Postgraduate Medicine

ISSN: 0032-5481 (Print) 1941-9260 (Online) Journal homepage: http://www.tandfonline.com/loi/ipgm20

Direct Gram staining and its various benefits in the

diagnosis of bacterial infections

Lyudmila Boyanova

To cite this article: Lyudmila Boyanova (2017): Direct Gram staining and its various benefits in the
diagnosis of bacterial infections, Postgraduate Medicine, DOI: 10.1080/00325481.2018.1398049

To link to this article: http://dx.doi.org/10.1080/00325481.2018.1398049

Published online: 01 Nov 2017.

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 POSTGRADUATE MEDICINE, 2018
https://doi.org/10.1080/00325481.2018.1398049

CLINICAL FEATURE
REVIEW

Direct Gram staining and its various benefits in the diagnosis of bacterial infections
Lyudmila Boyanova
Department of Medical Microbiology, Medical University of Sofia, Sofia, Bulgaria

ABSTRACT ARTICLE HISTORY

In the era of rapid development of molecular and other diagnostic methods, direct Gram staining (DGS) Received 9 August 2017
tends to remain in the background, although it can provide both microbiologists and clinicians Accepted 24 October 2017
numerous benefits. The aim of this review was to emphasize the importance of DGS for the diagnosis KEYWORDS
of many clinically important infections. A PubMed search was carried out using relevant keywords for Diagnostic method; infection
articles published primarily since 2010. The DGS can provide early information for a timely diagnosis of control; sensitivity and
infections, can reveal the causative agents of the infections even under suboptimal conditions of specificity; benefits; health;
Downloaded by [Cornell University Library] at 08:38 02 November 2017

specimen collection, transport or identification methods, can detect the presence of rare/unusual bacterial infections
pathogens, moreover, the method shows the specimen quality, by distinguishing between contamina-
tion and true infection, it can direct or change initial antibiotic treatment before the availability of
culture results, can indicate the need of other methods for pathogen identification and, in some cases,
can show the need for emergency attention such as urgent antibiotic therapy and surgical measures.
Briefly, the DGS remains an easy, rapid, inexpensive and important method, which use should be
encouraged in conditions of a standardized and controlled performance to avoid technical or inter-
pretation errors.

Introduction bacterial infections rather than on the technique itself and

The 133-year-old Gram staining technique reveals bacterial
morphology (cocci, rods, or spiral-shaped bacteria) and distin-
guishes between the gram-positive (violet-stained) and gram-
negative (red-stained) bacteria according to their cell wall
structure (the differences in the peptidoglycan layer thickness)
and permeability [1]. The method is used in many laboratories
due to its simplicity of execution and rapidity (within less than
5 min). Direct Gram staining (DGS), if correctly performed and
well interpreted, provides numerous benefits to both micro-
biologists and clinicians [1] (Table 1). However, in the years of
extensive use of rapid molecular and other diagnostic meth-
ods, the DGS can appear less important. Therefore, in this
review, recent data about the DGS utility are presented and
discussed, although the use of the method is not considered a
research subject anymore, particularly in molecular microbiol-
ogy and some papers which may have contributed are not
recorded in recent papers.
PubMed search was performed using relevant keywords
for articles published mainly since 2010, which constitute
for 80.0% (44/55) of the references. The search in the litera-
ture was made using the following key words: ‘Gram stain-
ing,’ ‘direct Gram stain,’ ‘microscopic examination,’
‘diagnosis,’ ‘infection,’ ‘antibiotic,’ ‘treatment,’ ‘sensitivity,’
‘accuracy,’ and ‘utility.’
This review focuses on diagnostic, clinical, and therapeutic
benefits of the DGS in the control of clinically important
discusses mostly the recent benefits of the method.
Biological fluids
DGS use is always important in evaluation of normally sterile
clinical specimens such as blood culture, cerebrospinal fluid,
pleural, peritoneal and joint fluids. In the normally sterile
specimens, the method reveals presumptive bacterial patho-
gens and even unusual or rare pathogens. The use of mole-
cular methods is highly accurate and informative, but
frequently cannot completely cover the spectrum of all possi-
ble pathogens, especially the rare or unusual pathogens. For
instance, multiplex PCR for pathogen detection in emergency,
trauma or burn surgery patients with suspicion of sepsis
missed or did not involve Acinetobacter baumanii, some
Bacillus spp. and Chryseobacterium meningioseptium [2].
However, in some studies, the DGS was helpful in the diag-
nosis of brain and spinal epidural abscesses caused by the
gram-positive anaerobic cocci, Parvimonas micra, as well as
Listeria bacteremia and meningitis and bacteremia caused by
the gram-negative spiral-shaped Campylobacter fetus [3–5]. In
such cases, the DGS can call the attention and can point to the
next necessary tests for the accurate diagnosis of the infection.
The DGS also can distinguish between bacterial and fungal
infections, for which the treatment agents are completely
different [6–8].

