EUCIC Local Module

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Athens, Greece 20 – 22 May 2019

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FORMS OF AUTOMATION (VARIOUS PLATFORMS FOR

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IDENTIFICATION OF PATHOGENS AND ANTIBIOGRAM)

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VASSILIKI PITIRIGA
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MD, PhD Assistant Professor of Microbiology
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Medical School of Athens
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National & Kapodistrial University of Athens

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E-mail: vpitiriga@med.uoa.gr
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• No conflict of interest

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 THE RATIONALE FOR AUTOMATED SYSTEMS

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Large self-contained incubator/reader device that
can incubate and analyze microdilution trays

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These systems are able to:

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At present automated systems have been widely • Decrease the in-laboratory TAT
used for ID of pathogens and AST due to:

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• More reliable AST results-correct ID

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 Increasing volumes of clinical specimens, • Reduced “user” errors

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 Perceived cost-effectiveness, • Increased number of agents tested

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 Convenient interfaces with hospital information • Improved laboratory workflow

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systems
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• Guide for optimal antimicrobial
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therapy
Each system has inherent strengths as
well as recognized limitations
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 Automated systems for the identification and

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AST of bacteria

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The automated systems available with

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FDA clearance include:

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 Vitek 2 (bioMérieux)

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 MicroScan (Beckman Coulter)

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 Phoenix (BD Diagnostic Systems)

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BD Phoenix™ M50

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 Dual AST technology: Turbidity and Redox thus

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ensuring reliability in detection of growth

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 Detection of delayed resistance with multiple

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algorithms

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 Detection of Resistance mechanisms

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 Urine-dedicated panels in two formulations
 Emerge panels with more antibiotics and /or

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M concentrations
 New CPO detect test for the confirmation of
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carbapenemase producing organisms
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 Compatibility with MALDI TOF

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 CHARACTERISTICS BD PHOENIXTM M50

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Principle of ID Chromogenic and fluorogenic biochemical substrates
Tests are modifications of classical biochemical micro-methods.

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Principle of AST MIC determination based on modified broth microdilution method
Each panel contains multiple wells with different antibiotics at serial two-fold dilutions

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Growth in different wells determines an MIC

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Dual AST technology: Turbidity AND Redox detection of organism growth and metabolism
Detection of delayed resistance with multiple algorithms

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Nr of ID Bacteria > 160 GN Taxa, > 140 GP Taxa

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Nr of ID Yeasts 64 Taxa
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Time to Result max. 16 hrs (average time to ID: 3hrs)
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OFF-LINE tests NO OFF-LINE TESTS are needed

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 CHARACTERISTICS BD PHOENIXTM M50

Inoculum for bacteria ID 0,5-0,6 McF

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Inoculum for yeasts ID 2-2,4 McF

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Type of Panels ID & AST Gram Negative (GN)
ID & AST Gram Positive (GP)

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ID & AST Streptococci No NH-ANC card

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ID Yeasts

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CLSI 5 panels (GN + GP + Streptococci)

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EUCAST 25 panels (GN + GP + Streptococci)

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Format of Panels ID/AST (combo)
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ID only No AST YEASTS
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AST only card

AST emerge (more antibiotics) – only EUCAST
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Nr of antibiotic dil. min. 3 dilutions

max. 8 dilutions
 CHARACTERISTICS BD PHOENIXTM M50

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New Antibiotics EUCAST panels w/ Ceftolozane/tazobactam, ceftazidime/avibactam

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Mechanism Resistance • ESBL + CPO screen (ID/AST format CLSI or EUCAST)

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Detection • ESBL + CPO screen (Emerge AST format CLSI or EUCAST)
GN including Ambler Class classification:

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KPC

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NDM
OXA

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Modularity Built-in modularity (increasing capacity with a small footprint)
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Compatibility EPICENTER (Data Management System)
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LIS
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Compatibility MALDI-TOFF BRUKER

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 Workflow

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1 2 3

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ID
4 M 5 6
1.Pick Colonies & Inoculate ID Broth
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2. Standardize suspension to 0.5-0.6 McF (2 McF for yeasts ID)
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3. Add one free-falling drop of the AST Indicator to the AST Broth
4. Transfer 25 ul of ID suspension to the AST Broth
5. Pour Broth into panel and cap
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VITEK 2 system BIOMERIEUX

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• Developed by McDonnell Douglas in
cooperation with NASA.

