sequences were subsequently aligned with RSV genotype A and
B reference sequences, edited and then used to construct Maximum Likelihood phylogenetic tree using
MEGA 6.0 software. The global patterns of RSV analyzed utilizing publically available data from 12
countries and newly sequenced data generated from collected samples during the study. Results: Out of
the 1,121 samples, 226 (20%) were found positive for RSV using real- time PCR. The proportion of RSV
cases in various months varied from 2% to 46%. The cases started increasing in the month of July (14%)
reaching the peak (43%) in September. Phylogenetic analysis revealed that all the samples clustered
with genotype B reference sequences indicating genotype B to be the most prevalent strain in the
infected children. Majority of the sequence formed a single monophyletic cluster, indicating that most
of the infection was carried out by a founder virus sharing similar genetic characteristics. Our findings
also inferred the high diversity of circulating RSV B strains globally. Conclusions: The study data shows
the high occurrence of RSV genotype B in children in Pakistan. Genetic analysis indicated that the RSV
infection is carried out by phylogenetically similar genotype B strains evolving from a common ancestor.
This information can further be explored to understand the epidemic dynamics of RSV and may help in
designing strategies for effective management and prevention of RSV infection in children.
Session Number: 401
Session Number: 401
Abstract Title:
Canine Parvovirus Types 2a and 2c Detection from Peruvian Dogs with Suspected Parvoviral Enteritis
R. Quino, L. Luna, R. Rímac, R. Rosadio, L. Maturrano; Univ. Natl. Mayor de San Marcos, Lima, Peru
Abstract Body:
Canine parvovirus type 2 (CPV-2) has been reported worldwide as the main agent related to acute
hemorrhagic enteritis of high morbidity and variable mortality in puppies. The detection and
characterization of this virus is essential for understanding the etiology of this disease and to develop
control measures. To characterize the strains circulating in Peruvian dogs and to provide new insights
into the local diversity of CPV-2, rectal swabs from 39 puppies with clinical symptoms and with no
history of previous vaccinations were analyzed. Total DNA was extracted by fast boiling method, and
PCR and sequencing were performed using specific primers that amplify a 1316 bp fragment
correspondent to the VP2 gene of CPV-2. CPV-2 was detected in 62% of analyzed samples. The 1316 bp
sequencing of the PCR product using bidirectional sequencing was possible in 9 samples. They were
edited using BLAST program and BioEdit 7.1.3.0 software and joined using Contig Assembly Program
application. We identified 4 samples as type 2a and 5 samples as type 2c. A phylogenetic analysis of
both variants circulating in Peruvian dogs showed similarities to Equatorian and Uruguayan strains. This
work constitutes the first report about genetic characterization of CPV-2 in Peru.
Session Number: 401
Session Number: 401
Abstract Title:
Human Papilloma Virus Prevalence and Risk Factors Associated with Cervical Cancer in A Rural
Population of Salvadoran Women
T. Ascencio1, M. I. Giménez1, V. Valle1, C. Flores1, A. Marroquín2, R. Rodriguez2, W. Guerra2, P.
Pineda2; 1Dr. José Matías Delgado, La Libertad, El Salvador, 2Ministry of Hlth., Chalatenango, El
Salvador
Abstract Body:
Background: In El Salvador, cervical cancer caused by High-Risk Human Papilloma Virus infection (HR-
HPV), is the most frequent malignancy in the female population. Every year, 823 women are diagnosed
and 388 die from it. Its incidence, 25/100,000 women per year is one of the highest worldwide.
Although HPV infection is a necessary cause to develop cervical cancer, most women infected with the
virus do not develop it. This means that other cofactors are required for HPV infection to progress into
cancer. In developing countries, cytological-histological tests are routinely used to detect the presence
of HPV-caused precancerous lesions; however, they do not detect small, early lesions. Molecular tests,
such as Hybrid Capture 2 (hc2), maximize the sensitivity of cervical cancer screening. Methods: We
studied HPV prevalence using hc2, which is novel in El Salvador, to detect the presence of viral DNA in
cervical cells infected with one or more of 13 HR-HPV genotypes. Besides, the main risk factors
associated with cervical cancer in rural women were also studied. From each participant, an utero-
cervical sample was swabbed and placed in a tube containing transport medium for processing with hc2
at the Molecular Biology Laboratory of Dr. Jose Matias Delgado University. Also, participants provided all
of their demographic information, which was recorded. The statistical analysis was done applying Excel
2016. Results: 223 women coming from three socially and demographically similar small communities
were included. From all participants, 81% were exposed to firewood/tobacco smoke, 59% started sexual
activity at age 17 or younger, and 61% were exposed to 3 or more risk factors. 15% (33) of the
participants tested positive for HR-HPV. From them, 73% started sexual activity at age 17 or younger,
and 73% were exposed to 3 or more risk factors. Conclusions: The high exposure to several risk factors
that was found, could be promoting HPV infection and its progress into cancer. These results seem to
reveal a high vulnerability of this population, which could be due to their socio-economic status, their
lack of knowledge on the risk factors involved, and a limited access to modern diagnostic methods. This
research intends to show some of the benefits of solving the diagnostic hurdle: early counseling and
treatment for patients who tested positive for HR-HPV, and to underline the importance of educating
population on the risk factors involved in cervical cancer development.
Session Number: 401
Session Number: 401
Abstract Title:
Viral Causes of Acute Febrile Jaundice in Suspected Cases of Yellow Fever in the Democratic Republic of
Congo
S. Makiala-Mandanda1, F. Le Gal2, S. Ahuka-Mundeke1, J. L. Abbate3, N. Ngwaka-Matsung4, E. Pukuta-
Simbu1, N. Berthet5, E. M. Leroy3, P. Becquart3, J-J. Muyembe-Tamfum1; 1Inst. Natl. de Recherche
Biomédicale, Kinshasa, Congo, Democratic Republic of the, 2
Abstract Body:
Background: For the majority (>95%) of patients with acute febrile jaundice identified through yellow
fever surveillance in the Democratic Republic of Congo (DRC) that test negative for antibodies against
yellow fever virus, no etiological investigation has ever been carried out. The aim of this study was to
investigate for potential causes of this syndrome, including hepatitis virus, arbovirus and herpes virus in
the yellow fever negative cohort. Methods: Of 652 patients included in the yellow fever surveillance
program from January 2003 to January 2012, 498 were screened for IgM antibodies (Ab) against
hepatitis A virus (HAV) and hepatitis E virus (HEV) and for antigens and antibodies against hepatitis B
virus (HBV: Ag HBs and anti-HBc IgM), hepatitis C virus (HCV) and hepatitis D virus (HDV) using ELISA
techniques. Viral loads and genotypes were determined for HBV and HDV. Real time PCR was performed
for the detection of arboviruses (dengue, West Nile, Chikungunya, O’Nyong Nyong, Rift Valley fever, Zika
and yellow fever viruses) and herpes viruses (cytomegalovirus (CMV), herpes simplex virus (HSV), human
herpes virus 6 (HHV-6) and varicella-zoster virus (VZV)). Results: The average age of patients was 24
years. Hepatitis viruses were the most important causes of acute febrile jaundice identified in this
cohort. The seroprevalence was 16.7% for HAV, 24.6% HBV, 2.3% HCV and 10.4% for HEV. HDV was
detected in 26.1% of HBV-infected patients. The median viral loads were 4.16 x 105 IU/ml for HBV
(range: 769 to 9.82 x 109 IU/ml) and 1.4 x 106 UI/ml for HDV (range: 3.1 x 102 to 2.9 x 108 IU/ml).
Genotypes A, E and D of HBV and genotype 1 of HDV were identified. The frequency of herpes viruses
was 13.0% for CMV, 6.2% for HHV6, 2.4% for HSV, and 2.4% for VZV. Sixteen cases of dengue (serotypes
1 and 2) and two cases of Chikungunya were also found. In total, 64% of acute febrile jaundice cases that
tested negative for yellow fever virus indicated a different viral infection that went undiagnosed.
Conclusions: These results highlight the need to extend routine diagnosis in the yellow fever
surveillance. This study also allowed the detection of a previously-unreported outbreak of hepatitis E in
Province of Equateur in 2006.
Session Number: 401
Session Number: 401
Abstract Title:
Poor Performance of Serology in Detection of Hepatitis C Virus in Blood Donors in Nigeria
N. F. Isong1, E. A. Ochang2, R. A. Bolarinwa3, A. O. Aboderin3; 1Univ. of Uyo Teaching Hosp., Uyo,
Nigeria, 2Univ. of Calabar Teaching Hosp., Calabar, Nigeria, 3Obafemi Awolowo Univ.Teaching Hosp.
Complex, Ile-Ife, Nigeria
Abstract Body:
Background: Sub-Saharan Africa has the highest estimated prevalence of hepatitis C virus infections.
Detection and donor blood screening continues to depend on serology which may miss several cases
and allow further transmission. We evaluated the serological and molecular prevalence and HCV
genotypes among blood donors in a tertiary hospital in South-West Nigeria. Methods: Study was cross
sectional and approved by the Obafemi Awolowo University Teaching Hospital (OAUTH) Ethics and
Research Committee (IRB/IEC/0004553). After initial screening for fitness to donate blood, blood
specimens were collected from all consenting donors. Demographic data and risk evaluation was done
using interviewer-administered questionnaire. Serum specimens were screened for anti-HCV antibody
using rapid test (Micro point Diagnostics,Nantong, China) and HCV antibody ELISA (Diagnostic Bioprobes,
Milano, Italy). Plasma specimens were screened for HCVRNA using Nested Reverse Transcriptase
Polymerase Chain Reaction (RT-PCR). RNA was extraction was done with ZR viral RNA kit while 5′
untranslated region of viral isolates were amplified using One Taq One Step RT-PCR and Quick load
Master Mix (New England Bio Labs Inc. Massachusetts). HCV positive samples were genotyped by direct
gene sequencing. Results: A total of 205 donors were recruited over a three month period. Twenty four
(11.7%) had detectable HCVRNA while 13 (6.3%) and 15 (7.3%) donors were positive for anti HCV by
rapid test and ELISA respectively. There was a significant difference between HCV detection by
molecular test and anti-HCV ELISA (F=3.213, p =0.031) as well as between molecular test and rapid test
(F-4.222, p <0.01). All HCVRNA were genotype 1 with 1b in 20 (83.3%) and 1a in four (16.3%) donors.
Significant risk factors for HCV infection were previous surgical procedures (x2=14.913, p<0.001) and
sexually transmitted infection (x2=4.930, p =0.026). Conclusion: Every ninth intending donor in this
environment is HCV infected and the current practice of serologic screening alone is allowing continued
transmission of HCV to recipients of blood and blood products. A review of National Guideline is
urgently required.
Session Number: 401
Session Number: 401
Abstract Title:
Sociodemographic Characterization and Molecular Profiling of Hepatitis A Virus Circulating in Most
Populous State (Uttar Pradesh) of India
S. Prakash, S. Shukla, A. Jain; King Georges Med. Univ., Lucknow, India
Abstract Body:
Background: Hepatitis A is the most common form of acute viral hepatitis in the world. Major
geographical differences in endemicity of hepatitis A are closely related to hygienic and sanitary
conditions and other indicators of the level of socioeconomic development. The anti-hepatitis A virus
(HAV) seroprevalence rate is presently decreasing in many parts of the world. The present study was
aimed to carry out the epidemiology, molecular detection and phylogenetic analysis of hepatitis A virus
strains circulating in northern state of India. Methods: Patients with acute viral hepatitis were enrolled.
Serum samples were collected over a period of one year from June 2016 till May 2017. Informed and
written consents were obtained. Serum samples were tested for anti-IgM HAV antibodies. The
seropositive samples were analysed by realtime-PCR. Sequencing of the VP1 region and phylogenetic
analysis were done; the results were analysed together with patient and geographical data. Results:
Total 1615 patients were enrolled and serum samples were collected and tested. The Male:Female ratio
was 1.3:1 with mean age 24.31 years (range 0-83years; S.D. ±17.02). Among those, 128 (7.93%) were
positive for anti-HAV IgM antibodies. Of all seropositive samples 59 (46.09%) were positive for HAV RNA.
Genotyping sequencing of 10 representative strains was carried out and the circulating genotype in our
region was found to be IIIA. The nucleotide sequences showed homology among the strains.