CONTACT Lyudmila Boyanova l.boyanova@hotmail.com Department of Medical Microbiology, Medical University of Sofia, Zdrave Street 2, 1431, Sofia,
Bulgaria
© 2017 Informa UK Limited, trading as Taylor & Francis Group
 2 L. BOYANOVA

Table 1. Benefits of the direct Gram staining. Bacteremia and meningitis

Benefits of the direct Gram
staining
Shows presumptive microbial
pathogens
Can establish the diagnosis
Detect unusual/rare, fastidious
or slowly growing pathogens
Determines quality of the
specimen
Distinguishes between true
infection or contamination
Helps with the choice of other Anaerobic infections, Campylobacter
identification methods
Helps with the choice of initial Bacteremia, ventilator-associated
antibiotic therapy
Indicates the need for
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Examples for the benefits in different
infections
Bacterial meningitis, bacteremia, pneumonia,
gonorrhea in men, campylobacteriosis,
bacterial vaginosis etc.
Gonorrhea in men, H. pylori infection,
pneumococcal pneumonia
Anaerobic infections, actinomycosis,
Campylobacter fetus bacteremia, Listeria
bacteremia and meningitis, gastric non-
pylori Helicobacter species, fungal
specimens
Pneumonia, wound infections, Helicobacter
pylori infection.
Pneumonia, urinary tract infections,
wound infections
infections
pneumonia
Meningitis, bacteremia, anaerobic
Bacteremic sepsis is a severe disease and both timeliness
and accuracy of preliminary blood culture results are impor-
tant for the clinical outcome [11]. Interpretation of blood
culture DGS has been usually correct [11–13]. Schifman
et al. [11] found an excellent (98.8%) agreement between
preliminary DGS and culture results among 5031 blood
cultures evaluated in 64 laboratories, with the highest dis-
crepancy among mixed cultures (20.8%). Time to report DGS
results ranged from 27 min to >2.5 h [11]. The authors
emphasized the need for continuous blood culture monitor-
ing and the benefit of reporting all preliminary blood cul-
ture results to patients’ caregivers [11]. Moreover,
Barenfanger et al. [12] found sepsis mortality to decrease
by 17% when DGS results were reported within 1 h of blood
culture positivity. In neonates in intensive care, DGS from

emergency attention infections, including gas gangrene
Indicates the need for antibiotic Meningitis, bacteremia, anaerobic
treatment/surgery infections, including gas gangrene
Can be a good adjunct of newer Gram staining and MALDI-TOF sequential
diagnostic methods technique for UTIs
It is simple, easy, and cost effective All infections
MALDI-TOF: matrix-assisted laser desorption ionization-time of flight mass spec-
trometry; UTIs: urinary tract infections.
To fully benefit the DGS, some recommendations about
the specimen collection and transport should be followed:
to take appropriate specimens (e.g. tissues and aspirates are
better than swabs) from the site of the infection (e.g. spu-
tum instead of saliva in case of pneumonia) and, whenever
possible, before the start of antibiotic therapy, to avoid the
specimen contamination by the normal flora of the infection
site, to use transport media for detection of anaerobic,
microaerophilic and fastidious bacteria or in case of delayed
transport (>2 h for most specimens, while immediate trans-
port is required for cerebrospinal fluid specimens), and to
provide data about the patient’s characteristics, infection,
comorbidities, and current treatment [9,10].
blood from catheters helped with diagnosis of catheter-
associated bloodstream infections with sensitivity and spe-
cificity of 80.0 and 99.4%, respectively [14]. Notably, by
blood culture DGS, bacteria or fungi were detected even
when the specimens were reported negative by automated
blood culture systems [15]. The data highlight both the
need and the usefulness of reporting positive DGS results
to the clinicians within minutes to hours.
The DGS is required in diagnosis of meningitis caused by
Streptococcus pneumoniae, Neisseria meningitidis, and
Haemophilus influenzae. Common DGS sensitivity for the diag-
nosis of bacterial meningitis was 60–>80% in untreated
patients, while it decreased to 40–60% in treated patients
[16]. The results stress the importance of sending the clinical
specimens to the microbiology laboratory before the start of
the antibiotic therapy.
In a study, both sensitivity and specificity of the DGS from
cerebrospinal fluid specimens were similar to those (>94%) of
real-time PCR [17]. Diagnostic parameters of several diagnostic
methods are given in Table 2.
The volume of the clinical specimen and the transport are other
factors that can influence the performance and the results of the