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• Released in the late 1970s

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• Intended for use in the direct detection of

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urinary pathogens by astronauts during space

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travel

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 Fifteen new microorganism IDs

Vitek-2 CARDS NH YST

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Riemerella
Cryptococcus gattii

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anatipestifer
Zygosaccharomyces s

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Histophilus somni
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• Menu expansion (More antimicrobials available for testing Actinobacillus
Candida auris

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pleuropneumoniae
of Streptococci sp.)
Candida
• 5-7 MIC doubling dilutions per antibiotic Actinobacillus suis
duobushaemulonii

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• Development of new antimicrobials (ex. Ceftaroline) Moraxella (Neisseria) Candida haemulonii

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ovis var vulnera
• Re-development of existing antimicrobials (imipenem and meropenem

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Neisseria weaveri
for improved detection of carbapenem resistance)

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• Addition of new phenotypes to AES database
– Bacterial AST results = 4 hours

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– Yeast AST results = 13 hours
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Table 1: Turbidity range for Card Inoculation

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BCL ID card
 Vitek- 2
Expert rules within the Advanced Expert System (AES)

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– Extensive Knowledge VITEK® 2 Systems 8.01 software

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Base • Updated Web-based, customizable software

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• Provides validation of every • Set up ID and AST cards remotely- Review and

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susceptibility test result approve isolate and patient information from any
web-enabled computer

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Knowledge base developed from

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– >100,000 references
– >2,000 described phenotypes

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– >20,000 MIC distributions
– >100 resistance mechanisms detected
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– >99 organisms
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• Deduced antibiotic results to meet
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formulary requirements
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Identification required for interpretation of BP &

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expert rules

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 Vitek-2: How is the MIC determined?

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The MIC is determined by comparing the growth of the
patient isolate to the growth of isolates with known MICs

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“Standard curves” in VITEK® 2:

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 Multidimensional
 Created using

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 >1800 gram-negative organisms

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 >1200 gram-positive organisms
 various resistance mechanisms

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• Does not provide a well for every MIC reported
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USE OF MULTIPLE PARAMETERS

TO CALCULATE THE REPORTED MICs
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 MicroScan WalkAway plus System

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(Beckman Coulter)

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• Large self-contained incubator/reader device that
can incubate and analyze 40–96 microdilution

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trays

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• Standard size microdilution trays inoculated

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manually and then placed in one of the incubator

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slots in the instrument.
• Periodical examination

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• Growth development is determined by photometer
or fluorometer
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 MicroScan WalkAway plus System

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• Conventional panels

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• Enterobacteriaceae, gram-negative non-
fermenters, staphylococci, enterococci

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• Combo, MIC-only, ID-only
• Rapid panels

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– 2 - 2.5 hour ID (pre-formed enzymes)

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– AST for Gram negative organisms (MIC < 4 hrs)

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• Synergies plus panels CLSI or EUCAST reporting guidelines
– 2 - 2.5 hour ID (pre-formed enzymes)
• Panels read by

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– Broth microdilution “Read-when-ready” MIC
– WalkAway® systems
– Resistance detection flagged in as few as 4.5
M – autoSCAN-4
hours – Manual
– All results finalized in 16/18 hours
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• Additional
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– Yeast ID, Anaerobe ID, HNID

– MICroSTREP plus ® - dry format
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– ESβL plus ® -ESβL Confirmatory Panel

 WalkAway – Panel Set-up

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1) Chose the panel 2) Print and label Panel with 3) Select colony 4) One suspension for ID and
barcode AST

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ID
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5) Pour Suspension 6) Purity control 7) Use RENOK to 8) Load Panel in the

into a seed tray inoculate panel MicroScan
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 Prompt Inoculation Preparation

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• One step inoculum preparation-

