Download Positive Blood Cultures Can We Always Trust the Gram Stain?

AJCP / EDITORIAL
Positive Blood Cultures
Can We Always Trust the Gram Stain?
Calvin L. Strand, MD
DOI: 10.1309/A3A734C91JHPHWB6
The Gram stain, a method for staining bacteria developed
and described by the Danish pathologist and bacteriologist
Hans Christian Gram in 1884,1 is still one of the most important
and useful clinical laboratory tests. This simple, rapid differential stain separates most clinically significant bacteria into 2
groups: gram-positive organisms, which appear blue or purple
when observed under the light microscope, and gram-negative
bacteria, which appear red or pink. The Gram stain reactions, in
conjunction with the morphologic types of bacteria (cocci vs
bacilli) and arrangement of the bacteria, can be used to make
presumptive identifications.
The Gram stain is used for several purposes2: to directly
examine specimens submitted for microbiologic examination,
to provide preliminary information to the clinician regarding
presumptive bacterial pathogens, to characterize the type of
bacteria growing in culture media (including blood cultures),
and to assess the quality of specimens submitted for culture (ie,
sputum specimens) to determine whether they are likely to yield
clinically useful information and whether they should be
processed (cultured). This specimen quality assessment is possible because the leukocytes and epithelial cells of the human
host also stain with the Gram stain.
Like other laboratory tests, the Gram stain has certain
inherent limitations and is subject to technical variation and
misinterpretation—the subject of the article by Rand and Tillan3
in this issue of the Journal. This retrospective study reviews
major errors, defined by the authors as errors in which the original Gram stain of a positive blood culture reports a single
organism whose Gram stain morphology is opposite (gram positive vs gram negative or vice versa) the Gram stain morphology of the final culture organism identification. The authors
found that in 57 (0.7%) of 8,253 patients with positive blood
cultures the Gram stain was misread, resulting in major errors
that fell into 3 categories: (1) Six cases where the original Gram
stain report was gram-positive cocci and the culture yielded
gram-negative bacilli, 3 of which were Acinetobacter species.
(2) Twenty-five cases where the original Gram stain report was
gram-negative bacilli and the culture yielded gram-positive
organisms; 9 of these were Bacillus species and 2 were
Clostridium species (this type error is probably due to overdecolorization, a common technical problem in Gram stains). (3)
Twenty-eight cases where the original Gram stain showed a single organism but the culture yielded multiple organisms; most
often in this group the original Gram stain showed gram-positive cocci. When the medical charts of these patients were
reviewed, there were 4 patients in whom delays of 14 hours to
3 days occurred in starting appropriate antibiotics, with 2
deaths, although the authors believed that the erroneous Gram
stain report was probably not contributory.3
Although the study by Rand and Tillan3 has limitations (eg,
the reasons for the errors in the Gram stain reports—technical
artifact, interpretation error, or clerical error—could not be
specifically determined in this retrospective study), it is still a
useful contribution to the field of clinical microbiology for several reasons. First, the data presented show that the overall performance of the clinical microbiology technologists in interpreting Gram stains (99.31% of the Gram stains were interpreted
accurately) over 23 months was very good, especially when one
considers that the Gram stain morphology of bacteria can be
affected by antibiotic therapy, technical variables including
overdecolorization and underdecolorization, and misinterpretation by those reading the Gram stains. Although 100% accuracy in reporting is always the goal of the laboratory, the accuracy of greater than 99% for reporting of positive blood cultures,
which are critical laboratory values, is to be commended.
Second, the authors provide important information to laboratory directors by identifying the most common types of major
errors in blood culture Gram stains so that the directors can
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© American Society for Clinical Pathology
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Strand / CAN WE ALWAYS TRUST THE GRAM STAIN?
emphasize these problems in training and continuing education
of the bench microbiologists responsible for interpreting Gram
stains. Third, the finding that a frequent error was reporting a
single organism when 2 or more were present prompts microbiologists to continue to vigorously search for other organisms,
even after identifying 1 organism in a blood culture Gram stain,
especially since gram-positive organisms are generally more
easily visualized than gram-negative organisms.
What are the principal teaching points that laboratory
directors and microbiology supervisors can apply from the
study by Rand and Tillan?3 I believe there are several:
1. Corrected report. When an error in interpretation of a
positive blood culture is discovered by the microbiology laboratory, the physician should be immediately notified by telephone and an additional report should be issued for posting in
the patient’s medical record. Of course, this is done because the
physician may change the diagnosis and/or treatment based on
the correct organism identification (and Gram stain reaction).