Table 2. Diagnostic parameters of direct Gram stain (DGS), culture, MALDI-TOF MS, and polymerase chain reaction (PCR) according to some studies.
No of patients/ Sensitivity Specificity % positive in culture-negative
Suspected disease Specimens Method samples (%) (%) cases/notes Reference
Bacteremia Blood culture DGS 5021 NA NA Discrepancy, 1.2 [11]
CRBSIs Blood samples (catheter-drawn) DGS 397 80.0 99.4 PPV, 93.3, [14]
NPV, 98.1
Meningitis CSF Culture 451 81.3 99.7 NA [17]
(S. pneumoniae, N. CSF DGS 451 98.2 98.7 5.9 (22/371) [17]
meningitidis, CSF RT-PCR 451 95.7 94.3 10.0 (37/371) [17]
H. influenzae)
VAP Bronchoalveolar lavage, DGS Meta-analysis 79.0 75.0 Good NPV (91), low PPV (40) [18]
endotracheal aspirate (21 studies)
UTIs Urine DGS 1000 81.3 93.2 2.5 [19]
(discrepancy, 10.0)
Urine MALDI-TOF 84 79.2 73.5 NA [19]
MS
UTIs Urine DGS NA (review) 91.0 96.0 NA [20]
(children)
Suspected intra- Amniotic fluid DGS 131 44.8 97.6 NA [21]
amniotic infection
Bacterial vaginosis Vaginal discharge DGS Multicenter study 89.0 83.0 NA [22]
CRBSIs: catheter-related bloodstream infections; CFS: cerebrospinal fluid; RT-PCR: real-time PCR; NA: non-available/applicable; VAP: ventilator-associated pneumonia;
UTIs: urinary tract infections; MALDI-TOF MS: Matrix-Assisted Laser Desorption (with extraction); NPV: negative predictive value; PPV: positive predictive value.
 POSTGRADUATE MEDICINE 3

diagnostic techniques. To obtain good DGS and culture results,
≥0.5–1 ml of CSF (up to 10 ml for mycobacteria or fungi) should be
taken in sterile containers and should immediately be sent to the
microbiology laboratory [16]. DGSs of CSF should be made after
centrifugation and positive results should be reported to the
clinicians without any delay [16].
Pneumonia
DGS of sputum/endotracheal aspirates can indicate common
causative agents of pneumonia. The method also reveals the
specimen quality, distinguishing between lower respiratory
tract specimens (scanty squamous epithelial cells and abun-
dant polymorphonuclear cells-PMNs by low-power field micro-
scopy) and oropharyngeal contamination (prevalence of
epithelial cells and scanty/absent PMNs) [23]. In a recent
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magnification of 1000×, corresponding to about 105 colony
forming units/ml [31].
In a recent study [19] about sequential performance of Gram
staining and MALDI-TOF MS mass spectrometry on 1000 urine
samples from patients with suspected UTIs, the Gram staining
and MALDI-TOF diagnostic accuracy was 90.0 and 78.3%, respec-
tively, and the results of both methods correlated with those of
the culture in 82.7% [19]. An advantage of the sequential tech-
nique was the availability of results within only 1 h [19].
Clinical utility of urine DGS is a still controversial topic [32].
Although DGS is not the preferable method to detect leuko-
cytes in urine specimens, it can reveal high numbers of Gram-
negative bacteria in patients, including children with UTIs
[16,20]. DGS from non-centrifuged urine had high sensitivity
(91%) and specificity (96%) to detect UTIs in children, when
pyuria and bacteriuria were associated with observation of
≥10 leukocytes/mm3 and bacteria [20]. However, it is neces-