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• Obtain standard inoculations
• Easier than creating a 0.5 McFarland-based

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turbidity inoculum

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• Requires fewest number of isolated

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colonies

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• Frequently one colony is

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enough (esp. in GNs) M
– Less likely to pick up mixed culture
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• Inoculum stable for 4 hours, compared to

turbidity preparations which is recommended for Problems with :-
30 minutes Mucoid organisms
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 AST CARDS MicroScan MIC are correlated with ISO 20776

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0.06 0.12 0.25 0.5 1 2 4 8 16 32 64

MIC

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 The inoculum tested in the MicroScan wells is identical to the ISO 20776

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5 X 105 CFU/ml for the final concentrations tested

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AST “Direct Growth MIC”
Growth-based Direct check of growth

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non-ID-dependent method
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Detection of emerging or low levels of resistance
Reduction of confirmatory tests
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 MicroScan WalkAway plus System

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• Expert rules within

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LabPro Alert system

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– Pre-loaded

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– Customisable

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 Standard Alert comments

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are also on board

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 Can be customized
 Instructional text in the M
comments can aid staff in
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Flexibility-manual
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determining corrective
action. intervention
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©
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 CLSI - EUCAST breakpoints
GRAM POSITIVE

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panel

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• Up to 28 antibiotics
• Cefoxitin screening methicillin-

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resistant S. aureus (MRSA),

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• Inducible clindamycin resistance

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detection, ICD

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– Staphylococcus spp.
– Streptococcus spp.
– β- Hemolytic group

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• Low MIC vancomycin (0.25-16 M
µg/mL) for S. aureus
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90% of MRSA: 6.5 h

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75% of VRE: 8 h
 GRAM NEGATIVE PANEL

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• Up to 31 antibiotics

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• 4 carbapenems
• CLSI – EUCAST breakpoints

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• Lower dilutions of select antibiotics

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support evaluation of alternative

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therapeutic dosing regimens

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• Cefazolin: Indicator for use of per os
cephalosporins in no complicated

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UTI from Enterobacteriaceae
• Antibiotics per os M
– Step-down therapy
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– Outpatients
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©
 or
COMPARISON OF AUTOMATED SYSTEMS

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Microscan Vitek 2 Phoenix
GPOS
GPOS

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GNEG GPOS
GNEG NH
ID available NH GNEG
AnO2

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AnO2 Yeast
Yeast
Yeast

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GPOS GPOS
GPOS
AST available GNEG GNEG

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GNEG
Yeast
Continuous automated
Reading Continuous automated Continuous automated
or manual

ID
MIC Y +/- +/-
Expert system M Published rules Phenotypic comparison Published rules
Biochemical ID   
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Epidemiological software   
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PERFORMANCE OF AUTOMATED

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SYSTEMS –CLINICAL TRIALS

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ID
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PERFORMANCE OF AUTOMATED SYSTEMS

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The accuracy of identification systems

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is influenced by several factors:

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 the proportion of clinical versus Comparison of data from

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reference isolates evaluated multiple studies, is

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 the constituency and overall inherently problematic

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diversity of organisms used for
diagnosis

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 the reference or comparator method
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 whether “low‐discrimination” values
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are considered “correct” if rapid
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off‐line testing permits accurate

classification
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 PERFORMANCE

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Phoenix GP Panels Phoenix GN Panels

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 Phoenix NID card - Conventional biochemical testing

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• Concordance to the species level for Overall agreement:
streptococci, staphylococci and enterococci • 95% to the genus level

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– >90% compared to other phenotypic methods,
• 94% to the species level

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– 85-90%- compared to MALDIToF/MS and/or
WGS ID - Carroll K. et al. J. Clin. Microbiol. 44:3506–3509.

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 Phoenix NID card- MALDI TOF

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80–85% Accuracy
-Saffert, R. et al. 2011. J. Clin. Microbiol. 49: 887–892.