Conclusions: Our results showed hepatitis A still a disease of young children with IIIA as a circulating
genotype in this region. The mutations at VP1 region warrant further analysis.<p><a
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Session Number: 401
Session Number: 401
Abstract Title:
Seroprevalence of Hepatitis B and Delta Viruses among Hiv Infected Population Attending Antiretroviral
Clin. in Selected Hlth. Facilities in Abuja Nigeria
I. M. Ifeorah1, S. A. Bakarey2, J. A. Adeniji2, N. F. Onyemelukwe1; 1Univ. of Nigeria Enugu Campus,
Enugu, Nigeria, 2Univ. of Ibadan, Ibadan, Nigeria
Abstract Body:
Background: Tri- infection with human immunodeficiency virus (HIV), hepatitis B virus (HBV), and
hepatitis D virus (HDV) is rare. There is limited data on the seroprevalence of HIV/HBV/HDV tri-infection
especially in Nigeria. The aim of this study was to determine the seroprevalences of HBsAg and HDV
among HIV infected individuals attending Anti-retroviral (ARV) clinics in Abuja, Nigeria Methods: In this
cohort study, blood samples were collected from 1102 (Male=450; Female= 652) age ranged <20 to
<u>></u>51years (Mean age= 34.0; S.D=11.5) consenting HIV infected population attending ARV clinics
at selected health facilities in Abuja, Nigeria between April and October, 2016. A well-structured
questionnaire was used to capture demographic information from the respondents. Enzyme Linked
Immunosorbent Assay (ELISA) was used to determine the seroprevalence of Hepatitis B surface antigen
and Anti HDV. The result was a interpreted according to manufacturer’s instruction and statistical data
were analyzed using SPSS software version 21 and Chi square (χ ‐2 ) test was used to determine
association and p < 0.05 was considered significant. Results: Overall seroprevalences of 10.3%, 7.1% and
0.7% for HBV, HBV/HDV and HIV/HBV/HDV were found among the study population respectively. The
infection rate (13.3%) peaked at age range 31-40 years for HBV (p=0.002), 50% at <20 years for
HBV/HDV (p=0.049) and 1.5% at 31-40years for HIV/HBV/HDV (p=0.202). By gender, the rate was higher
in male (10.9%, 10.2%, 1.1%) than female (9.8%, 4.9%, 0.5%) participants for HBV, HBV/HDV and
HIV/HBV/HDV infections respectively. However there was no significant association between infection
rate and gender. Conclusions: This study has established that HBV and HDV prevalence is still high in the
population studied and that the rate of Tri infection is low. We advocate for more robust control
measures for HBV control and by extension HDV in HIV population through screening and vaccination.
Session Number: 401
Session Number: 401
Abstract Title:
Aetiology of Hepatitis among Jaundiced Patients Presenting to A Tertiary Hosp. in Ghana
M. Owusu1, J. K. Bonney2, A. A. Annan3, G. Mawuli2, M. Mutocheluh3, J. Aryeequaye2, N. K. Adjei4, M.
Afihene5, K. Spangenberg4, J. Sylverken4, E. Owusu-Dabo3, C. Drosten6, Y. Adu-Sarkodie3; 1Kumasi Ctr.
for Collaborative Res. in Tropical Med., kumasi, Gh
Abstract Body:
Background: Viral hepatitis continues to play significant role in causing morbidity and mortality in sub-
Saharan Africa. Apart from the few population based studies available, not many have investigated the
burden of these viruses in jaundiced patients. Among the few studies, hepatitis E is the least studied
among jaundiced patients. This study was aimed at describing the frequency, distribution and risk of the
different hepatitis viruses among jaundiced patients reporting to the second largest teaching hospital in
Ghana Methods: From November, 2015 to April, 2017, a cross-sectional study was conducted among
jaundiced patients attending the Komfo Anokye Teaching Hospital. Between 3-5 ml of blood was
collected from each patient and screened for various causes of viral hepatitis using both serologic and
molecular-based assays. Environmental, socio-demographic and clinical information were also collected
from patients and others extracted from their medical records Results: In the 155 patients recruited,
hepatitis B was the most prevalent [54.2% (95% CI = 46.0% - 62.2%)] followed by hepatitis E [32.9% (95%
CI = 25.6 - 40.9%)]. Most cases of hepatitis E occurred as co-infections with hepatitis B (18%), with the
predominant clinical feature being hepatocellular carcinoma. Risk factor variable analysis showed
middle and older aged individuals were more at risk of hepatitis B exposure whereas younger age
groups (<18 years) were more at risk of hepatitis E virus infection Conclusions: Hepatitis viruses are still
important in the viral aetiology of jaundice in Ghana. Hepatitis B and hepatitis E co-infections could play
significant roles in causing severe disease. A more aggressive approach needs to be adopted in order to
reduce the morbidity and mortality associated with hepatitis causing viruses in Ghana and other
developing countries
Session Number: 401
Session Number: 401
Abstract Title:
Enterovirus D68: Distinct Strains Circulating in the United States, 2014-2017
V. L. Vilrane, J. Zhuge, W. Huang, A. Budhai, S. M. Nolan, J. T. Fallon, G. Wang; New York Med. Coll.,
Valhalla, NY
Abstract Body:
Background: Enterovirus D68 (EV-D68) infection has been associated with severe respiratory illness and
acute flaccid myelitis. Following a nationwide outbreak in the US in 2014, only limited EV-D68 detection
was reported in 2016 and no data are available to date on EV-D68 infection in 2017. The objectives of
this study were to monitor if EV-D68 continued circulating in 2017 and to compare the viral strains
recognized since 2014 at regional, national and global levels. Methods: Nasopharyngeal (NP) specimens
collected from 2013 through 2017 from patients in the Lower Hudson Valley, New York were examined
for Rhinovirus/Enterovirus (RhV/EV) by the FilmArray Respiratory Panel. Selected RhV/EV-positive and
negative NP specimens were analyzed using two EV-D68-specific rRT-PCR assays, Sanger sequencing and
a RNA-Seq-based metagenomic next-generation sequencing on the Illumina MiSeq platform. Also,
genomic sequences of all EV-D68 strains since 1962 were retrieved from the GenBank and analyzed for
strain genotypes (clades) using genome-based algorithms. Results: A total of 1,094 NP specimens from
2013 through 2017 were examined for EV-D68. EV-D68 was detected in 29.4%, 26.6% and 1.6% NP
samples collected in 2014 (94 of 320), 2016 (160 of 602) and 2017 (9 of 562), respectively, but not in
those collected in 2013 (n=25) or 2015 (n=199). Comparative genomic analysis confirmed that distinct
EV-D68 strains were circulating and caused outbreaks in the Lower Hudson Valley, New York in 2014
(subclades B1 and B2) and 2016 (subclade B3) with a relatively high viral load in patient specimens. In
contrast, only low levels of clade D strains were detected in 2017 by a laboratory-developed RT-PCR with
improved sensitivity and were confirmed by DNA sequencing. Bioinformatic analysis of 2,746 sequences
worldwide (1962-2017) revealed temporal and spatial diversity in EV-D68 population but a shared global
evolutionary trend. Conclusions: We report patients infected with a new EV-D68 clade D strain in the
Lower Hudson Valley, New York in 2017 and demonstrate the necessity of EV-D68 surveillance using
molecular assays that are capable of detecting low-level virus in circulation. The establishment of
distinct viral strains and variable levels of circulation provides essential information for surveillance,
diagnosis and control of EV-D68 infection in the US and worldwide.
Session Number: 401
Session Number: 401
Abstract Title:
Hepatitis C Virus (Hcv) and Human Immunodeficiency Virus (Hiv) Genotypes Circulate in Hiv/Hcv-
Coinfected Patients in Pakistan
A. Iqbal Khan, S. Khan; Dow Univ. of Hlth.Sci., Karachi, Pakistan
Abstract Body:
Background: Human immunodeficiency virus (HIV) and hepatitis C virus (HCV) are major public health
concerns. Because of shared routes of transmission, HIV-positive individuals are at risk of coinfection
with HCV infections. The prevalence rates of coinfection with HCV in HIV- patients have been variable
worldwide depending on the geographic regions, and the type of exposure. It is likely that certain
genotypes or genetic variants in this study the HIV and HCV genotype in coinfected and single infected
patients will be analyzed in Pakistani patients. In this study we investigate the HIV and HCV genotype in
co-infected patients. Methods: In this study total sample size 150 comprising of 25 HIV and HCV co-
infected cases and 70 HIV, 55 HCV single infected cases. Hepatitis C virus (HCV) RNA and Human
immunodeficiency virus (HIV) DNA extracted from patient Sample was amplified and after their
sequencing genotype was analyzed using bioinformatics tools for this study. Results:Blood samples of
150 subjects were analyzed. Total of 11(7.3 %) were <20 years, 82 (54.6%) were between 21-40 years
and 57 (38 %) were >40 years. There were 112 (74.7%) male and 38 (25.3%) female patients and total of
70 (46.6%) patients suffered from HIV mono infection, 55 (36.7%) HCV mono infection and 25(16.6%)
HIV and HCV co-infection. In HIV and HCV coinfection 52% of patient’s co circulate HIV-1 Subtype A1 and
HCV genotype 3a and 24 % of Patients was co circulate HIV-1 subtype C and HCV genotype 3a.
Conclusions: In the present study the most prevalent genotype was HIV-1 subtype A and HCV genotype
3a which co circulate in co infected patients. We also found HIV-1 subtype C in our population which is
new finding and most of our patients were male and of younger age.
Session Number: 401
Session Number: 401
Abstract Title:
Incidence of Transmitted Drug Resistance among Treatment NaïVe Hiv-1 Infected Patients in the Eastern
Cape of South Africa
T. O. Digban, B. C. Iweriebor, L. C. Obi, U. U. Nwodo, A. I. Okoh; Univ. of fort hare, Alice, South Africa
Abstract Body:
Background: Transmitted HIV drug resistance (TDR) remains a significant threat to individuals unexposed
to antiretroviral treatment. South Africa has the highest number of patients infected with HIV-1 and also
has the largest number of patients initiated into the antiretroviral programme globally. Although the
introduction of combined antiretroviral therapy (ART) has reduced the death related to HIV among
infected individuals, the emergence of drug resistance is gradually on the rise. There is a dearth of
information on the prevalence of transmitted drug resistance among newly diagnosed patients in the
Eastern Cape of South Africa. This study is aimed to determine the prevalence of drug resistance
mutations and the subtypes of HIV-1 among ART drug naive patients in the Eastern Cape of South Africa.
Methods: Viral RNA was extracted from the blood of 101 newly diagnosed HIV-1 patients attending
three HIV testing and counselling clinics from July 2016 to May 2017. Partial pol gene fragment were
amplified with specific primers by RT-PCR and Sanger sequencing. The generated sequences were edited
with the Geneious 10.2 bioinformatics, analysed phylogenetically for evolutionary relationship to all
representatives of the HIV-1 subtypes in the HIV Databases. Thereafter drug related resistance
mutations (DRMs) analysis was performed on all the derived sequences according to the 2009 WHO list
of surveillance drug resistance mutations by submitting the generated sequences to Stanford HIV drug
resistance database for query of drug resistance associated mutations. Phylogenetic analysis of the
partial pol gene showed that all sequences belonged to HIV-1 subtype C virus. Results: Four major PI
related mutations (I54V, V82A/L, L76V and L90M) were observed in seven patients while several other
minor and accessory PIs were also identified. A total of 11(25.0%) patients had NRTIs mutations while
NNRTIs were observed among 14(31.8%) patients. K103N, V106M and M184V were the most common
mutations identified in the drug naïve patients studied. Conclusion: These findings show high prevalence
of transmitted drug resistance among the newly infected drug naive individuals that can compromise
first line of therapy. Results suggest that HIV-1 subtype C is still driving the epidemic in Eastern Cape of
South Africa with the upsurge in the prevalence of transmitted drug resistance. However, more samples
from various parts of the province need to be accumulated and assayed to provide current information
on drug resistance from this province of South Africa.
Session Number: 401
Session Number: 401
Abstract Title:
Molecular Detection of Occult Hepatitis C Infection in Cryptogenic Liver Disease - A North Indian Tertiary
Care Ctr. Experience
S. Khullar, M. Brijwal, D. Shalimar, B. Nayak, L. Dar, A. Choudhary; All India Inst. of Med. Sci. (AIIMS),
New Delhi, India
Abstract Body:
Background: Occult Hepatitis C Infection (OCI) is the persistence of Hepatitis C Virus (HCV) in liver
without detectable virus in the blood by a sensitive molecular test. OCI is a potential source for HCV
reactivation in scenarios like HIV infection, immunosuppressive therapy etc. Due to ensuing damage to
the liver, it could lead to liver cirrhosis and hepatocellular carcinoma. Diagnosis of OCI is established by
demonstration of HCV RNA in the liver tissue (gold standard) by RT-PCR. The other samples that have
been studied include peripheral blood mononuclear cells (PBMCs) and ultracentrifuged plasma. There is
paucity of data on OCI both globally and from India. This study was planned to ascertain the prevalence
of OCI in cryptogenic liver disease (CrLD) in the Indian population. Methods: Thirty three patients with
CrLD (median age = 35 years) were enrolled over a period of one and a half years. Liver biopsy samples
were collected from all these patients. One portion of the biopsy was sent for histological evaluation
and the other was immersed in an RNA preserving solution (RNAlater). Disruption of liver tissue was
carried out using liquid nitrogen. PBMCs and plasma were separated from blood samples of 31 patients
by Ficoll-hypaque density gradient centrifugation. Plasma sample was ultracentrifuged on 10% sucrose
cushion at 100,000g for 17 hours at 4°C. All samples were tested by real time RT-PCR for HCV RNA using
a commercial kit. Results: HCV RNA was not detected in the liver tissues, PBMCs or ultracentrifuged
plasma samples of all 33 patients included in the study. Conclusions: Our work suggests that OCI is not a
frequently encountered entity amongst patients with cryptogenic liver disease in our population.
Nevertheless, it would be unjust to assume complete absence of OCI. Our findings can be attributed to
various factors. One important factor is that only 2/33 (6%) of our patients were seropositive anti-HCV
positive (these patients have higher incidence of OCI). Other factors include overall low seroprevalence
of HCV in our population, the less number of patients included in the study, small amount of liver tissue
available etc. The present study is the first study on OCI in cryptogenic liver disease from this part of the
world where testing of all the three samples (liver tissue, PBMCs and ultracentrifuged plasma) has been
thoroughly carried out. More such studies over a larger time frame with a larger number of patients are
needed to ascertain the actual scenario of OCI in India.