study, the Gram staining revealed Streptococcus pneumoniae

sary to refrigerate the urine specimen at 4°C if the transport to
(gram-positive diplococci) in only 31% of 105 cases with
the laboratory is delayed for >1 h and to minimize perineal
pneumococcal pneumonia, however, when the specimens
flora contamination [16].
were adequate and the patients received antibiotics for
Notably, DGS is highly (near 100%) specific to detect gonor-
≤24 h, the proportion of correct diagnosis increased to 63%
rhea (Neisseria gonorrhoeae infection) in men. For diagnosis of
[24]. The concordance between DGS and culture results for
gonococcal urethritis in men, the DGS exhibited both sensi-
some specific organisms/groups is given in Table 3.
tivity and specificity of >97% in the study of Taylor et al. [28].
Moreover, in some cases, the technique can help with the
Overall concordance between DGS and PCR results for diag-
use of narrow-spectrum instead of broad-spectrum antibiotics,
nosis of gonorrhea in men was 99.4% in a study in Kuwait [33].
thus reducing the risk of appearance of multidrug resistant
It should be taken into account, however, that in women, the
bacteria. In patients with ventilator-associated pneumonia,
DGS sensitivity was much lower (≤32.0%) compared with that
antibiotic treatment based on bedside DGS results of endo-
in men (≥95.4%) [34]. Furthermore, the use of meatal swabs is
tracheal aspirates was successfully used to choose appropriate
unadvisable to perform DGS of urethral swabs from men
initial antibiotic therapy, thus avoiding broad-spectrum anti-
because they do not collect enough material [35].
biotic administration [29]. In patients with aspiration pneumo-
For bacterial vaginosis, DGS has been reported to be more
nia, the presence of PMNs, fusiform and coccobacillary
specific than the culture or probe hybridization and this is
bacteria and cocci can indicate the presence of anaerobic
important because many of the associated microorganisms
infections [30]. Therefore, the DGS can show the need for
cannot be cultured [16].
additional diagnostic methods such as anaerobic evaluation
Amniotic fluid is considered to be sterile and testing of
of the clinical specimen.
amniotic fluid with culture, molecular methods and interleu-
kin-6 concentrations are recommended for the diagnosis of
intra-amniotic infections [36]. Given that intra-amniotic infec-
Urinary tract, sexually transmitted, and intra-amniotic tion is a major cause of sepsis and that clinicians may only
infections have a Gram stain in low resource countries, they can use the
Urine DGS has been used to detect urinary tract infections method exhibiting an excellent specificity (97.6–98.5%)
(UTIs) if exhibiting ≥1–2 bacterial cells per field at a although a low sensitivity (23.8–44.8%) [21,36].

Table 3. Direct Gram stain results associated with culture results for some specific microorganisms/groups.
No. of Sensitivity Specificity
Infection/conditions Specimen Microorganisms cases (%) (%) Reference
Meningitis CSF Neisseria meningitidis 451 98.7 99.6 [17]
CSF Streptococcus 451 96.4 99.0 [17]
pneumoniae
Pneumococcal pneumonia with Sputum (adequate specimens, all patients) Streptococcus 74 45.0 NA [24]
bacteremia pneumoniae
Sputum (adequate specimens, untreated Streptococcus 15 80.0 NA [24]
patients) pneumoniae
VAP Endotracheal aspirates Gram-positive cocci 114 90.5 82.5 [25]
Endotracheal aspirates Gram-negative rods 114 69.6 77.8 [25]
Gastroduodenal diseases Gastric biopsy (untreated patients) Helicobacter pylori 1441 72.2–74.2 77.8–79.7 [26]
Gastric biopsy (treated patients) Helicobacter pylori 270 58.8–65.7 83.8–92.9 [26]
Diarrhea Stool samples Campylobacter spp. 585 63.6 100.0 [27]
(children)
Suspected gonococcal urethritis Male urethral specimens Neisseria gonorrheae 307 97.3 99.6 [28]
CFS: cerebrospinal fluid; VAP: ventilator-associated pneumonia.
 4 L. BOYANOVA

Wound infections H. pylori isolation strongly depends of gastric biopsy num-

ber, time and conditions of transport and microaerophilic
In wound infections, DGS can detect either infection by pre-
incubation, use of several media for isolation, previous
sence of inflammatory cells and bacteria or superficial contam-
patient’s use of antibiotics or proton pump inhibitors, never-
ination by predominance of squamous epithelial cells [16]. The
theless, gastric biopsy DGS can reveal the presence of
method is especially useful in wound infections, given that
Helicobacter-like organisms even under suboptimal conditions.
only about 10% of microbes can be detected by culture [37].
In our previous studies [26,44], the accuracy of direct stain was
It is important to consider DGSs when bacteria of the
>73% in both nontreated and treated adults and untreated
normal skin/mucosal flora are detected, before discarding
children versus >63% for that of an in-house rapid urease test.
them as contaminants, especially if neutrophils also are
It is important, however, to observe many (if necessary, >10)
observed [38,39]. The presence of Cutibacterium (ex-
microscopic fields because of patchy H. pylori distribution in
Propionibacterium) acnes and Corynebacterium striatum in CSF
the gastric mucosa [45].
shunt specimens, implant-associated infections and sterile
Furthermore, by DGS, we observed gastric non-pylori
sites, as well as that of Cutibacterium (ex-Propionibacterium)
Helicobacter species (NPHS) in 0.3% of 1762 gastric biopsy
avidum and Actinomyces spp. in wound specimens should be
specimens from patients with gastroduodenal diseases [these
carefully considered [38,39].
are the author’s unpublished data]. These bacteria of animal
origin did not grow on the media for H. pylori but revealed 4–6
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spiral shapes and larger size compared with H. pylori. Similar