Also, the blood culture should be reviewed with the technologist involved as a teaching or quality improvement exercise,
especially if there is evidence of a technical problem or misinterpretation of the Gram stain.
2. Quality improvement. Errors, although uncommon, can
provide excellent opportunities for performance improvement.
As we know, reliable analytic results require organizing staff,
instruments, methods, and reagents into a well-controlled system
where protocols and procedures are strictly followed. The Gram
stain reporting of positive blood cultures, a critical laboratory
report that will provide important diagnostic information to clinicians for patients with life-threatening diseases, is certainly a
test that should be monitored carefully in the laboratory’s quality improvement program. The institution where I serve as director, a community teaching hospital, has monitored the positive
blood culture Gram stain results as a quality indicator for 10
years. When we first implemented this indicator, there were
some problems or opportunities for performance improvement
identified. However, after correcting these problems, the accuracy of blood culture Gram stain reports has been comparable to
that reported by Rand and Tillan.3 We have chosen to monitor
this indicator for 1 month per year, with the aggregate data for
2001 to 2006 showing greater than 99% accuracy of the Gram
stain using the criteria of Rand and Tillan. It should be pointed
out that we have included in our definition of a major error Gram
stains showing gram-positive cocci in which the initial report is
gram-positive cocci in clusters and the culture shows
Streptococcus species (not Staphylococcus species as expected)
or when the initial report is gram-positive cocci in chains and the
culture shows Staphylococcus species (not Streptococcus
species as expected). The clinical implications of gram-positive
cocci in clusters (presumed Staphylococcus aureus, possibly
methicillin-resistant or coagulase-negative Staphylococcus) vs
gram-positive cocci in chains (presumed Streptococcus species
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DOI: 10.1309/A3A734C91JHPHWB6
or Enterococcus species) are quite different. Thus, laboratories
may wish to include lack of concordance of Gram stain and culture for gram-positive cocci as a major error in blood culture
Gram stain interpretation in quality improvement studies.
3. Overdecolorization of gram-positive bacilli. The group
of 25 cases where the original Gram stain report was gram-negative bacilli and the culture yielded gram-positive organisms is
very important, because a gram-negative bacillus critical value
report will likely lead a physician to immediately order empiric
antimicrobial therapy to cover a broad spectrum of organisms,
whereas a report of gram-positive bacilli may cause the clinician to consider blood culture contamination and withhold
antimicrobial treatment until further information is available,
because gram-positive bacilli are much more likely to be classified as presumed contaminants by clinicians than gram-negative
bacilli. For example, analysis of blood culture data at the
University of Colorado Medical Center4 found that 94% of
Bacillus species cases, 79% of Corynebacterium species cases,
and 50% of Clostridium perfringens cases were considered contaminants. This problem shows how important it is to maintain
good technical performance of the Gram stain to prevent
overdecolorization of gram-positive bacilli. An article by
McClelland5 lists 8 causes of overdecolorization of the Gram
stain: bacterial cell wall damage due to host response or antimicrobial effect, excessive heat during fixation, low concentration
of crystal violet, excessive washing between steps, insufficient
iodine exposure, lack of available iodine, prolonged decolorization, and excessive counterstaining.
Finally, what is the answer to the question can we always
trust the Gram stain? In the case of positive blood cultures,
because of the important contribution of Rand and Tillan,3 the
answer is yes, almost always. However, only when the laboratory directors and supervisors carefully monitor this important
laboratory test and continue to educate and train bench personnel about all the pitfalls that can lead to spurious results, can this
high-quality level of performance be maintained.
From the Department of Pathology, Jersey City Medical Center,
Jersey City, NJ.
References
1. Madani K. Dr. Hans Christian Joachim Gram: inventor of the
Gram stain. Prim Care Update Ob Gyn. 2003;10:235-237.
2. Baron EJ, Peterson LR, Finegold SM, eds. Bailey & Scott’s
Diagnostic Microbiology. 9th ed. St Louis, MO: Mosby; 1994:69-70.
3. Rand KH, Tillan M. Errors in interpretation of Gram stains from
positive blood cultures. Am J Clin Pathol. 2006;126:686-690.
4. Weinstein MP, Reller LB, Murphy JR, et al. The clinical
significance of positive blood cultures: a comprehensive
analysis of 500 episodes of bacteremia and fungemia in adults,
I: laboratory and epidemiologic observations. Rev Infect Dis.
1983;5:35-53.
5. McClelland R. Gram’s stain: the key to microbiology. MLO
Med Lab Obs. 2001;33:20-22, 25-28.
© American Society for Clinical Pathology