Session Number: 401
Session Number: 401
Abstract Title:
Prevalence & Molecular Epidemiology of Hcv & Hbv Infection in Sukkur Sindh, Pakistan
A. Qureshi, S. Khan; Dow Univ. of Hlth.Sci., Karachi, Pakistan
Abstract Body:
Background: Viral hepatitis is the major cause of serious liver diseases such as cirrhosis, Hepatocellular
carcinoma and liver failure. Hepatitis B virus (HBV) and Hepatitis C virus (HCV) shares the similar route of
transmission & can cause chronic infection which is the most common reason for the fetal liver diseases.
Globally 180 million peoples are infected with HCV & 350 million are infected with HBV. Pakistan in
among the highest carrier countries for HCV & HBV infection with the prevalence rate of 8% & 5%
respectively which is continuously boosting at alarming rate. As a result Pakistan is declared as a
cirrhotic state in international health circles. Several studies indicates that the rate of HCV & HBV
infection in much higher in rural areas which represents the 66% of the Pakistani population. Therefore
epidemiological studies are required to determine risk factors & actual prevalence in these areas.The
aim of our study is to determine the prevalence of HBV & HCV infection in Sukkur Sindh. Methods:Total
205 Blood samples were collected from the male & female participants & are screened for the HBV &
HCV by ICT/ELISA followed by PCR . Results are analyzed by using SPSS. Results: Out of 205 participants,
24 (11.7%) were positive for Anti-HCV in which 13 (6.3 %) were male and 11 (5.3 %) were females. On
the other hand 12 (5.8 %) were positive for HBsAg in which 9 (4.3%) were males and 3 (1.4 %) were
females. Whereas only 1 (0.4 %) was positive for both Anti-HCV & HBsAg.Out of these 24 Anti-HCV
positive samples 17 (8.2 %) were also positive for HCV by PCR in which 8 were males (3.9%) & 9 (4.3%)
were females & on the other hand out of 12 HBsAg positive samples 9 (4.3%) samples were also positive
for HBV by PCR in which 7 (3.4%) were males & 2 (0.9%) were females. Only 1 (0.4 %) sample were
positive for both HCV & HBV by PCR.Conclusions:Our results indicates very high prevalence of HCV &
HBV in this region. Which needs to be address immediately. Another interesting finding of this study is
that the frequency of these infections are more in males as compare to females which might be due to
the fact that males are more exposed to the high risk activities. The concern authorities should take
notice of the situation & should make strategies to reduce the burden of this diseases.
Session Number: 401
Session Number: 401
Abstract Title:
Assessment of Hcv Genotypes with Gene Flow Hybridization and its Comparison with Conventional Pcr
M. Uzair1, M. F. Ali1, R. Ghani2; 1Dadabhoy Inst. for Higher Ed., Karachi, Pakistan, 2Rubina Ghani
Molecular and Pathological labratory, Karachi, Pakistan
Abstract Body:
Background: Nucleotide sequence analysis of hepatitis C virus (HCV) strains showed substantial
variability leading to a classification into several genotypes and subtypes. The data correlating HCV
genotypes and subtypes with hepatitis C viremia levels, demographic characteristics and severity of liver
diseases are conflicting. Objective: the objectives of the present study were the knowledge of the HCV
genotypes and its comparison with Gene Flow -through Hybridization, cost effective technique in
Pakistan. Material and Methods: during the course of this study, HCV-RNA positive sera samples from
200 chronically infected patients were characterized by genotyping assay with conventional method
utilized type specific primers as well as using hybridization technique. Results: During our study we came
across the genotype 3a was the most prevalent (36%) followed with 3b (26%), 2a(12%), 3d (4%),
3c(3.5%), 1a(2.5%) 1c(2.5%), 4a(1.5%), 1b(1%), 1d(0.5%), 2b(0.5%), 2c(0.5%), 4(0.5%). Genotypes 3d, 1c,
1d and 2c were reported for the first time from Pakistan. Conclusion: Advances in the field of molecular
biology have provided rapid diagnostic tools that have reduced the turnaround times for detecting HCV
genotype by using “Flow-through” hybridization in Pakistan.
Session Number: 401
Session Number: 401
Abstract Title:
Perception and Use of Influenza Antigen and Molecular Testing
N. Anderson, S. Lawrence; Washington Univ. in Saint Louis Sch. of Med., Saint Louis, MO
Abstract Body:
Background: Influenza antigen testing using rapid influenza diagnostic tests (RIDT’s) is common, though
has low sensitivity in comparison to molecular testing. During influenza season negative RIDT results
should not be used to rule out influenza in symptomatic patients. Methods: We administered a
voluntary electronic survey via email to providers involved with the care of patients with influenza at 2
academic hospitals, 6 community hospitals, and multiple outpatient clinics in the Saint Louis, Missouri
area (estimated 6,000 providers surveyed). Questions were designed to quantify the use of different
influenza diagnostics, assess knowledge of RIDT limitations, and assess barriers to molecular test
implementation. Statistically significant response differences were calculated using Fisher’s exact test
(p>0.05). Results: A total of 283 providers participated (38% community, 62% academic) resulting in an
estimated participation rate of 5%. RIDT’s were the primary diagnostic used by 32% of providers
(molecular testing primarily used by 55% of providers). Those who used RIDT’s primarily were more
likely to be from community settings (59%) vs. academic (16%) (p<0.05), and outpatient facilities (51%)
versus inpatient (16%) (p<0.05). Regarding interpretation, 18% of providers responded that a negative
RIDT rules out a diagnosis of influenza. Of those with this response (n=50), there was no significant
difference between the practice setting (community vs. academic, p=0.52), location (outpatient vs.
inpatient, p=1.0) or training (attending vs. resident, p=0.07). Providers rated RIDT testing as high
importance to their practice (4 or 5 on 5 point scale) 35% of the time while they rated molecular testing
as high importance 57% of the time. Turnaround time was given as the greatest benefit of RIDT’s (61%),
whereas sensitivity was given as the greatest limitation (51%). Sensitivity (46%) and specificity (26%)
were given as greatest benefits of molecular testing whereas turnaround time (42%) was given as the
greatest limitation. When asked what would be the longest time they would be willing to wait for a test
with 100% accuracy, 81% of participants would be satisfied with a result within 1 hour. Conclusions:
RIDT testing is commonly used in outpatient clinics and community settings. Misconceptions regarding
RIDT negative results exist and are not specific to setting or training level. The greatest limitation of
molecular testing was identified as turnaround time, though tests with result availability within 1 hour
would satisfy most providers.
Session Number: 402
Session Number: 402
Session Title: CPHM13 - Laboratory Informatics
Abstract Title:
Evaluation of the Impact of Pooling Data from Multiple Institutions When Generating Antibiograms
Using the Lab. R-Based Analytics (Larana) Program
W. Lainhart1, M. Nowak2, C-A. D. Burnham1; 1Washington Univ. Sch. of Med. in St. Louis, Saint Louis,
MO, 2Emory Univ., Atlanta, GA
Abstract Body:
Background: Clinical microbiology laboratories are required to generate an antibiogram annually. The
objective of this study is to evaluate the impact of population selection on antibiogram preparation.
Methods: The Laboratory R-based Analytics (LARANA) program is a method of rapid laboratory
information system (LIS) data extraction from Cerner Millennium (Kansas City) for automated generation
of antibiograms. Using LARANA, we evaluated the utility of unit-specific, inpatient only, hospital system-
wide, and pediatric only antibiograms for a clinical microbiology laboratory serving 5 hospitals, as well as
outpatient clinics. Comparisons of percent susceptibility for each species/antibiotic combination were
evaluated using Chi-squared statistics with Bonferroni adjusted p-values. Results: Evaluation of a unit-
specific (emergency department) vs. academic hospital (AH, all patients) antibiogram revealed one
statistically significant difference (170 comparisons, 21 species) for S. aureus and clindamycin
(padj<0.05). One difference (126 comparisons, 19 species) each was noted between the AH (all patients)
vs. AH (inpatient only) antibiograms in S. aureus and E. coli (both padj<0.05). Nine differences were
noted when comparing the AH (all patients) to the system-wide (all patients) antibiogram (220
comparisons, 31 species), with the majority seen in E. coli. The susceptibility of E. coli to 7 different
agents and P. aeruginosa to 2 agents was significantly lower in the AH antibiogram (all padj<0.05).
Finally, of 96 comparisons (15 species) between system-wide (all patients) and system-wide (pediatric
only) antibiograms, 18 differences in rates of susceptibility were noted across a variety of organisms,
including S. aureus (n=3), E. coli (n=6), P. aeruginosa (n=6), Achromobacter spp. (n=1), K. pneumoniae
(n=1), and P. mirabilis (n=1) (all padj<0.05). Across all antibiograms, rates of susceptibility were highest
in pediatrics, outpatients and community hospital patients, and lowest in AH inpatients. Conclusions:
The hospital system-wide antibiogram only differed from that of the AH in rates of susceptibility of two
organisms (both Gram negative). In system-wide analysis of all patients vs. pediatric patients,
differences in susceptibility were noted among both Gram positive and negative organisms, suggesting
that there is utility in generation of pediatric only antibiograms.
Session Number: 402
Session Number: 402
Abstract Title:
Prevalence of Meningitis/Encephalitis Pathogens Using Cloud Based Epidemiology Network
J. Dien Bard1, R. Selvarangan2, J. Jones3, J. Nawrocki3, FilmArray Trend Working Group, L. Meyers3;
1Children's Hosp. Los Angeles; Univ. of Southern California, Los Angeles, CA, 2Children's Mercy Hosptial;
Univ. of Missouri, Kansas City, MO, 3BioFire Dia
Abstract Body:
Background: Real-time monitoring of pathogens can identify and facilitate management of outbreaks.
The BioFire FilmArray® Meningitis/Encephalitis (ME) Panel offers detection of 14 viral, bacterial, and
fungal targets in total. Due to the low prevalence of certain bacterial and viral infections of the central
nervous system (CNS), current performance data ability of the FilmArray ME Panel is limited to the
clinical trial evaluations. This study represents the largest dataset of ME results from 12 institutions
currently offering the FilmArray ME Panel and reports an estimated prevalence of CNS pathogens within
the U.S. Methods: Using the FilmArray® Trend epidemiology tool, de-identified FilmArray ME Panel
results are directly sent to a secure, HIPAA-compliant, cloud-based database. Data were compiled
between January 2016 and January 2018 from 12 U.S. medical facilities, two of which were children’s
hospitals. To account for quality control and proficiency testing, samples with >2 targets detected were
omitted from the analysis. Results: A total of 4,831 cerebrospinal fluid (CSF) samples were tested on the
FilmArray ME Panel and up to 2 targets were detected in 859 (746) 15.4%= 1 target, (113) 2.3% = 2
targets) samples; a 13.8% (179/1291) positivity rate was observed at children’s hospitals compared to
19% (680/3540) in hospitals for adults and children. Viral pathogens represented the majority of
positives at 66.6% (650). All 14 targets in the FilmArray ME Panel were detected during this time period
with the most common pathogens being enterovirus (267), human herpesvirus 6 (HHV-6; 116) and S.
pneumoniae (95) for all institutions and children’s hospitals. Detection of less common bacterial
pathogens were also found including, N. meningitidis (8), S. agalactiae (40), L. monocytogenes (5), H.
influenzae (37), and E. coli K1 (10). A high number of other viral targets were also detected including,
HSV-1 and 2 (134), VZV (80) human parechovirus (28), and CMV (25). Surveillance of enterovirus
demonstrated a seasonal effect with predominance in the late summer/fall. Conclusions: The FilmArray
Trend software is a powerful epidemiological tool utilized by clinical laboratories to survey CNS
pathogens detected by the FilmArray ME Panel within their institution. This study demonstrates
widespread target detection and a high diagnostic yield achievable with molecular testing of CSF
specimens. Real-time monitoring of FilmArray ME Panel data provides valuable information on
prevalence and distribution of ME pathogens in different patient populations.
Session Number: 402
Session Number: 402
Abstract Title:
Semantic Lab. Data Interoperability: Encoding Microbiology Tests and Test Results Using Loinc® and
Snomed Ct® from An Ivd Manufacturer Perspective
M. Le Gall1, R. Vachon1, B. Cellière2, M. Dante3, F. Holder4, M. Mary2, M. Olléon2, X. Gansel1;
1bioMérieux, Grenoble, France, 2bioMérieux, La Balme-les-Grottes, France, 3bioMérieux, Saint Louis,
MO, 4bioMérieux, Marcy l'étoile, France
Abstract Body:
Background: Each IVD systems transmits test descriptions (Observations) and test results (Observation
Values) to the Lab. Information Systems that use distinct vocabularies pose interoperability challenges.
At the recent Public Workshop on ‘Promoting Semantic Interoperability of Laboratory Data’, the FDA
recommended that LOINC® be implemented by IVD manufacturers to describe Observations. On its side,
the IVD Industry Connectivity Consortium (IICC) defined and published the LIVD
(http://ivdconnectivity.org/livd/) format for digital publication of LOINC code mapping by IVD
manufacturers. This study aims to, from an IVD manufacturer perspective, demonstrate if LOINC® &
SNOMED CT® are capable of encoding our menu of microbiology IVD observations & observation values.