Anaerobic microbiology
NPHS prevalence was found in Iran [46]. The NPHS are clini-
DGS is of high value in anaerobic microbiology. Some anaero- cally important, being associated with a 10-fold higher (1.5–
bic species have a specific and easily distinguishable morphol- 10%) risk for development of MALT lymphoma compared with
ogy. For instance, Fusobacterium nucleatum are thin needle- that of H. pylori infection (<1%) [47].
shaped Gram-negative cells with tapered ends, Bacteroides/
Parabacteroides spp. can present as pleomorphic Gram-nega-
tive bacteria with swellings or vacuoles and Actinomyces spp. Other infections
are branching gram-positive rods, although a similar appear-
Campylobacter is the most common bacterial cause of human
ance can be shared by Bifidobacterium or Cutibacterium/
gastroenteritis worldwide and the causative agent of ¼ of all
Propionibacterium spp. [40].
diarrheal diseases [48]. Although DGS is usually not performed
The DGS is of particular importance in cases of suspected
on stool specimens, in the case of Campylobacter infections, it
gas gangrene that requires emergent surgical and empirical
can be helpful because of the characteristic spiral shape of the
antibiotic treatment [30]. The most common causative agent
bacteria. In the study of Ghosh et al. [27], DGS had a higher
of the rare but rapidly progressing and life-threatening infec-
(63.6%) sensitivity than that of the culture (37.2%), although
tion, Clostridium perfringens, shows large boxcar-shaped
lower than that of PCR (96.7%). Similar results (DGS sensitivity
square gram-positive rods without visible spores [30,40].
of 64.3% with a specificity of 93.4%) have been reported by
However, in contrast to other infections, in the case of gas
Mushi et al. [49]. Therefore, the detection of abundant gram-
gangrene, no PMNs are visible because of the powerful toxi-
negative spiral-shaped bacteria and inflammatory cells in
genic and enzymatic activities of the clostridia [30,41].
DGSs from feces can be suggestive for campylobacteriosis.
In case of detection of bacterial morphotypes suggesting
This is of value because treatment of Campylobacter infections
anaerobic infection, anaerobic incubation of the specimen and
is different from that for other causative agents of enterocolitis
use of appropriate media are required. Moreover, detection of
and the drugs of choice for severe Campylobacter infections
clostridia-like bacteria in association with clinical data for
are macrolides such as azithromycin [50,51].
severe soft-tissue infection implies surgical debridement and
The DGS also can reveal fungal infections and the most
prompt empirical treatment by penicillin and clindamycin for
common fungal causative agents, Candida spp. appear as vio-
gas gangrene or by broad-spectrum antibiotics for mixed
let-stained microbes larger than the staphylococci. However, for
infections, and the early treatment can be life-saving [42].
other fungal genera such as Cryptococcus spp., different staining
This is highly important since mortality rate in patients with
methods such as India ink staining are recommended [52].
gas gangrene with bacteremia was >50% [42].
DGS can reveal the presence of branching gram-positive
rods and the so-called sulfur granules (Actinomyces microcolo-
Errors and optimization of the technique
nies) in patients with persistent/recurrent abscess and draining
sinuses, suggesting the rare infection, actinomycosis [43]. In this Reasons for DGS errors or misinterpretation can be the poor
case, a prolonged (7–14-day) incubation of the clinical speci- quality or contamination of the clinical specimen, characteristics
men in anaerobic or microaerophilic atmosphere is needed, of the bacteria themselves, mixed infections, antibiotic treat-
and, furthermore, the therapy should be between 1 and ment of the patient, technical reasons (in fixation, over- or
4 weeks to even 6 and 12 months in order to be curative [43]. under-decolorization) and reader misinterpretation [1,13,53].
Standard Gram staining technique is performed for anae- Rand and Tillan [13] detected misreading of Gram stains
robes; however, the safranin is better to be substituted by from blood cultures in only 0.7% of 8253 patients, most often
0.1% basic fuchsine [40]. in mixed infections or with gram-positive cocci. However, the
Helicobacter pylori and non-pylori Helicobacter infections DGS misinterpretation errors led to serious clinical