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Problems with :-
Mucoid organisms M  Phoenix NID card- MALDI‐ToF and/or 16S rRNA gene
sequencing
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> 1000 clinical isolates recovered from urine sources
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99.5% accuracy to the genus level

U. EIGNER, et all 11th ECCMID 2001 98.2% to the species level
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-Yan et al. J Clin Microbiol. 2011 Nov; 49(11): 3936–3939

 BD Phoenix Yeast ID Panel

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(Becton Dickinson, Sparks, MD)

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Posteraro B, et al. J Clin Microbiol. 2013 Nov; 51(11): 3841–3845.

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• The BD Phoenix system was evaluated for species-level identification of yeasts

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• 250 clinical isolates

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• Compared with the Vitek 2 system,
• Ref Meth: (ITS) sequence analysis

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• Results: Correct ID M
– BD Phoenix 96.3% (236/245)
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– Vitek 2: 91.4% (224/245)

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 VITEK ID Correct taxonomic assignment

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Vitek GP card

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• 85–95% of all strains tested

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– Eigner, U., et al J. Clin. Microbiol. 43: 3829–3834.

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• 92%-100% [44] for Enterococcus spp.

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– Gavin, P . 2002. Eur. J. Clin. Microbiol. Inf. Dis. 21: 869–874.

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Vitek GN card

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• > 90%
– Crowley E et al.J AOAC Int. 2012 May-Jun;95(3):778-85
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Vitek NH card
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• 85–90% of all strains tested

– Rennie et al. J Clin Microbiol. 2008 Aug; 46(8): 2681–2685.
©
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Bacteroides fragilis 97.5%
Anaerobes and Corynebacterium • 50 QC strains Clostridium ramosum 90.9 %

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Clostridium septicum 87.5 %
• McFarland #3 • 365 clinical isolates Clostridium tertium 71.4 %

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• Fresh colonies • Reference method: 6S Fusobacterium necrophorum 71.4 %

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Prevotella intermedia 66.7 %
• Anaerobe blood agar plate after rRNAgene sequencing Prevotella melaninogenica 75 %

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– 24 h - fast growing anaerobes Propionibacterium acnes 97.1 %
– 48–72 h - slower growing strains

ID
M
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ES
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 Vitek 2 YST system (bioMerieux)

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Candida auris

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Candida albicans
Candida boidinii Cryptococcus albidus

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Candida catenulata Cryptococcus laurentii
Candida colliculosa Cryptococcus neoformans
Correct identification: 85–98% Candida dubliniensis Cryptococcus terreus

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Candida famata Cryptococcus uniguttulatus
Candida freyschussii Geotrichum klebahnii
Misidentifications Candida glabrata Kloeckera spp.

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Candida guilliermondii Kodamaea ohmeri
 S. cerevisiae Candida haemulonii Malassezia furfur

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 C. tropicalis C. glabrata Candida inconspicua/Candida Malassezia pachydermatis
Millerozyma farinosa (Pichia farinosa)
lambica

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 C. krusei, Candida intermedia Prototheca wickerhamii
Candida kefyr Prototheca zopfii
 C. guilliermondii, C. parapsilosis

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Candida krusei
 Trichosporon sp. Candida lipolytica
Candida lusitaniae
Rhodotorula glutinis/Rhodotorula mucilaginosa
Rhodotorula minuta
Saccharomyces cerevisiae

ID
Candida magnoliae Saprochaete capitata (Geotrichum capitatum)
– The highest identification rate: Columbia Candida norvegensis Sporobolomyces salmonicolor
agar with sheep blood (86.8%) M Candida parapsilosis Stephanoascus ciferrii
– The lowest identification rate: Candida pelliculosa Trichosporon asahii
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Candida pulcherrima Trichosporon inkin
CHROMagar
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Candida rugosa Trichosporon mucoides

Candida sake Zygosaccharomyces bailii
Candida spherica
J Clin Microbiol. 2007;45(4):1087-92. Candida tropicalis
©

Candida utilis
J Clin Microbiol. 2008; 46(11): 3784–3787. Candida zeylanoides
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PERFORMANCE: Microscan GP-GN cards

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• Not enough data from literature

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• Coagulase-negative Staphylococci: 82.5%,

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– Misidentification was the main problem in MicroScan (10.8%)