Methods: bioMérieux® Observations for VITEK2®, VITEK MS®, Etest® and VIDAS® were LOINC® encoded
in tight collaboration with the LOINC® team. Corresponding ordinal observation values and nominal
observation values (i.e. micro-organisms identifications) were mapped to SNOMED CT® using a
combination of string matching algorithm and manual curation of the hits involving expert biologists.
Results: In total 378 observation were considered for a LOINC mapping. Collaboration with the LOINC®
team allowed to encode 99% (375) of our observations, when appropriate LOINC® codes where created.
Remaining 3 observations are under analysis with the LOINC® team. Regarding observation values, we
were able to map to SNOMED CT * 59% (10) of 17 unique observation values. Each used for more than
one Observation * 89,6% (1333) of the 1487 nominal observation values. Those nominal observation
values (i.e. micro-organisms identifications) mapping covered 97,8% (1303) of consensual taxa (100% -
82 of genus; 97% - 1141 of species and 98% - 59 subspecies ) and far less for non-consensual taxa such
as ‘x OR y’ (22% - 11), variants (25% - 12) and groups (19% - 7). We also observed wide differences in
code coverage between bacteria, yeasts and filamentous fungus. Conclusions: Our study shows that
encoding Observation with LOINC® is achievable in cooperation from the LOINC group, especially when
new codes are required. Our LOINC® code mappings are now published on the companies’ Technical
Libraries using the LIVD format. We had more limited success identifying observation values in SNOMED
CT® . Most of missing code were for (i) non-consensual taxa (ii) filamentous fungus and yeasts. We are
hopeful that we can fill in the gaps through collaboration with SNOMED International.
Session Number: 402
Session Number: 402
Abstract Title:
Ethics in Clin. Metagenomics Data Sharing: High Correlation of Sample Collection Date and Patient
Admission Date in Microbiological Testing
A. Greninger, R. Shean; Univ. of Washington, Seattle, WA
Abstract Body:
Background: Infectious pathogens are known for their rapid evolutionary rates with new mutations
arising over days to weeks. The ability to rapidly recover whole genome sequences and analyze the
spread and evolution of viral pathogens using metagenomics and sample collection dates has lead to
interest in real-time tracking of infectious transmission and outbreaks. However, the level of temporal
resolution afforded by these analyses may conflict with definitions of what constitutes protected health
information (PHI) and privacy requirements for de-identification for publication and sharing of such
data. In the United States, dates and locations associated with patient care that provide greater
resolution than year or the first three digits of the zip code are generally considered patient identifiers;
admission and discharge dates are specifically named as identifiers in Department of Health and Human
Services guidance. Methods: To understand the degree to which one can impute admission dates from
specimen collection dates, we examined sample collection dates and patient admission dates associated
with more than 270,000 unique microbiological results from the University of Washington Laboratory
Medicine Department between 2010 and 2017. Cumulative distribution curves were plotted and
compared using two-sample Kolmogorov-Smirnov tests. Results: Across all positive microbiological tests,
the sample collection date exactly matched the patient admission date in 68.8% of tests. Collection
dates and admission dates were identical from emergency department and outpatient testing 86.7%
and 96.5% of the time, respectively, with more than 99% of tests collected within one day from the
patient admission date. Samples from female patients were significantly more likely to be collected
closer to admission date that those from male patients. Conclusions: We show that PHI-associated dates
such as admission date can confidently be imputed from deposited collection date. We suggest that
publicly depositing microbiological collection dates at greater resolution than the year may not meet
routine Safe Harbor-based requirements of patient de-identification. At the UW Virology lab, we have
made a decision to deposit only the collection year for the more than 700 viral genomes deposited in
NCBI to date. We recommend the use of Expert Determination to determine definitions of PHI for a
given study and/or direct patient consent if clinical laboratories or phylodynamic practitioners desire to
make higher resolution data available.
Session Number: 402
Session Number: 402
Abstract Title:
Can Standardized Comments for Culture Results in A Lab. Information Sys. Decrease Call Volume in the
Microbiology Laboratory?
H. M. Ruff, H. Poonawala, C. Sebastian, D. R. Peaper; Yale Sch. of Med., Yale New Haven Hosp., New
Haven, CT
Abstract Body:
Background: Phone calls to the microbiology laboratory can clarify important information for patient
care and offer opportunities to educate providers. However, they can interrupt workflow. Laboratory
information systems (LIS) can be used to communicate important information to clinicians. Methods:
Microbiology laboratory calls were logged from, and standard comments to address common call
subjects were created and implemented in the LIS. Calls were logged during the same time period the
subsequent year, and data pre- and post-implementation were analyzed.<table border="1"
cellpadding="1" class="DisplayTable" id="{66DE2D94-8A12-433D-973F-
035A4F354BEA}"><caption></caption><tr><td rowspan="1" colspan="1"></td><td rowspan="1"
rowspan="1" colspan="1"></td><td rowspan="1" colspan="1">Pre-Comment</td><td rowspan="1"
colspan="1">Post-Comment</td><td rowspan="1" colspan="1">Total</td></tr><tr><td rowspan="1"
colspan="1">Total</td><td rowspan="1" colspan="1">496 (100%)</td><td rowspan="1"
colspan="1">419 (100%)</td><td rowspan="1" colspan="1">915 (100%)</td></tr><tr><td rowspan="1"
colspan="1">Level of training (p < 0.05)</td><td rowspan="1" colspan="1"></td><td rowspan="1"
colspan="1">Fellow</td><td rowspan="1" colspan="1">154 (31.0%)</td><td rowspan="1"
colspan="1">92 (22.0%)</td><td rowspan="1" colspan="1">246 (26.9%)</td></tr><tr><td rowspan="1"
colspan="1">Attending</td><td rowspan="1" colspan="1">107 (21.6%)</td><td rowspan="1"
colspan="1">113 (27.0%)</td><td rowspan="1" colspan="1">220 (24.0%)</td></tr><tr><td
rowspan="1" colspan="1">Resident</td><td rowspan="1" colspan="1">114 (23.0%)</td><td
rowspan="1" colspan="1">81 (19.3%)</td><td rowspan="1" colspan="1">195 (21.3%)</td></tr><tr><td
rowspan="1" colspan="1">PharmD</td><td rowspan="1" colspan="1">61 (12.3%)</td><td rowspan="1"
colspan="1">Other Non-MD</td><td rowspan="1" colspan="1">60 (12.1%)</td><td rowspan="1"
colspan="1">Call Type (N.S.)</td><td rowspan="1" colspan="1"></td><td rowspan="1"
colspan="1">Sensitivity</td><td rowspan="1" colspan="1">253 (51.0%)</td><td rowspan="1"
rowspan="1" colspan="1">Mixed ID</td><td rowspan="1" colspan="1">95 (19.2%)</td><td
rowspan="1" colspan="1">Special Requests</td><td rowspan="1" colspan="1">59 (11.9%)</td><td
rowspan="1" colspan="1">Interpretation</td><td rowspan="1" colspan="1">40 (8.1%)</td><td
rowspan="1" colspan="1">Results not available</td><td rowspan="1" colspan="1">37 (7.5%)</td><td
rowspan="1" colspan="1">Order Guidance</td><td rowspan="1" colspan="1">12 (2.4%)</td><td
rowspan="1" colspan="1">Laboratory Action (p < 0.005)</td><td rowspan="1" colspan="1"></td><td
colspan="1">No action taken</td><td rowspan="1" colspan="1">201 (40.5%)</td><td rowspan="1"
rowspan="1" colspan="1">Additional data released</td><td rowspan="1" colspan="1">51
(10.3%)</td><td rowspan="1" colspan="1">70 (16.7%)</td><td rowspan="1" colspan="1">121
(13.2%)</td></tr><tr><td rowspan="1" colspan="1">Additional testing performed</td><td
rowspan="1" colspan="1">244 (49.2%)</td><td rowspan="1" colspan="1">217 (51.8%)</td><td
rowspan="1" colspan="1">461 (50.4%)</td></tr></table> Results: Call volume decreased from 496 to
419 after comment implementation (Table 1; 15.5% decrease, p < 0.01). There was a significant
difference in the level of training of individuals calling the laboratory (p < 0.005), but the nature of calls
pre- and post-implementation did not change significantly. There was a significant difference in
laboratory action with an increase in the release of previously generated laboratory data. Comments
that were specifically developed to address common antibiotic susceptibility questions related to known
intrinsic resistance and susceptibility patterns had no impact on call volume. Conclusion:
Implementation of standardized comments regarding laboratory workflow and culture results in LIS
systems can decrease call volume in the microbiology laboratory, but targeted comments were less
effective than anticipated.
Session Number: 402
Session Number: 402
Abstract Title:
Assessing Customer Satisfaction Using Web Tools : the Salvation of the Lab. Quality Manager
J-F. Carod; West French Guiana Hosp. Ctr., Saint-Laurent-du-Maroni, French Guiana
Abstract Body:
Background: In clinical laboratory service, customer satisfaction is a major component of a quality
management system, and a significant focus in the International Organization for Standardization (ISO)
standards. But a manual data collection is time consuming, a source of transcript and typing errors and
customer misunderstandings. The objective of the study is to assess clients’ and clinicians’ satisfaction
using online tools. Methods: Benefiting from a 8 years background, we summarized the main current
options available online and their characteristics. The tools have been divided into two groups : the
open ones who are entirely customizable from the data entry to the data extraction and graphic display.
They offer unlimited surveys and respondents but require a higher time investment to design the initial
template and analyze the data. They are mostly free. The closed tools have ready to use templates
which may midly be customized and do have options that may require a paid subcription. They decrease
the time dedicated to the survey creation, publishing and analyzing. Exporting data is often limited to
the paid versions, so does the Technical support. Moreover, some offer continuous evaluation.<br
/>Results:<br /><table class="AbstractTable" id="{F16141A7-B8C9-4611-9881-
8E47D00ED504}"><caption class="AbstractTableCaption"></caption><tr><td rowspan="1"
rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1">Non-exhaustive list</td><td
rowspan="1" colspan="1">Pro’s</td><td rowspan="1" colspan="1">Con’s</td><td rowspan="1"
colspan="1">Comments</td></tr><tr><td rowspan="1" colspan="1">Open tools</td><td rowspan="1"
rowspan="1" colspan="1">1.Excel Online spreadsheet survey</td><td rowspan="1"
colspan="1">Microsoft ecosystem</td><td rowspan="1" colspan="1">Security concerns (due to the
creation of a Microscoft Windows account)</td><td rowspan="1" colspan="1">An Excel Survey is a Web
form.</td></tr><tr><td rowspan="1" colspan="1">2.Google forms</td><td rowspan="1"
colspan="1">Free skip logic</td><td rowspan="1" colspan="1">Aesthetics</td><td rowspan="1"
colspan="1">More options offered</td></tr><tr><td rowspan="1" colspan="1">Closed tools</td><td
colspan="1"></td></tr><tr><td rowspan="1" colspan="1">3.Survey Monkeys</td><td rowspan="1"
colspan="1">The most used</td><td rowspan="1" colspan="1">Basic free version : not exporting
data.</td><td rowspan="1" colspan="1"></td></tr><tr><td rowspan="1"
colspan="1">4.Evalandgo.com</td><td rowspan="1" colspan="1">Ergonomics and intuition.</td><td
rowspan="1" colspan="1">Basic free version : not exporting data.</td><td rowspan="1"
colspan="1"></td></tr><tr><td rowspan="1" colspan="1">5.Typeform</td><td rowspan="1"
colspan="1">Aesthetic, ergonomics and intuition. The core plan offers Unlimited questions and answers
plus Data export.</td><td rowspan="1" colspan="1">The core plan doesn’t include logic jumps</td><td
rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1">6.Client Heartbeat</td><td
rowspan="1" colspan="1">The more advanced reporting features.</td><td rowspan="1"
colspan="1">No free version.</td><td rowspan="1" colspan="1">Warning alerts. Deep analytic
possibilities.</td></tr><tr><td rowspan="1" colspan="1">7.Zoho survey</td><td rowspan="1"
colspan="1">Unlimited surveys</td><td rowspan="1" colspan="1">Piping logic on paid versions</td><td
rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1">8.Survey Gizmo</td><td
rowspan="1" colspan="1">Unlimited surveys and questions. Attractive design.</td><td rowspan="1"
colspan="1">Paid version for more than 50 respondents</td><td rowspan="1"
colspan="1"></td></tr><tr><td rowspan="1" colspan="1">9.Survey planet</td><td rowspan="1"
colspan="1">Unlimited surveys, questions, Unlimited respondents</td><td rowspan="1"
colspan="1">Basic free version : not exporting data.</td><td rowspan="1"
colspan="1"></td></tr></table><br />Conclusion: Online tools have considerably saved time allowing to
spend the energy in designing and following up a precise and accurate Corrective Action Plan. They may
also be used for the assessment of the employee satisfaction rate.
Session Number: 402
Session Number: 402
Abstract Title:
Lab Competency Assessment Made Easy: Using Free Online Tools
J-F. Carod; West French Guiana Hosp. Ctr., Saint-Laurent-du-Maroni, French Guiana
Abstract Body:
Background: Competency assessment is used to ensure that the laboratory personnel are fulfilling their
duties as required by federal regulation or in compliance with International Standards. Competency
assessment, which includes at least six procedures as mentionned by CLIA, must be performed for
testing personnel for each test that the individual is approved by the laboratory director to perform. If
the on-the-bench job verification is mandatory, many items can be verified, for both primary or re-
assessment, using free online tools. Thoses tools offer the following benefits : 1 / traceability 2 /
objectivity 3 / evidence-prooves recording 4 / efficiency 5 / time saving 6/ repeatability, reproducibility.