consequences such as treatment delay (in four cases) and a
fatal outcome (in two cases) [13].
In a recent multicenter study [53] on 6115 clinical specimens
from four medical centers, discrepancy between Gram stain and
culture results were found in 4–6% of all specimens. Reader
errors were found in 24% (9–45%) of the discrepant results [53].
Some examples of misinterpretations have been: reporting
Enterococcus faecalis as S. pneumoniae in blood culture,
Acinetobacter baumannii as Moraxella catarrhalis in sputum,
Prevotella and Porphyromonas spp. as Haemophilus influenzae,
etc. [54]. Because of their needle-shaped morphology, micro-
aerophilic Capnocytophaga and Leptotrichia spp. resemble the
anaerobic F. nucleatum [40,54]. Importantly, DGS results
should be considered in the context of all clinical and diag-
nostic data available.
On the other hand, DGS should not be overlooked as a
routine method in the microbiology laboratory since it can
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easily be improved. For instance, using centrifuged instead of
un-concentrated body fluid smears can increase DGS sensitiv-
ity by almost 2 logs [31]. Sputum DGS detects better pneu-
mococcal pneumonia if specimens are not heavily
contaminated and are collected before the start of antibiotic
therapy [24]. It is also crucial to optimize the technique by
spreading the specimens onto the glass to obtain cell mono-
layers instead of clumps (which can hinder the decolorization
process) by the recommended methanol fixation instead of
heat fixation and by using basic fuchsine as a counterstain if
an anaerobic infection is suspected [31,40,53]. The control of
the DGS is easy if a mixed culture of Staphylococcus spp. and
Escherichia coli is used in parallel [55].
Importantly, the usefulness of the GDS depends on the
efforts of both microbiologists and clinicians and their con-
stant and good communication.
Conclusion
In conclusion, if well performed and interpreted, DGS provides
many benefits to both microbiologists and clinicians: it facil-
itates the early diagnosis of most bacterial infections, can
detect presumptive bacterial pathogens, including rare or
unusual microorganisms, determines the quality of the speci-
men and distinguishes between true infection and specimen
contamination, helps with choosing initial antibiotic therapy
and avoiding unnecessary broad-spectrum antibiotic use or, in
some cases, indicates the need for urgent treatment. Even in
the era of rapidly emerging new diagnostic methods, the DGS
remains an important helping tool in the infection control.
Funding
This work has no funding.
Declaration of interests
The author has no relevant affiliations or financial involvement with any
organization or entity with a financial interest in or financial conflict with
the subject matter or materials discussed in the manuscript. This includes
employment, consultancies, honoraria, stock ownership or options, expert
testimony, grants or patents received or pending, or royalties.
POSTGRADUATE MEDICINE 5
References
1. Thairu Y, Nasir IA, Usman Y. Laboratory perspective of gram stain-
ing and its significance in investigations of infectious diseases. Sub-
Saharan Afr J Med. 2014;1:168–174.
2. Tran NK, Wisner DH, Albertson TE, et al. Multiplex polymerase chain
reaction pathogen detection in patients with suspected septicemia
after trauma, emergency, and burn surgery. Surgery. 2012;151:456–463.
3. Wagenaar JA, Van Bergen MA, Blaser MJ, et al. Campylobacter fetus
infections in humans: exposure and disease. Clin Infect Dis.
2014;58:1579–1586.
4. McKinney JS. Listeria infections in neonates. NeoReviews. 2016;17:
e515–e520.
5. Shtaya A, Schuster H, Riley P, et al. Oesophageal pleural fistula
presenting with Parvimonas micra infection causing cervical and
brain abscesses. Anaerobe. 2017;47:233–237.
6. Deepa R, Banu ST, Jayalakshmi G, et al. Pleuropulmonary nocardio-
sis due to Nocardia otitidiscaviarum in a debilitated host. Indian J
Pathol Microbiol. 2016;59:240–242.
7. Gassiep I, Douglas J, Playford EG. First report of monomicrobial
Candida parapsilosis necrotizing fasciitis. Transpl Infect Dis.
2016;18:752–755.
8. Spec A, Santos CA, Pence MA. The Brief Case: dyspnea in a pro-
foundly immunocompromised man. J Clin Microbiol. 2016;54:2410–
2412.
9. Baron E, Thomson R. Specimen Collection, Transport, and
Processing: bacteriology. In: Versalovic J, Carroll K, Funke G, et al.,