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• BMC Microbiology 2008, 8:233

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• Enterobacteriacae: 91,6% M
• Enterococcus spp.: 92,3%
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– https://www.medigraphic.com/pdfs/invdis/ir-2017/ir173b.pdf
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 Microscan Rapid Anaerobe Panel-

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performance

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• Correct identifications

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166/237 strains (70%)

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• Misidentifications:

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Bacteroides fragilis,

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Pigmented anaerobic
Gram‐negative rods,
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M
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Anaerobic Gram‐positive
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bacilli
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– Stoakes et all. J of Clin Microbiol 990,p.1135-1138

 Yeast ID MICROSCAN performance

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St. Germain, G. et al. 1991. J. Clin. Microbiol. 29: 2296–2299

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• 357 isolates – Microscan compared to the API

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• ID of 42 yeasts and
yeast‐like organisms 20 C AUX system,
Results determined after 4 h

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• Chromogenic and modified
• 94.1% correlation

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conventional tests
– Candida,
• Mc Farland >3

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– Hansenula,
• Fresh yeast isolate – Pichia,

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– Rhodotorula, and
• Incubation 4 h at 35–37°C. – Saccharomyces

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• Manually readings - • 65% correlation for fastidious species,
Autoscan instrument M – Trichosporon spp., and yeast‐like organisms,
– Microscan Rapid Yeast Results obtained after 72 h
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Biotype Codebook • The overall correlation: 85%
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– 78% / isolates with no supplementary tests.

– 96.6% with supplementary tests,
©
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Examined160 clinical isolates and 50 reference strains.

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Correctly identified

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• Clinical strains: VITEK 2, MicroScan, and Phoenix at the species level

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93.7%, 82.4%, and 93.0%

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• Reference strains: VITEK 2, MicroScan, and Phoenix correctly identified

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55.3%, 54.4%, and 78.0% of the at the species level

ID
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• Carried out: 2002 – 2014/ United States

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• 11,020 multidrug-resistant clinical isolates

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– A. baumannii, K. pneumoniae, P. aeruginosa, E. coli, and MRSA

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• ID /AST platforms Vitek- Microscan- Phoenix
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• Reference Method: Pulsed-field gel electrophoresis, multilocus sequence
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typing, PCR, and whole genome sequencing (WGS)

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Identification

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• All 3 platforms agreed at the species level for more than 99%

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• MicroScan and Phoenix misidentified 52/11.149

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• Vitek-2 misidentified 5/11.149

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• MicroScan misidentified A. baumannii more often than Vitek-2 or Phoenix
– reporting Shigella species in 16/1363 (1.2%)

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• MRSA was the least likely to be misidentified by any platform
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©
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ID
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AST GRAM NEGATIVES

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 ANTIMICROBIAL SUSCEPTIBILITY

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 Overall, MicroScan had the highest

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number of discrepancies // a 2-fold higher E. coli / Nitrofurantoin/ Vitek-2
MIC: IR, SI  P. aeruginosa/ Ciprofloxacin / Phoenix

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A. baumannii/ Tobramycin/ Vitek (VMD error rate)
 Cefepime more MDs and VMDs on the

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K. pneumoniae/ Aztreonam/ Phoenix (VMDs)
MicroScan and Vitek-2
K. pneumoniae/ Imipenem/ MicroScan

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 Vitek-2 reported a higher proportion of K. pneumoniae/ Ciprofloxacin/ Trimethoprim-

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VMD with E. coli against sulfamethoxazole/Vitek-2
sulbactam/ampicillin, aztreonam,

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ceftazidime, and ceftriaxoneM
 Vitek-2 often underestimated the MIC
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values of E. coli and K. pneumoniae
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©
 AST GRAM POSITIVES

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MRSA

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• Most errors were mDs and were drug, not platform, dependent.
For MDs, M
• MicroScan and Phoenix  more likely than Vitek-2 to show discrepancies for tetracycline and vancomycin
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• MicroScan > MDs for rifampicin, moxifloxacin, levofloxacin, erythromycin, and clindamycin
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• Vitek-2: higher MD rate for daptomycin