Methods: a 5 years experience feedback with the use of several tools will allow to summarize the main
currently available tools at the disposal of laboratory directors. Those tools enable problem-solving
quizzes that may include pictures, photos or videos.<br />Results: <table class="AbstractTable"
id="{51C1DA80-C267-4820-BA21-AA2921E619B7}"><caption
rowspan="1" colspan="1"></td></tr><tr><td rowspan="1" colspan="1">Software</td><td rowspan="1"
colspan="1">Customizable ?</td><td rowspan="1" colspan="1">Assessment methods ?</td><td
rowspan="1" colspan="1">Graduation ?</td><td rowspan="1" colspan="1">Freeware
?</td></tr><tr><td rowspan="1" colspan="1">ClassMarker.com</td><td rowspan="1"
colspan="1">Yes</td><td rowspan="1" colspan="1">tests and quizzes</td><td rowspan="1"
colspan="1">Instantly and final</td><td rowspan="1" colspan="1">Free version</td></tr><tr><td
rowspan="1" colspan="1">Easytestmaker.com</td><td rowspan="1" colspan="1">Yes</td><td
rowspan="1" colspan="1">multiple choice, fill-in-the-blank, matching, short answer and true or false
questions</td><td rowspan="1" colspan="1">Instantly and final</td><td rowspan="1"
colspan="1">Free version</td></tr><tr><td rowspan="1" colspan="1">Hot Potatoes
(http://web.uvic.ca/</td><td rowspan="1" colspan="1">Yes/limited</td><td rowspan="1"
colspan="1">multiple-choice, short-answer, gap-fill, matching/ordering and jumbled-sentence</td><td
rowspan="1" colspan="1">Instantly and final</td><td rowspan="1" colspan="1">Freeware, no technical
support</td></tr><tr><td rowspan="1" colspan="1">Moodle.org</td><td rowspan="1"
colspan="1">Yes</td><td rowspan="1" colspan="1">multiple choice, true-false, and short answer
questions</td><td rowspan="1" colspan="1">Instantly and final</td><td rowspan="1"
colspan="1">Free platform</td></tr><tr><td rowspan="1" colspan="1">Google.com/forms/</td><td
rowspan="1" colspan="1">Yes</td><td rowspan="1" colspan="1">multiple choice, true-false, and short
answer questions</td><td rowspan="1" colspan="1">Instantly and final</td><td rowspan="1"
colspan="1">Free access</td></tr></table><br />Discussion: Teaching is the pre-requisite to evaluation,
many websites offer to build customizable elearnings such as easygenerator.com ; ispringsolutions.com ;
moodle.org... Assessment may be done by quizzes testing knowledge, know-how or reflection. Our
experience shows a 50-75% time saving and an engaging and motivating approach for the staff.
Session Number: 458
Session Number: 458
Session Title: Diagnosing the Ancient Scourge of Tuberculosis
Abstract Title:
Xpert Mtb/Rif Assay for the Diagnosis of Mycobacterium Tuberculosis and its Rifampicin Resistance At
Felege Hiwot and Debre Tabor Hospitals; Northwest Ethiopia: A Preliminary Implementation Research
A. Habteyohhanes; Bahir Dar Univ., Bahir Dra, Ethiopia
Abstract Body:
Background: The World Health Organization in 2010 indorsed Xpert MTB/RIF (Xpert) assay for the
diagnosis of tuberculosis and multidrug resistant tuberculosis. However, the use of this novel diagnostic
method is still limited in a high TB and human immunodeficiency virus burden settings including
Ethiopia. Therefore, we conducted this study to describe the first implementation result of Xpert assay
in the diagnosis of TB, TB/HIV and MDR-TB at Felege Hiwot Referral Hospital (FHRH) and Debre Tabor
General Hospital (DTGH), Northwest Ethiopia. Methods: We analyzed the records of 1922 (FHRH=544
and DTGH=1378) presumptive TB patients diagnosed using Xpert test from 2015 to 2016 at FHRH and
DTGH, Northwest Ethiopia. Information on the demographic and clinical data was collected. Data were
entered, cleared, and analyzed using SPSS statistical software package; p < 0.05 was considered to be
significant. Results: Overall Xpert assay properly diagnosed 14.6% of the cases (258/1922). Among this
rifampicin (RIF) resistance was detected at 9.3% (24/258) of the cases. In the studied region, clinical data
reported that 81.0% (1556/1922) of the cases were MDR- TB. Among the study subjects, 888 (46.2 %) of
them were HIV positive. TB-HIV coinfection rate was at 41.9% (108/258). Of the total patients
registered, 1005 (52.3%) of whom were males. The mean age of patients was 31.1 years with SD of 17.5.
Significant predictors of the Xpert test were: age (p=0.000), sex (p=0.009), HIV (p=0.003) and
presumptive MDR-TB (p=0.000). Conclusions: In the studied areas, large proportion of clinically TB
suspected patients were wrongly diagnosed with multidrug resistant TB. Therefore, the use of Xpert
assay in health settings with no culture facility will decrease the unnecessary use of anti-TB drugs and
improve rapid TB, TB/HIV and MDR-TB detection and proper management of the cases.
Session Number: 458
Session Number: 458
Abstract Title:
High Resolution Melting Curve Analysis (Hrm) for Differentiation of Mycobacterium Tuberculosis And
Common Non Tuberculous Mycobacteria (Ntm)
K. Sharma, M. Modi, A. Sharma, M. Sharma, P. Ray; PGIMER, Chandigarh, India
Abstract Body:
Background: India constitutes nearly 25% of the world’s tuberculosis burden. Non-tuberculous
mycobacteria (NTM) are also gaining importance as opportunistic pathogens responsible for a variety of
clinical diseases. There is need for rapid, accurate and cost effective identification of Mycobacterium
species to aid in diagnosis and for initiating appropriate therapy. We aimed to evaluate high-resolution
melting curve (HRM) analysis to identify and differentiate clinical isolates of Mycobacterium spp. To
evaluate Heat shock protein 65 (hsp65) gene HRM analysis for identification of M.tuberculosis (MTB)
and NTM from clinical mycobacterial isolates. Methods: A total of 170 mycobacterial isolates were
evaluated. MPT64 antigen detection was performed to identify MTB while the identification of the
MPT64 antigen negative isolates (NTM) was confirmed by hsp65 sequencing. All isolates were subjected
to hsp65 real time PCR- HRM analysis for species identification. Results: Among the 170 isolates, 100
were MPT64 positive (MTB) while 70 were MPT64 negative (NTM). There was 100% concordance
between HRM analysis and MPT64 test. Amongst the 70 MPT 64 negative isolates 100% concordance
was observed between hsp65 HRM analysis and hsp65 sequencing results. Of the 70 NTM isolates, the
majority was constituted by M. intracellulare 20 (28.57%) followed by M. avium 18 (25.71%), M.
fortuitum 13 (18.57%), M. abscessus 11 (15.75%), M. gordonae 6 (8.57%) and M. kansasii 2 (2.85%).
Conclusions: By analyzing both the melting temperature and melting profile of the hsp65 gene, were
able to discriminate 7 different mycobacterial species simultaneously in 90 minutes. The hsp65 real-time
HRM assay is therefore a rapid, sensitive and cost effective method for identifying commonly prevalent
NTM and MTB in the clinical laboratory with subsequent utility in patient management.
Session Number: 458
Session Number: 458
Abstract Title:
Xpert Mtb/Rif Assay for Detection of M. Tuberculosis and Rifampicin Resistance in Extrapulmonary
Specimens
M. Tadesse1, A. Bekele1, M. Bezabih1, D. Yilma1, G. Abebe1, L. Rigouts2; 1Jimma Univ., Jimma,
Ethiopia, 2Inst. of Tropical Med., Antwerp, Belgium
Abstract Body:
Background: Ethiopia has an extremely high rate of extrapulmonary tuberculosis (EPTB). However, the
diagnosis of EPTB is often made on clinical suspicion alone, and many people receive the wrong
diagnosis leading to unnecessary TB treatment or poor outcomes from untreated EPTB. In this study, we
evaluated the clinical utility of the Xpert MTB/RIF (real time PCR technology) for detection of M.
tuberculosis and rifampicin resistance in routinely collected extra-pulmonary specimens in Ethiopia.
Methods: This study was carried out at Jimma University Specialized Hospital, Southwest Ethiopia from
September 2015 to June 2017. Extrapulmonary specimens were collected from 572 patients with
clinically presumed of EPTB. These comprised 250 fine-needle aspirates (FNA), 45 cerebrospinal fluid
(CSF), 248 other fluids and 29 pus specimens. All specimens were tested for M. tuberculosis using liquid
culture (BACTEC 960 MGIT) and Xpert MTB/RIF at Mycobacteriology Research Center of Jimma
University. The sensitivity and specificity of Xpert MTB/RIF was calculated compared to culture. If
rifampicin resistance was detected by the Xpert MTB/RIF, further drug susceptibility testing by the
GenoTypeMTBDRplus line probe assay was performed on DNA extracted from a positive culture.
Results: Overall, 226 (39.5%) specimens were positive for M. tuberculosis by culture and 242 (42.3%) by
Xpert MTB/RIF. Xpert MTB/RIF gave positive results in 17 culture negative and in 9 culture contaminated
cases and in all these cases, TB was clinically confirmed. The pooled sensitivity and specificity of Xpert
MTB/RIF were calculated to be 91% and 90.6% respectively. Xpert MTB/RIF has the highest sensitivity
(94%) in FNA and moderate sensitivity (75%) in CSF. The sensitivity of Xpert MTB/RIF was lowest (60-
66%) in pleural or peritoneal fluids, with only around 35% of probable TB cases being detected. A
negative Xpert MTB/RIF on fluid specimens does not exclude the diagnosis of pleural or abdominal TB.
Xpert MTB/RIF detected rifampicin resistance in 13 patients in perfect agreement with line probe assay.
Conclusions: Our study showed that Xpert MTB/RIF is likely to be of greatest utility when testing FNA
and CSF specimens. Moreover, Xpert MTB/RIF can directly detect rifampicin resistant strains in extra-
pulmonary specimens and improves management of drug resistant EPTB patients.
Session Number: 458
Session Number: 458
Abstract Title:
Magnetic Bead Based Concentration and Fluorescence Staining in A Single Tube for Capturing
Mycobacterium Tuberculosis in Sputum Samples
M. Kumar1, R. K. Verma1, S. Verma2, T. N. Dhole3; 1UP Univ. of Med. Sci., Etawah, India, 2King George's
Med. Univ., Lucknow, India, 3Sanjay Gandhi Post Graduate Inst. of Med. Sci., Lucknow, India
Abstract Body:
Background: Direct sputum smear microscopy is the mainstay of TB diagnosis in most low and middle
income countries, and is highly specific for Mycobacterium tuberculosis (M. Tuberculosis) in such
settings. However it is fast and inexpensive technique but limited by low sensitivity. Concentration by
centrifugation has been reported to be more sensitive than direct smear preparation, but is only
suitable for referral laboratories. An easier concentration methods are urgently needed to improve M.
Tuberculosis detection rates at peripheral center. Methods: The feasibility of a ligand-coated magnetic
bead technology to concentrate M. tuberculosis in single tube (decontamination, concentration and
staining) prior to detection by fluorescence microscopy was observed in this study. We compared the
sensitivity of this method with concentrated Ziehl-Neelsen (ZN) and fluorescence microscopy (FM) in
sputum samples. The kappa coefficient (Kc) analysis was performed for positive correlation between the
tests and LJ culture was taken as a gold standard for evaluation to sensitivity and specificity of
performed tests. Results: By active screening in all performed tests, the concentrated magnetic bead-FM
possess showing higher positivity rate (61%), concentrated FM microscopy (46.5%) and ZN microscopy
(40.5%) among all 200 sputum samples. The concentrated magnetic bead-FM having significantly higher
sensitivity (91.3%) over the concentrated FM (75.7%) and ZN microscopy (72.8%) in compare with LJ
culture. The specificity of magnetic bead FM, FM and ZN microscopy were 71.1%, 84.5% and 93.8% were
observed. The fair correlation found between culture and concentrated ZN microscopy (Kc = 0.66,),
concentrated magnetic bead-FM (Kc = 0.63) and Kc = 0.60 with concentrated FM. Conclusion: Magnetic
bead concentration of M. tuberculosis from sputum led to significant improvement in the sensitivity of
microscopy. Therefore, the national programs in high TB burden countries may consider incorporating
the technique into their guidelines at least in the district and higher level laboratories to improve case
finding strategy.
Session Number: 458
Session Number: 458
Abstract Title:
Point of Care Diagnosis of Tuberculosis Using Loop Mediated Isothermal Amplification
M. Nimesh, D. Joon, M. Varma-Basil, D. Saluja; Univ. of Delhi, Delhi, India
Abstract Body:
Background: Rapid detection of tuberculosis (TB) is essential to improve management of infected
patients and avoid transmission in the community. Automated real-time PCR based Xpert MTB/RIF assay
has been endorsed by WHO for detecting the presence of Mycobacterium tuberculosis. There is need
for sensitive diagnostic test with minimal infrastructure, cost and training. Therefore, present study was
carried out to design and evaluate the diagnostic performance of loop-mediated isothermal
amplification (LAMP) assay combined with lateral flow dipstick (LFD) as point of care method. Methods:
LAMP assay targeting sdaA gene for detection of M. tuberculosis was modified for sequence specific
detection by lateral flow dipstick. The LAMP-LFD assay was evaluated in a cross-sectional study using
respiratory specimens collected from patients visiting Vallabhbhai Patel Chest Institute, Delhi, India.