ed. Manual of Clinical Microbiology. 10th ed. Washington, DC: ASM
Press; 2011. p. 228–271.
10. Spichler A, Hurwitz BL, Armstrong DG, et al. Microbiology of dia-
betic foot infections: from Louis Pasteur to “crime scene investiga-
tion.”. BMC Medicine. 2015;13:2.
11. Schifman RB, Meier FA, Souers RJ. Timeliness and accuracy of
reporting preliminary blood culture results: a College of American
Pathologists Q-probes study of 65 institutions. Arch Pathol Lab
Med. 2015;139:621–626.
12. Barenfanger J, Graham DR, Kolluri L, et al. Decreased mortality
associated with prompt Gram staining of blood cultures. Am J
Clin Pathol. 2008;130:870–876.
13. Rand KH, Tillan M. Errors in interpretation of Gram stains from
positive blood cultures. Am J Clin Pathol. 2006;126:686–690.
14. Deleers M, Dodémont M, Van Overmeire B, et al. High positive
predictive value of Gram stain on catheter-drawn blood samples
for the diagnosis of catheter-related bloodstream infection in
intensive care neonates. Eur J Clin Microbiol Infect Dis.
2016;35:691–696.
15. Peretz A, Isakovich N, Pastukh N, et al. Performance of Gram stain-
ing on blood cultures flagged negative by an automated blood
culture system. Eur J Clin Microbiol Infect Dis. 2015;34:1539–1541.
16. Baron EJ, Miller JM, Weinstein MP, et al. A guide to utilization of the
microbiology laboratory for diagnosis of infectious diseases: 2013
recommendations by the Infectious Diseases Society of America
(IDSA) and the American Society for Microbiology (ASM)(a. Clin
Infect Dis. 2013;57:e22–121.
17. Wu HM, Cordeiro SM, Harcourt BH, et al. Accuracy of real-time PCR,
Gram stain and culture for Streptococcus pneumoniae, Neisseria
meningitidis and Haemophilus influenzae meningitis diagnosis.
BMC Infect Dis. 2013;13:26.
18. O’Horo JC, Thompson D, Safdar N. Is the gram stain useful in the
microbiologic diagnosis of VAP? A meta-analysis. Clin Infect Dis.
2012;55:551–561.
19. Burillo A, Rodríguez-Sánchez B, Ramiro A, et al. Gram-stain plus
MALDI-TOF MS (Matrix-Assisted Laser Desorption Ionization-Time
of Flight Mass Spectrometry) for a rapid diagnosis of urinary tract
infection. PLoS One. 2014;9:e86915.
20. Saadeh SA, Mattoo TK. Managing urinary tract infections. Pediatr
Nephrol. 2011;26:1967–1976.
21. Romero R, Emamian M, Quintero R, et al. The value and limitations
of the Gram stain examination in the diagnosis of intraamniotic
infection. Am J Obstet Gynecol. 1988;159:114–119.
 6 L. BOYANOVA
22. Schwebke JR, Hillier SL, Sobel JD, et al. Validity of the vaginal gram
stain for the diagnosis of bacterial vaginosis. Obstet Gynecol.
1996;88:573–576.
23. Murdoch DR, Morpeth SC, Hammitt LL, et al. Microscopic analysis
and quality assessment of induced sputum from children with
pneumonia in the PERCH Study. Clin Infect Dis. 2017;64(suppl_3):
S271–S279.
24. Musher DM, Montoya R, Wanahita A. Diagnostic value of micro-
scopic examination of Gram-stained sputum and sputum cultures
in patients with bacteremic pneumococcal pneumonia. Clin Infect
Dis. 2004;39:165–169.
25. Gottesman T, Yossepowitch O, Lerner E, et al. The accuracy of Gram
stain of respiratory specimens in excluding Staphylococcus
aureus in ventilator-associated pneumonia. J Crit Care.
2014;29:739–742.
26. Boyanova L. Detection of Helicobacter pylori infection in sympto-
matic Bulgarian adults. Clin Microbiol Infect. 2007;13:908–914.
27. Ghosh R, Uppal B, Aggarwal P, et al. A comparative study of
conventional and molecular techniques in diagnosis of
Campylobacter gastroenteritis in children. Ann Clin Lab Sci.
Downloaded by [Cornell University Library] at 08:38 02 November 2017
2014;44:42–48.
28. Taylor SN, DiCarlo RP, Martin DH. Comparison of methylene blue/
gentian violet stain to Gram’s stain for the rapid
diagnosis of gonococcal urethritis in men. Sex Transm Dis. 2011;38:
995–996.
29. Yoshimura J, Kinoshita T, Yamakawa K, et al. Impact of Gram stain
results on initial treatment selection in patients with ventilator-
associated pneumonia: a retrospective analysis of two treatment
algorithms. Crit Care. 2017;21:156.
30. Baron E. Approaches to identification of anaerobic bacteria. In:
Jorgensen J, Pfaller M, Carroll K, et al., editors. Manual of Clinical
Microbiology. Eleventh ed. Washington, DC: ASM Press; 2015. p.
905–908.
31. Thomson RB Jr. One small step for the Gram stain, one giant leap
for clinical microbiology. J Clin Microbiol. 2016;54:1416–1417.
32. Cantey JB, Gaviria-Agudelo C, McElvania TeKippe E, et al. Lack of