©
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• 81 clinical CRE isolates 2011-2012

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• Presence of carbapenemase genotypes-Ref. Method: PCR and sequencing

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• The sensitivities/specificities of

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– MicroScan WalkAway 93.8/42.4%,

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– Phoenix 100 54.2/66.7%,

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– Vitek-2 75.0/36.4%
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MicroScan – Vitek 2 systems are more reliable in clinical identification of CRE-
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additional tests are required for the Phoenix 100 system
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©
 ANAEROBES AND AUTOMATION

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General Limitations …

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PREPARATION OF THE INOCULUM

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• Use of nonselective media

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– Trypticase soy agar,

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– Brucella, brain heart infusion,
– Columbia;

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– 5% sheep blood (only).

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• No use of selective media that contain antibiotics

ID
PRIOR TO TESTING
M
• Test for aerotolerance, purity, Gram‐stain
morphology
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ES

• Supplementary tests may be necessary

• Fastidious anaerobes require several blood agar
©

plates in order to provide Mc Farland=3

 CANDIDA AURIS DETECTION?

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ORGANISM C. AURIS CAN BE
IDENTIFICATION METHOD MISIDENTIFIED AS

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Vitek 2 YST Candida haemulonii
Candida duobushaemulonii

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BD Phoenix yeast ID Candida haemulonii

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Candida catenulata

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MicroScan Candida famata
*

ID
Candida guilliermondii
*
M Candida lusitaniae
*
Candida parapsilosis
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Centers for Disease Control and Prevention, National Center for Emerging and Zoonotic Infectious Diseases
©

(NCEZID), Division of Foodborne, Waterborne, and Environmental Diseases (DFWED)

 ONLY BMD should be used for

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colistin MIC determination

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AST of Colistin?? (ISO-20776)

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• Technical challenges

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• Propensity to adsorb to
polystyrene,

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• Addition of a surfactant such as

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polysorbate 80; may act
synergistically with the

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polymyxins  artificially LOWER

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MIC
• Polymyxins diffuse poorly

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through agar
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– Agar dilution: to be evaluated
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– Disk diffusion: false
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susceptibility (up to 35%)

– Gradient Diffusion: false
susceptibility up to 32%
©

J Clin Microbiol. 2017 Sep;55(9):2609-2616

Tigecycline MIC can vary depending on test method

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o Vitek 2 and Etest vs reference BMD with 48 KPC+

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K. pneumoniae
o Vitek 2 overestimates TIG MICs (10% of major errors, 25% of minor
ID
errors) and underestimates MER and FEP MICs (27% and 67% of very
M
C
major errors)
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o Etest slightly overestimates TIG MICs (10% of minor errors)

©
 Tigecycline MIC can vary depending

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on test method

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o Vitek 2 and Etest vs reference BMD for TIG testing with 241 MDR Gram-negative
pathogens (ESBL, CRE, CRA)

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o Vitek 2 overestimates TIG MICs (26% of major errors and 47% of minor errors with CRE)

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o Etest slightly overestimates MICs (1% of major errors and 34% of minor errors with CRE)
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Vitek 2 TIG results require confirmation with BMD
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when testing MDR Gram-negatives

©
 CONCLUSIONS

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BENEFITS HOWEVER....

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 Only for standardized bacteria

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• Latest ID panels offer an enhanced testing
 Not for fastidius bacteria
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 Discrepancies in ID of atypical phenotype:

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• Lower dilutions of select antibiotics support mucous morphology of colonies

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evaluation of alternative therapeutic dosing  Vitek - not visual MIC
regimens  No detection of heterogeneity

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• Additional oral drugs provide options for  Limited MIC range

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step-down therapies and outpatient  Technical issues should be promptly
management recognized

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• Multiple options of MIC panel selections
M  McFarland 0.5 turbidity-ID issues especially in
complement workflows for laboratories GP
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 ID/AST performance relies on
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– Correct ID,
– Correct AST
– Appropriate expert interpretation
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• Incorrect ID can lead to incorrect AST

Ερωτήσεις ;
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ID
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Ευχαριστώ !

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