Pulmonary TB diagnosis using sputum smear microscopy, culture, GeneXpert MTB assay and sdaA LAMP
assay combined with LFD was carried out. LAMP assay targeting rpoB gene of M. tuberculosis was also
standardized to amplify the rifampicin resistance determining region and combined with LFD to develop
multiplex assay. Results: The LAMP assay was standardized for sequence specific detection of LAMP
amplified products in user friendly and rapid format. The LAMP-LFD assay was tested in 18 culture
confirmed specimens for pulmonary tuberculosis. All the specimens showed positive result with LAMP-
LFD assay. The diagnostic accuracy of the method was also evaluated in comparison with GeneXpert
MTB/RIF assay using 107 clinical specimens from patients with clinical symptoms of pulmonary
tuberculosis. Out of 107, 15 specimens were positive with both the methods showing high concordance.
LAMP assay targeting rpoB gene combined with LFD in multiplex format was successfully tested as
proof-of-concept for diagnosis of TB and screening for drug resistance. Conclusions: Lateral flow dipstick
method has provided an excellent detection format with LAMP method. The sdaA LAMP-LFD assay
showed high diagnostic accuracy in comparison to other methods and can be used as point of care test.
The increasing resistance to drugs used in anti-tubercular treatment is cause of concern to global TB
control efforts. The rpoB LAMP assay multiplexed with sdaA LAMP assay for simultaneous amplification
of both targets leads to diagnosis of tuberculosis as well as screening for drug resistance.
Session Number: 458
Session Number: 458
Abstract Title:
Moderator
Reynolds Salerno; 1
Abstract Body:
Session Number: 466
Session Number: 466
Session Title: Beyond Identification: Novel Applications of MALDI-TOF Analysis
Abstract Title:
Beyond Maldi-Tof's Score Values...Use of Bionumerics Software to Overcome Maldi-Tof Limitations.
Identification of Classical Bordetella Species
J. C. Zintgraff, C. S. Lara, M. Arias, G. A. Ayala, M. Santos; INEI-ANLIS Dr Carlos G Malbran, Ciudad
Autonoma de Bs As, Argentina
Abstract Body:
Background: Differentiation of classical Bordetella species by MALDI-TOF MS is well known to be
challenging. This study used the Bruker MALDI Biotyper system in addition to mass spectra model
analysis generated with the BioNumerics software package. Methods: This study used Bruker MALDI
Biotyper system in addition to a mass spectra model analysis generated by reference strains of
Bordetella pertussis, Bordetella parapertussis andBordetella bronchiseptica in the BioNumerics software
package (Applied Maths, Belgium) to identify clinical isolates. The results were compared with those
generated by MALDI Biotyper system alone.Different classifiers were evalated such as SVM and Basic
Similarity among others. PCA plotters were also performed. Results: The percentages of correct species
level identification using MALDI Biotyper system alone and following manufacturer recommendations
was 62.2% (66/106) with the additional Bionumerics mass spectra analysis the percentage of correct
identification increased to 99.1% (105/106) according the use of different classifiers. Conclusions: This
new workflow may improve the accuracy of classical Bordetella identification significantly. However
validation and verification procedure for accuracy and precision need to be evaluated in further
analysis.<p><a
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src="http://files.abstractsonline.com/CTRL/55/b/113/c1b/d90/42d/a81/5e0/767/c0e/fd8/38/g2091_1.p
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Session Number: 466
Session Number: 466
Abstract Title:
Multilocus Sequence Typing and Maldi-Tof Ms Fingerprinting for Discrimination of Cronobacter Sakazakii
Isolates from Environmental Surveillance Samples
I. Sulaiman, N. Miranda, S. Simpson, K. Kerdahi; FDA, Atlanta, GA
Abstract Body:
Background: Cronobacter species are considered as opportunistic foodborne pathogenic bacteria
causing acute meningitis and necrotizing enterocolitis in neonates, and linked to powdered infant
formula worldwide. Of these, three Cronobacter species (C. sakazakii, C. malonaticus and C. turicensis)
have been reported to be more virulent, and isolated often from infant meningitis cases. Species
identification of Cronobacter is critical for the detection of foodborne pathogen and source tracking of
contaminated foods Methods: A total of 314 environmental surveillance samples were analyzed to
isolate Cronobacter, initially by two-step enrichment followed by streaking on a selective agar. Initial
identification of recovered isolates was completed using bioMerieux VITEK 2 System and real-time PCR
analysis. Afterwards, the isolates were analyzed on the bioMerieux VITEK MALDI-TOF MS system.
Multilocus sequence typing (MLST) was performed based on the seven housekeeping genes (atpD, fusA,
glnS, gltB, gyrB, infB, and pps) using ABI 3500XL Genetic Analyzer. Results: All ten recovered Cronobacter
isolates were identified as Cronobacter sakazakii with a high confidence value (99.9%) by the VITEK MS
system. The MLST analysis identified three distinct clonal complex (2/CC1, 2/CC4 and 8/CC64) for the
recovered Cronobacter sakazakii isolates. All the three-clonal complex have been previously recovered
from Powdered Infant Formula, Infant Formula Production Factory, and patient with Neonatal
Meningitis. Conclusions: Results of this study suggest that MALDI-TOF MS and MLST based analysis can
be used for species-identification of Cronobacter isolated from environmental surveillance samples of
public health importance.
Session Number: 466
Session Number: 466
Abstract Title:
Detection of Community Acquired Antibiotics from Cholera and Non-Cholera Patient Urine and Stool
Samples Using Mass Spectrometry
E. J. Nelson1, L. Alexandrova2, F. Haque3, V. Ramachandran2, P. Rodriguez1, A. Creasy1, C. Adams2, S.
A. Siddique4, A. I. Khan4, F. Qadri5, M. Rahman3, A. Chien2; 1Univ. of Florida, Gainesville, FL, 2Stanford
Univ., Stanford, CA, 3Inst. of Epidemiology D
Abstract Body:
Antibiotics impact infection and transmission of gastrointestinal pathogens like Vibrio cholerae.
However, it remains difficult to monitor antibiotic impact because self-reported medication use is
unreliable and analytic assays are often too expensive, or lack sensitivity and specificity. To address this
problem, we developed a unified liquid chromatography-mass spectrometry (LC-MS) method that
minimizes cost by qualitatively targeting sixteen clinically relevant antibiotic and non-antibiotic
medications for diarrheal disease treatment and streamlining preparation and analysis. We validated
the method by prospectively collecting and testing urine and diarrheal samples from a cohort of patients
in Bangladesh. Due to differences in analyte excretion patterns in urine and stool, the spectra were
analyzed for both parent and known metabolites. A total of 30 paired urine and diarrheal samples were
tested (10 non-cholera and 20 cholera). All samples contained at least one antibiotic and 80% (N=24/30)
had at least two antibiotics independent of self-reports. Metronidazole (80%), ciprofloxacin (60%),
ondansetron (Zofran; 36%), azithromycin (33%) and paracetamol (20%) were the most common
medications. The primary limitation is stool analysis showed less sensitivity due to filtration, dilution
because increased methanol was needed to accommodate for high protein concentration, and likely
different excretion and degradation. We advocate that this technique be deployed to monitor for
antibiotics in research designed to elucidate critical determinants of diarrheal disease severity and
transmission. This platform can also be a gold-standard to assess biologic antimicrobial detection assays
and diagnostic performance in the setting of antibiotics.
Session Number: 466
Session Number: 466
Abstract Title:
Maldi-Tof Ms As An Alternative Tool for Rapid Detection of Vancomycin-Resistant “Enterococcus
faecium”
R. L. Ribeiro, T. C. A. Pinto, J. M. Morais, F. S. P. Rocha, L. M. Teixeira; Univ.e Federal do Rio de Janeiro,
Rio de Janeiro, Brazil
Abstract Body:
Vancomycin-resistant Enterococcus faecium (VREfm) has been appointed by the World Health
Organization as one of the current twelve most important antimicrobial resistance threats for public
health worldwide. VREfm is a leading cause of opportunistic infections, especially in hospital
environments, being associated with therapeutic challenges and a high ability to disseminate. Thus,
rapid detection of this microorganism, in both colonized and diseased patients, is essential to prevent,
control and properly treat enterococcal infections. Nevertheless, identification of VREfm can be slow,
expensive or even inaccurate by the existing phenotypic (conventional and automated) and genetic
methods. In the present study, we evaluated the ability of MALDI-TOF MS to discriminate between
VREfm (91 strains) and vancomycin-susceptible E. faecium (VSEfm; 31 strains) isolates recovered from
human sources (intestinal colonization and enterococcal infections) in Brazil. All 122 strains were
previously identified and evaluated regarding vancomycin susceptibility by phenotypic and/or genotypic
methods. For MALDI-TOF MS analysis, fresh colonies were directly applied to the target plate and
covered with CHCA matrix solution. Measurements were performed with a Microflex LT mass
spectrometer using default parameters and generating spectra in the range of 2,000-20,000 m/z.
Spectra were then analyzed with the BioNumerics software v7.6. All 122 strains were correctly identified
to the species level, with scores >2.0. By using BioNumerics a comparative analysis of the spectra was
performed. Overall, 30 peaks (biomarkers) ranging from 2,212 to 10,225 m/z were detected. Twelve of
these biomarkers were exclusively associated with VREfm: four of them were present in 100% and eight
were present in more than 90% of the 91 VREfm strains and absent in all 31 VSEfm isolates. Our results
indicate that MALDI-TOF MS is a promising alternative procedure to discriminate between VREfm and
VSEfm isolates from human sources, highlighting its usefulness for rapid and cost-effectiveness routine
application in clinical laboratories.
Session Number: 466
Session Number: 466
Abstract Title:
Detection of Methicillin Resistance in Staphylococcus aureus by Maldi-Tof Mass Spectrometry Using
Direct-On-Target Microdroplet Growth Assay (Dot-Mga) Directly from Positive Blood Cultures
E. Idelevich1, L. Storck1, K. Sparbier2, M. Kostrzewa2, K. Becker1; 1Univ. Hosp. Muenster, Muenster,
Germany, 2Bruker Daltonik GmbH, Bremen, Germany
Abstract Body:
Background: Direct-on-target microdroplet growth assay (DOT-MGA) based on MALDI-TOF MS has
recently been suggested for rapid antimicrobial susceptibility testing. This proof-of-principal study aimed
to investigate this method for detection of methicillin-resistant Staphylococcus aureus (MRSA) directly
from positive blood cultures (BCs). Methods: After pilot tests with agar cultures, six MRSA and six
methicillin-susceptible S. aureus isolates were added to 10 ml human blood in final concentration 10
cfu/ml. The inoculated blood was introduced into BACTEC aerobic bottles and incubated in the BACTEC
system. Each positive BC sample was processed by (i) dilution protocols followed by testing of serial
dilutions in cation-adjusted Mueller-Hinton broth (CA-MHB), and (ii) centrifugation protocols with
differential centrifugation and lysis/centrifugation, followed by dissolving the pellet and standardization
of inoculum to 5x105 cfu/ml in CA-MHB. 6-µl microdroplets containing S. aureus with or without 4 µg/ml
cefoxitin were spotted in triplicate onto MBT Biotarget96 target (Bruker). The targets were incubated at
36°C for 4, 5 and 6 hours, followed by medium removal. After adding matrix, MALDI-TOF MS spectra
were generated (Bruker) and identification scores ≥1.7 were designated as resistance. Broth
microdilution and mecA PCR were used as standard methods. Results: Initial testing of agar cultures
showed principle feasibility of DOT-MGA for S. aureus and reduction of test time by adding 1 µl of formic
acid to each spot, which was adopted for BC experiments. Testing 1:100 dilution from positive BCs
demonstrated best performance: 100% valid results (i.e., growth control detected) already after 5 hours.
At this time point, 100% sensitivity and 100% specificity were reached. Centrifugation protocols revealed
poorer performance achieving with differential centrifugation only 41.7% valid results after 6 hours.
Conclusions: This study demonstrated feasibility of rapid phenotypic detection of methicillin resistance
by MALDI-TOF MS-based direct DOT-MGA from positive BCs. Further optimization and standardization
of sample processing, measurement and evaluation algorithms is warranted.
Session Number: 466
Session Number: 466
Abstract Title:
Detection of Ctx-M- Producing Enterobacteriaceae Using Maldi-Tof
R. Figueroa Espinosa, B. Ghiglione, V. Rumi, C. Barberis, C. Vay, J. Di Conza, G. Gutkind; Univ. de Buenos
Aires, Buenoa Aires, Argentina
Abstract Body:
Background: The CTX-M enzymes are the most prevalent extended-spectrum β-lactamases (ESBL), both
in nosocomial and in community settings. Fast, easy to perform sensitive and specific methods for ESBLs
detection are needed to avoid treatment failure and prevent dissemination. MALDI-TOF mass
spectrometry is frequently used as a routine bacterial identification tool to categorize bacterial species.