clinical utility of urine Gram stain for suspected urinary tract infec-
tion in pediatric patients. J Clin Microbiol. 2015;53:1282–1285.
33. El-Gamal AHF, Al-Otaibi SR, Alshamali A, et al. Fouzan A.
Polymerase chain reaction is no better than Gram stain for diag-
nosis of gonococcal urethritis. Indian J Dermatol Venereol Leprol.
2009;75:101.
34. Bartelsman M, Straetemans M, Vaughan K, et al. Comparison of two
Gram stain point-of-care systems for urogenital gonorrhea among
high-risk patients: diagnostic accuracy and cost-effectiveness
before and after changing the screening algorithm at an STI clinic
in Amsterdam. Sex Transm Infect. 2014;90:358–362.
35. Jordan SJ, Schwebke JR, Aaron KJ, et al. Meatal swabs contain less
cellular material and are associated with a decrease in Gram Stain
smear quality compared to urethral swabs in men. J Clin Microbiol.
2017;55:2249–2254.
36. Romero R, Yoon BH, Mazor M, et al. A comparative study of the
diagnostic performance of amniotic fluid glucose, white blood cell
count, interleukin-6, and gram stain in the detection of microbial
invasion in patients with preterm premature rupture of mem-
branes. Am J Obstet Gynecol. 1993;169:839–851.
37. Grice EA. The skin microbiome: potential for novel diagnostic and
therapeutic approaches to cutaneous disease. Semin Cutan Med
Surg. 2014;33:98–103.
38. Leal SM Jr, Jones M, Gilligan PH. Clinical significance of commensal
Gram-positive rods routinely isolated from patient samples. J Clin
Microbiol. 2016;54:2928–2936.
39. Scholz CF, Kilian M. The natural history of cutaneous propionibac-
teria, and reclassification of selected species within the genus
Propionibacterium to the proposed novel genera
Acidipropionibacterium gen. nov., Cutibacterium gen. nov. and
Pseudopropionibacterium gen. nov. Int J Syst Evol Microbiol.
2016;66:4422–4432.
40. Jousimies-Somer HR, Summanen P, Citron DM, et al., editors. Chapter 3.
Processing clinical specimens and isolation procedures. Wadsworth-Ktl
Anaerobic Bacteriology Manual, Star Publishing Company, Belmont,
California, 2002. p. 43–54.
41. Sathyanarayana H, Rani L, Rajendran R, et al. Iatrogenic infection of
Clostridium welchii following intramuscular injection of sodium
diclofenac. J Lab Physicians. 2013;5:58–59.
42. Riefler J, Kosov M, Belotserkovskiy M. The treatment of a gang-
renous leg. Cid. 2015;61:1032–1033.
43. Boyanova L, Kolarov R, Mateva L, et al. Actinomycosis: a frequently
forgotten disease. Future Microbiol. 2015;10:613–628.
44. Boyanova L, Koumanova R, Lazarova E, et al. Helicobacter pylori and
Helicobacter heilmannii in children. A Bulgarian study. Diagn
Microbiol Infect Dis. 2003;46:249–252.
45. Boyanova L. Microbiology and characteristics of H. pylori. In:
Boyanova L, editor. Helicobacter pylori. Norfolk, UK: Caister
Academic Press; 2011. p. 29–69.
46. Bahadori A, Esmaeillu M, Bahadori F, et al. Non H. pylori
Helicobacter identified as H. heilmannii in gastric biopsy samples
in humans with gastric disorders by PCR and microscopic methods
in Iran (First Report). Euro J Zool Res. 2014;3:92–96.
47. Boyanova L. Genus Helicobacter. In: Boyanova L, editor. Helicobacter
pylori. Norfolk, UK: Caister Academic Press; 2011. p. 9–27.
48. World Health organization. Campylobacter. Fact sheet. 2016. [cited
2017 Jul 30]. http://www.who.int/mediacentre/factsheets/fs255/en/
49. Mushi MF, Paterno L, Tappe D, et al. Evaluation of detection
methods for Campylobacter infections among under-fives in
Mwanza city, Tanzania. Pan Afr Med J. 2014;19:392.
50. Rezaizadeh H, Olson E. Specific GI Microbial Infections. In: Wu G,
eds. Pocket handbook of GI Pharmacotherapeutics. Clinical
Gastroenterology. Cham: Humana Press; 2016.
51. Malik OAA. Role of antimicrobials in the treatment of adult patients
presenting to the emergency department with acute gastroenter-
itis - a mini review. Pakistan J Med Sci. 2017;33:488–492.
52. Schelenz S, Barnes RA, Barton RC, et al. British Society for Medical
Mycology best practice recommendations for the diagnosis of
serious fungal diseases. Lancet Infect Dis. 2015;15:461–474.
53. Samuel LP, Balada-Llasat JM, Harrington A, et al. Multicenter assess-
ment of Gram stain error rates. J Clin Microbiol. 2016;54:1442–1447.
54. Nagata K, Mino H, Yoshida S. [Usefulness and limit of Gram staining
smear examination]. Rinsho Byori. 2010;58:490–497.
55. Cheesbrough M. District laboratory practice in tropical countries.
Cambridge CB2 8RU, UK: Cambridge University Press; 2006. p. 1–
234. Part 2.Cheesbrough M. ed.