Direct detection of ß-lactamases production in Enterobacteriaceae by MALDI-TOF is another important
diagnostic challenge for clinical microbiology lab. In this study, an easy, fast and direct MALDI-TOF MS
protocol was designed to detect CTX-M-producing Enterobacteriaceae. Methods: 49 clinical isolates of
Escherichia coli (15), Salmonella enterica (15), Klebsiella pneumoniae (8), Serratia marcescens (8) and
Proteus mirabilis (3) were included in this study. Susceptibility tests were performed according to CLSI.
Isolates were analyzed by PCR and sequencing, and grouped according to the CTX-M variant.
Subsequently, CTX-M- detection was carried out by MALDI-TOF (Bruker) after proteins extraction with
organic solvents. MALDI-TOF spectra were recorded by MALDI Biotyper system and analyzed using
ClinProTool software (Bruker). E. coli transformants producing CTX-M-2, CTX-M-15 and CTX-M-9
enzymes were used as positive controls and isolates lacking any blaCTX-M- as negative controls. Results:
Operating at a mass range of 17,000 to 50,000 Da, three peaks at approximately m/z 28,287, 28,093 and
27,950 Da were detected by MALDI-TOF and they were correlated with the mature protein of CTX-M-2,
CTX-M-15 and CTX-M-14, respectively. After statistical analysis of peaks, two groups of isolates could be
distinguished: the CTX-M-producing enterobacteria (28) (clinical isolates and transformants) and non-
CTX-M-producing enterobacteria (24) (including susceptible strains and CMY-2, KPC-2 and TEM
producers). Furthermore, the 25 CTX-M-producing isolates were correctly classified into CTX-M-1 (6),
CTX-M-2 (15) and CTX-M-9 (7) groups. Conclusions: Our MALDI-TOF assay was able to identify the most
frequent CTX-M ß-lactamase groups reported in Argentina in different enterobacterial species.
Considering that our results could be obtained as early as bacterial identification time, and together
with previous communications on other β-lactamases, they may constitute the basis for designing a
database of β-lactamases peaks that could be detected in a single event, which may have deep impact
on resistance characterization.
Session Number: 466
Session Number: 466
Abstract Title:
Rapid and Reagent-Free Typing of Vancomycin-Resistant Enterococcus faecium by Attenuated Total
Reflectance-Fourier Transform Infrared (Atr-Ftir) Spectroscopy As A New Tool for Outbreak
Investigations
T. Tsutsumi1, L. Lam1, C. Frenette2, N. Doherty2, J. Sedman1, A. Ismail1; 1McGill Univ., Sainte-Anne-de-
Bellevue, QC, Canada, 2McGill Univ. Hlth.Ctr., Montreal, QC, Canada
Abstract Body:
Whole-organism fingerprinting by ATR-FTIR spectroscopy is a rapid, reagent-free technique for bacterial
identification and classification with subspecies-level discriminatory capabilities. ATR-FTIR methods for
identification of nosocomial pathogens developed by our group include the identification of
vancomycin-resistant (VR) Enterococcus faecium, based on the capability of ATR-FTIR spectroscopy to
discriminate between antibiotic-resistant and susceptible strains in the absence of antibiotic. The
present study extends this work to examine the possibility of employing ATR-FTIR spectroscopy as a
rapid method for discriminating among VRE clonal groups. As pulsed-field gel electrophoresis (PFGE) is
the gold-standard method for strain typing in VRE outbreak investigations, a set of 93 clinical isolates of
VRE. faecium previously typed by PFGE was employed in this study; these isolates were distributed
between two pulsotypes. FTIR spectra were acquired by transferring isolated colonies directly from
blood agar plates onto the sampling surface of a portable ATR-FTIR spectrometer; spectral acquisition
time was 1 min. Triplicate spectra were collected per isolate, each from different colonies on the same
culture plate. Spectral data analysis was performed by hierarchical cluster analysis and principal
component analysis (PCA) in conjunction with the use of a feature selection algorithm. The spectra of all
isolates were correctly identified as VRE. faecium using an ATR-FTIR spectral database for identification
of Gram-positive pathogens associated with nosocomial infection, developed in our previous work. PCA
showed successful discrimination between the two clonal groups, yielding 99% concordance with PFGE
results and indicating that ATR-FTIR spectroscopy may provide a rapid and easily implemented typing
method suitable for intrahospital surveillance of VRE outbreaks. Furthermore, this study illustrates that
the ATR-FTIR spectrum of a clinical isolate can be used to identify it, classify it as antibiotic resistant, and
assign it to a clonal group, ultimately reducing the time and cost of labor and reagents required,
compared to current laboratory procedures that require multiple analyses.
Session Number: 466
Session Number: 466
Abstract Title:
Media Makes A Difference in the Detection of Surveillance Isolates of Methicillin Resistant
Staphylococcus aureus Using the Bruker Maldi-Tof Mass Spectrometry Mrsa Psm-Mec Detection Module
D. Boulton; Grand River Hosp., Kitchener, ON, Canada
Abstract Body:
Background: Methicillin resistant Staphylococcus aureus (MRSA) is a major nosocomial pathogen
worldwide and associated with high rates of mortality and morbidity. Recently, Bruker developed a
software package to be used with their MALDI Biotyper System (MBT) allowing for direct detection of
phenol soluble moduline (PSM-mec), a staphylococcal toxin produced by a part of MRSA strains. This
toxin, coupled with antibiotic resistance genes allows a simultaneous species identification and MRSA
positivity alert by detection of the PSM-mec signal in MALDI-TOF mass spectra. Methods: Stored MRSA
isolates from surveillance samples were used to investigate the performance of the PSM detection
module. Isolates were originally confirmed from typical morphological growth on BioRad MRSASelect
Agar culture media and/or a positive Alere PBP2a SA Culture Colony Test. Each strain was plated onto
Columbia agar with 5% sheep blood (BA), BioRad MRSASelect Agar, and Alere Colorex MRSA Agar plates
and incubated at 35°C for 18-24 hours. Typical colonies from each plate were inoculated onto a target
plate before adding formic acid and matrix for protein extraction and run in the Bruker MALDI Biotyper
System. Ability to detect PSM-mec between chromogenic media was compared, using the BA as a non-
chromogenic comparison. Results: A total of 200 confirmed MRSA isolates from March to October 2017
were analyzed. Of the 200 samples, 78 (39%) detected PSM-mec when run from the BA plate, all 200
samples (100%) detected PSM-mec when run from the Alere Colorex MRSA Agar plates, and none of the
samples detected PSM-mec when run from the Bio-Rad Select Chromogenic plates. Conclusion: The
performance of the PSM MBT Subtyping module is influenced by the type of chromogenic media from
which isolates are run. Alere Colorex MRSA Agar plates outperformed both MRSASelect Agar (BioRad)
and BA plates.
Session Number: 466
Session Number: 466
Abstract Title:
Moderator
Karissa Culbreath; The Univ. of New Mexico, Albuquerque, NM
Abstract Body:
Session Number: 501
Session Number: 501
Session Title: Outcomes Impacting Income: Cost Effective Clinical Diagnostics
Abstract Title:
Micro-Emulsion Droplets As A Novel Platform to Study Evolution of A Synthetic E. coli Predator-Prey
Population
R. Ganiga Prabhakar, R. Alnahhas, M. R. Bennett, Y. Shamoo; Rice Univ., Houston, TX
Abstract Body:
Competition for resources is one of the most fundamental driving forces of natural selection and has
markedly influenced the evolution of microbial communities. As a consequence of competition, bacteria
have accumulated the ability to produce various bioactive secondary metabolites under specific
environmental conditions. Some of these pathways are activated only under certain stress conditions or
remain largely silent. Activation of these cryptic pathways can provide us with insights into how
microbial communities interact as well as access to potentially novel bioactive metabolites for industrial
and biomedical use. Encapsulation of bacteria in emulsion droplets allows us to study single cells and all
the metabolites secreted by those cells. We have used approaches from synthetic biology to engineer a
predator-prey system in E. coli to study how environmental stress can be tied to the activation of a
cryptic pathway in a predator to gain resources from another strain of E. coli that serves as the prey. As
a proof of principle, predator and prey are co-cultured under nutrient limitation conditions within
emulsion droplets. A predator evolved to activate the engineered cryptic pathway produces quorum
sensing (QS) molecules that communicates with the prey cells present within the droplet. Prey is
engineered to activate suicide lysis genes in response to the detection of QS molecules present in the
media. We have introduced a single nucleotide polymorphism (SNP) within the predator to knockout
production of the essential QS molecule required for killing prey. A predator population that reverses
the SNP can grow to a higher density compared to unevolved predator within the droplets by killing the
prey cells. Spatial segregation imparted by the emulsion droplet prevents the diffusion of common
resources between droplets carrying evolved and unevolved predators. By iterating the growth of
evolved predator in emulsion, we can increase its population density to detection level. If successful,
this system of competition based directed evolution can be extended for harnessing the evolutionary
power of bacteria to produce molecules of interest under defined synthetic stress conditions.
Session Number: 501
Session Number: 501
Abstract Title:
Evaluation of A Rapid Highly Multiplexed Molecular Diagnostic Lower Respiratory Panel for Clin. Impact
and Antibiotic Stewardship
H. Mopuru, K. Powell, M. Sims; Beaumont Hlth., Royal Oak, MI
Abstract Body:
Background: The Unyvero system is a rapid molecular diagnostics platform currently being reviewed for
IVD clearance by the FDA for a cartridge designed to detect lower respiratory tract (LRT) pathogens from
endotracheal aspirates and bronchoalveolar lavages (BAL). While there are clear benefits to a rapid and
sensitive diagnostic system, there is debate as to whether such a system would lead to overuse of broad
spectrum antibiotics versus improving stewardship. Methods: A retrospective chart review was
performed for the 442 patients included in the clinical trial of the Unyvero LRT cartridge. Culture results,
antibiotic treatment given, and outcomes were analyzed and a determination was made as to whether
knowing the results of the Unyvero test, which detects 20 pathogens and 16 antibiotic resistance genes,
would have led to no change in the antibiotics, a change favoring expanding the antibiotic coverage, a
change favoring stewardship by narrowing the antibiotic coverage, or a change that potentially missed a
pathogen detected on culture. Results: Of the 442 patient specimens the majority, 163 (37%) had a
negative result which agreed with culture results. 65 (15%) additional specimens had results favoring no
change in treatment, 91 (21%) had results favoring expanding the antibiotic treatment, 111 (25%) had
results favoring stewardship by narrowing the antibiotic treatment, 7 (1%) would have missed a
potential pathogen found on culture, and 5 (1%) did not have sufficient data to make a determination.
The difference in time to results from Unyvero to standard culture is >2 days (5 hours vs. 2.7 days).
Conclusions: The Unyvero system can significantly impact the time to optimization of antibiotics in
patients with lower respiratory tract infections in which an endotracheal aspirate or BAL is obtained. The
system had excellent agreement for negative cultures and could lead to both expansion and narrowing
to more appropriate antibiotics and the frequency in which a potential pathogen was missed was low
(1%). Thus the Unyvero platform and the LRT cartridge has significant potential to improve the
management of lower respiratory tract infections and can improve antibiotic stewardship at the same
time.
Session Number: 501
Session Number: 501
Abstract Title:
Fungal Blood Cultures for the Detection of Candidemia: Time to Retire?
S. Altamimi, M. Jamal, O. Rayes, L. Samuel, G. Alangaden; Henry Ford Hlth.System, Detroit, MI
Abstract Body:
Background: Blood cultures (BC) have sensitivities of 40-70% for detection of bloodstream infection due
to Candida (BSI-CA). The poor sensitivity has led to the use of the isolator fungal blood culture (IFC)
system (Alere, Waltham, MA, USA) that utilizes lysis-centrifugation to improve recovery of candida. IFC
has not been extensively evaluated and is labor-intensive compared to automated BC (Trek Diagnostic
Systems, Oakwood, OH, USA). The aim of this study is to determine if IFC provide significant information
compared to BC for detection of BSI-CA at a tertiary care center with patient populations at high risk for
BSI-CA. Methods: Retrospective review was done of all positive IFC and BC between 1-1-2013 and 9-30-
2016. Cultures were reviewed for corresponding results with the alternate method +/- 7 days. Fungal
biomarker results either Fungitell (Associates of Cape Cod, Falmouth, MA, USA) or T2Candida panel
(T2Biosystems, Lexington, MA, USA) were reviewed. Patient characteristics were obtained by review of
medical records. Results: Overall, 121 IFC and 730 BC were positive for yeast. Both IFC and BC were done
in 175 cases that Candida spp. was isolated: 159/175 (92%) were detected by BC and 14/175 (8%) by IFC
alone. Of 121 IFC positive for Candida, 76/121 had corresponding BC: 62/76 (82%) IFC and BC were both
positive, and 14/76 (18%) IFC was positive and BC was negative. In the 14 IFC positive/BC negative cases
C. albicans (6), C glabrata (3) and C. parapsilosis (3) were the common species isolated. In episodes that
were IFC positive/BC negative, 13/14 (93%) IFC had <5 CFU/ml whereas for episodes that were IFC
positive/BC positive, 39% had >5 CFU/ml. Fungal biomarker results were available in 13/14 cases of the
IFC positive/BC negative group and was positive in 12/13 (92%): Fungitell positive 11/13 and T2Candida
positive 1/2 cases. Clinical characetristics of the 14 patients in the IFC positive/BC negative group were:
100% had undifferentiated sepsis; 86% were in the ICU; 21% colonized with candida; 87% had Infectious
Diseases consultation, and all-cause 30-day mortality of 31%. Conclusions: These results suggest that
over 90% of BSI-CA can be detected using routine BC. IFC may complement BC for detection in patients
with low levels of candidemia (<5 CFU/ml). Fungal biomarkers including Fungitell and T2Candida Panel
are highly sensitive for the detection of BSI-CA in the BC negative cases. Utilization of BC together with a
fungal biomarker can eliminate the need for IFC in the detection of BSI-CA.
Session Number: 501
Session Number: 501
Abstract Title:
Impact of Rapid Influenza Molecular Testing on Diagnosis and Patient Management
N. Mercuro, S. Krupp, R. Tibbetts, E. Hadley, G. Sharma, I. Rubinfeld, J. Bongiorno, K. Callahan, G.
Alangaden, L. Samuel; Henry Ford Hlth.System, Detroit, MI
Abstract Body:
Background: The rapid influenza antigen test is limited by poor sensitivity, which may result in diagnostic
uncertainty and overtreatment. In 2017, the FDA reclassified rapid influenza antigen tests as class II
devices and mandated use of assays with improved performance characteristics by January 2018. The
purpose of this study was to evaluate the impact of on-site CLIA waived rapid influenza PCR
implementation, in conjunction with appropriate training. Methods: This was a multicenter, quasi-
experimental study including patients who were tested for influenza in four Emergency Department (ED)
locations of the Henry Ford Health System between December 2015 and April 2017. A series of
educational interventions and a testing algorithm for influenza was developed and all ED sites were
transitioned from antigen testing to rapid onsite PCR testing in December 2016 Patients who received
influenza antigen testing were compared to those with PCR testing . Microsoft SQL Server 2016® was
used to extract subjects tested for influenza along with baseline demographics, admission information,
diagnoses, and receipt of antiviral. Continuous variables were compared with Mann-Whitney U or t-test,
as appropriate, and Chi-squared was used to assess categorical variables. Logistic regression was used to
determine predictors of hospital admission from the ED. Results: During two consecutive flu seasons,
20157 patients were tested with the rapid antigen (n=12,672) or PCR (n=7,485). Tests were positive in
1,358 (10.7%) rapid antigens vs 1,700 (22.7%) PCRs (p<0.001). Subsequently, 7.3% vs 16.5% (p<0.001)
had primary diagnosis of influenza and there were no differences between sepsis or pneumonia
diagnoses. Patients tested with PCR were more likely to receive oseltamivir (15.8% vs 21.6%) and were
also less likely to receive inappropriate antivirals if tested negative (9.1% vs 6.9%, p <0.001). Admission
rates from the ED for patients tested with rapid antigen and PCR were 34.5% vs 42.3% (OR=1.31 [1.22-
1.48]), respectively. After using logistic regression to control for confounders, PCR use and influenza
positivity were protective against hospital admission, while age, sepsis, and hospital location predicted
hospital admission. Of those admitted, subjects tested with PCR had an overall shorter length of stay
(4.9 vs 4.4 days, p <0.001). Conclusions: The influenza PCR had an improved diagnostic yield and was
associated with greater odds of receiving appropriate antivirals. Results should be validated in a
prospective analysis.
Session Number: 501
Session Number: 501
Abstract Title:
Procalcitonin-Guided Treatment for Inpatients with Pneumonia Reduces Antibiotic Exposure
O. Henig, R. Putler, K. Rao; Univ. of Michigan Med. Sch., Ann Arbor, MI
Abstract Body:
Background: Antibiotic stewardship programs (ASPs) are crucial in reducing antimicrobial resistance.
Randomized controlled trials show procalcitonin (PCT) use reduces antibiotic exposure, but real-world
data are lacking. This observational study evaluated the impact of PCT use on antibiotic duration in
hospitalized patients with pneumonia. Methods: Adults admitted to Michigan Medicine from 2013-2015
with diagnostic codes for pneumonia were included. Data were extracted from the electronic medical
record using structured query. If ≥1 PCT was ordered, treatment was considered PCT-guided. A
PCT<0.25ng/mL was low. Antibiotic exposure was quantified by days of treatment (DOT). Unadjusted
and adjusted models of DOT were built using generalized linear models. A stratified analysis by intensive
care unit (ICU) stay was performed. Results: There were 2213 patients (2574 admissions) included; mean
age was 62.6±17.7 and 47.1% were female. Mean Elixhauser comorbidity score was 4.9±2.5. PCT was
used in 1443 admissions (56%) and was low in 692 of first measurements (48%). ICU stay greatly
increased DOT (+7.35 days) and interacted with several other covariates, prompting us to stratify our
analyses. In the ICU-stay cohort, PCT use associated with fewer DOT (−2.62 days; 95% CI −4.13 to −1.11;
P<.001) after adjustment for covariates (Table). A low first PCT associated fewer DOT (−2.78 days; 95%
CI −4.97 to −0.58, P=.01, unadjusted). Among Non-ICU patients, PCT use did not associate with DOT
(Table). However, a low first PCT associated with fewer DOT (−1.27 days; 95% CI −0.73 to −0.81; P<.001,
unadjusted). PCT-guided treatment was not associated with in-hospital mortality. Conclusions: In nearly
half of admissions, PCT was not used to guide treatment for pneumonia. When treatment was PCT-
guided, this independently associated with fewer DOT in ICU patients. In the entire cohort, a low first
PCT associated with fewer DOT and PCT-guidance did not increase in-hospital mortality.<br /><p><a
href="http://files.abstractsonline.com/CTRL/a8/7/da5/8f9/1b1/47b/586/58c/50d/17b/9c3/0e/g5522_2.
src="http://files.abstractsonline.com/CTRL/a8/7/da5/8f9/1b1/47b/586/58c/50d/17b/9c3/0e/g5522_2.J
Session Number: 501
Session Number: 501
Abstract Title:
Moderator
Abstract Body:
Session Number: 501
Session Number: 501
Abstract Title:
Assessing the Clin. Utility of An Algorithmic Approach to Molecular Testing for C. Difficile At A Tertiary
Care Hospital
A. Balla, A. Theiss, A. Ross, D. Francis, C. Wojewoda; Univ. of Vermont Med. Ctr., Burlington, VT
Abstract Body:
Background: Clostridium difficile is the causative agent for antimicrobial-associated diarrhea and
pseudomembranous colitis and is a major contributor to morbidity and mortality in the United States.
Diagnosis depends on identifying appropriate signs and symptoms which are then supported by
laboratory testing. Methods for identifying organism in stool include molecular platforms, enzyme
immunoassays, and culture. Controversy persists over whether molecular tests are too sensitive at
identifying C. difficile and have raised questions about how additional laboratory information could
inform clinical management and reduce over treatment. The aim of this study was to assess whether
clinical factors were related to toxin status of patients and whether information about toxin status could
potentially inform clinical management of patients. Methods: Stool from patients positive for C. difficile
testing by PCR at the University of Vermont Medical Center between June 2016 and May 2017 were
subjected to additional testing for toxin production by an enzyme immunoassay method. Clinical and
laboratory information including white blood cell count, fever, duration of diarrhea, concurrent laxative
use, prior antibiotic use, history of inflammatory bowel disease, and immune status were collected. The
percentage of PCR+/toxin+ patients and PCR+/toxin- patients was calculated. Data were analyzed for
categorical variables using a chi-square test, a t-test was done to compare means for continuous
variables and a binary logistical regression using the clinical variables was performed. Results: A total of
201 adult patients that were PCR positive for C. difficile were analyzed. Of the 201 samples, 94 (47%)
were toxin positive and 107 (53%) were toxin negative. Although PCR+/toxin+ patients were more likely
to have had a prior C. difficile infection (P=.015), there was no statistical difference between the
additional demographic or laboratory variables that correlated with a positive toxin test result.
Conclusions: We were unable to show that patients with a PCR+/toxin+ result had worse clinical
parameters than those with a PCR+/toxin- results, and concluded that establishing a testing algorithm
that included both PCR and toxin testing would not change the clinical management of patients at our
hospital. One C. difficile infection complication resulting in death occurred in a PCR +/ toxin- patient in
our study further supporting the notion that toxin status does not necessarily equate with clinical
severity of disease.
Session Number: 501
Session Number: 501
Abstract Title:
Fully Automated Disk Diffusion Susceptibility Testing by Adagio Wasplab Expert Sys. Compared to Vitek2
and Manual Disk Diffusion in Urines from Daily Clin. Practice
R. Verschuijten1, T. Liebregts2, L. B. J. Velden van der2, A. R. Jansz2, N. L. A. Arents2; 1FONTYS
Hogeschool voor toegepaste natuurwetenschappen, Eindhoven, Netherlands, 2PAMM, Veldhoven,
Netherlands
Abstract Body:
Background: Decreasing reimbursements stimulate clinical laboratories to search for the most cost-
effective diagnostic approaches. Over the past years PAMM has implemented COPAN WASPLab which
provides automated streaking, dispersion of antibiotic disks, incubation and digital imaging of incubated
culture media. In this study, we investigated the next step: fully automated disk diffusion interpretation
by ADAGIO WASPLab Expert System (WASPLab DD) of urine cultures from daily clinical practice.
Methods: From october 2017 to the end of 2017 culture positive urine samples derived from daily
clinical practice were considerd for this study. If these cultures showed Gram negative rods (GNR),
Staphylococci (STAP) or Enterococci (ENCO), demanding antibiotic susceptibility testing (AST) according
to our laboratory SOP, the strains were enrolled in the study. GNR AST was performed by VITEK2 and
compared to WASPLab DD for amoxicillin-clavulanic acid, cefotaxim, ceftazidim, ciprofloxacin,
fosfomycin, nitrofurantoin, trimethoprim and thrimethoprim-sulfamethoxazol. STAP AST was performed
by VITEK2 and compared to WASPLab DD for cefoxitin, ciprofloxacin, clindamycin, erythromycin and
thrimethoprim-sulfamethoxazol. ENCO AST by manual disk diffusion was compared to WASPLab DD for
nitrofurantoin, norfloxacin, tetracyclin and fosfomycin. Ampicillin AST in ENCO was performed by agar
dilution and compared to WASPLab DD. All ASTs were interpreted according to EUCAST breakpoints
except for ENCO and tetracyclin, which was interpreted by CLSI breakpoints. A cost-effectiveness
analysis comparing hands-on time and material cost per sample is ongoing. Results: In total 886 strains
(810 GNR, 41 STAP, 35 ENCO) were derived from 862 urine samples. For GNR and STAP concordance
between VITEK2 and WASPLab DD was 95,2% and 99,5% respectively. For ENCO concordence between
manual disk diffusion/agar dilution and WASPLab DD was 98,9%. The most frequently encounterd
discrepancy was a very major error when testing Enterobacteriaceae for AMCL (127 strains; 2,4% from
total). These strains were retested by AMCL Etest (with a variable clavulanic acid ratio concentration)
and proved to be Etest sensitive. Conclusions: In conclusion, ADAGIO WASPLab Expert System disk
diffusion reading and interpretation showed excellent overall results (concordance >95%) compared to
VITEK2 and manual disk diffusion based AST. The well known discrepancy between AMCL AST with a
fixed and a variable concentration of clavulanic acid accounted for 40% of all errors.
Session Number: 501
Session Number: 501
Abstract Title:
Reduction of Blood Culture Contamination Using Initial Specimen Diversion Device
J. Blakeney; Beebe Hlth.care, Lewes, DE
Abstract Body:
Background: Contaminated blood cultures remain a significant problem at our institution despite
repeated sterile technique education efforts. Our historical blood culture contamination (BCC) rate is
2.8% (about 250 contamination events/year), with higher rates (>4%) in some months. False positive
blood culture results can lead to unnecessary antibiotic treatment, longer stays, and increased costs
(estimated to be $3,500/event). The SteriPath (SP) device (Magnolia Medical Technologies) is designed
to reduce contamination by diverting and isolating the initial 1.5-2 mL of blood, which is most likely to
contain skin contaminants. We instituted a trial of this device in an effort to reduce the incidence of
contamination events. Methods: The study was conducted at Beebe Healthcare over 16 weeks. Blood
cultures drawn in the ED, ICU, and by the phlebotomy team were collected using either the SP device or
standard method (SM). The collection method was recorded for each sample, and the number of false
positive events was recorded for each method. A culture was considered contaminated if normal skin
bacteria grew in only one culture set within a three-day period. Results: A total of 2639 blood cultures
were collected between 5/30/207 and 9/16/2017, with a total of 38 contamination events (1.44%). SP
was used to collect 1837 cultures; SM was used for the remaining 802. The SP group had 14
contamination events (14/1837, 0.76%). The SM group had 24 contamination events (24/802, 2.99%).
There was a 75% reduction in the BCC contamination rate in the SP group compared to the SM group
(χ2=18.029, p<.0001) Conclusion: Use of the SP device led to a significant decrease in the BCC rate in all
groups: ER, ICU, and phlebotomy team. The overall contamination rate for the 16-week period was
1.44% (using both SP and SM), a 49% reduction compared to the historical rate of 2.8%. These results
led us to adopt the SP device as standard practice. With SP we have maintained an average BCC rate of
1.5% in the months following the study.

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Session Number: 24

Session Type: Poster Talk

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