Download Limitations of PCR-Based Assays Performed on Formalin

AJCP / Editorial
PCR vs “PCAren’t”
Limitations of PCR-Based Assays Performed on Formalin-Fixed,
Paraffin-Embedded Mucosal Biopsy Specimens
Laura Lamps, MD
DOI: 10.1309/AJCPWFN9D0EGDEUK
Clostridium difficile remains the most common nosocomial cause of diarrhea and is becoming an increasingly important
community-acquired infection.1,2 Antibiotic resistance, as well
as the recently identified BI/NAP1/027 strain, is responsible
for more cases of severe C difficile–associated diarrhea worldwide.3 The diagnosis of C difficile infection is usually based
on a combination of history of antibiotic use, the presence of
pseudomembranes on colonoscopy, appropriate histology on
biopsy, and confirmatory laboratory stool tests for either the C
difficile toxin or bacterial toxin genes. Most clinical laboratories employ a C difficile–associated toxin enzyme immunoassay (EIA). These assays are technically easy to perform and
have a rapid turnaround time. Although they have specificities
ranging from 65% to 100%, the sensitivities are limited, ranging from 31% to 99%.4 Recently, US Food and Drug Administration–approved, polymerase chain reaction (PCR)–based
molecular assays performed on stool lysates have been increasingly recognized as the most sensitive and specific methods
of diagnosing C difficile. These assays target C difficile toxin
genes rather than the toxins themselves, with a sensitivity from
77% to 100% and a specificity from 93% to 99%.5-7
The tissue diagnosis of C difficile in a gastrointestinal mucosal biopsy specimen is generally straightforward,
although some infections (eg, enterohemorrhagic Escherichia
coli, Shigella, and toxin-producing Clostridium perfringens)
may produce similar features both macroscopically and morphologically. Ischemia is frequently in the histologic differential diagnosis as well, and the distinction between the two
is extremely important because the treatments are different.
In their article in this issue of the American Journal of
Clinical Pathology, Wiland and colleagues8 examine the
degree of interobserver agreement in the histologic diagnosis
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of C difficile colitis and correlate the histologic findings with
clinical data. They also evaluate the utility of a PCR-based
assay for C difficile when applied to formalin-fixed, paraffinembedded mucosal biopsy specimens. To our knowledge
and that of the authors, this article is the first to evaluate the
feasibility and usefulness of PCR in detecting C difficile in
fixed, processed mucosal biopsy specimens. The authors analyzed 20 mucosal biopsy specimens from patients who had C
difficile colitis, with the diagnosis based on histology plus a
positive EIA or PCR test, and/or were clinically suspected of
having C difficile colitis or responded to treatment for C difficile. Twenty biopsy specimens from patients with ischemic
colitis were evaluated in comparison. Punch biopsy specimens from the paraffin blocks of most cases were subjected
to DNA extraction and PCR, although a few lacked enough
residual tissue for further testing.
Wiland et al8 demonstrated overall excellent interobserver agreement in distinguishing between ischemic colitis and C
difficile colitis, and there was also good correlation between
the consensus histologic interpretation and the clinical impression. The results of the PCR assay performed on the fixed,
paraffin-embedded tissue, however, were underwhelming.
Only 3 of the 15 cases categorized as C difficile colitis were
positive by PCR performed on the tissue block.
This study nicely confirms the reliability and reproducibility of the histologic diagnosis of C difficile colitis and
emphasizes the good correlation between histologic diagnosis
and clinical impression in most cases. It also highlights many
of the challenges in using PCR-based assays on fixed, processed biopsy specimens. Analysis of fixed, paraffin-embedded tissue samples with PCR assays is fraught with difficulty,
particularly when applied to small biopsy specimens. Wiland
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Lamps / PCR vs PCAren’t
et al8 do not state the size of the target DNA in the commercial
assay that they used, but typically the target DNA must be less
than 500 base pairs due to DNA fragmentation caused by fixation. Fixatives other than formalin (such as mercuric or picric
acid–based fixatives) may limit DNA yield even more. Commercial assays are usually designed for use on fresh or frozen
specimens, and the extraction and detection kits may not be
designed to handle the tiny quantities of DNA present in many
mucosal biopsy specimens. The authors emphasize this technical limitation themselves—the average DNA concentration
(ng/L) for their tested formalin-fixed, paraffin-embedded
biopsy cases was 70.05 ng/L, yet the average DNA concentration for a comparison group of 10 random stool lysates
was significantly greater at 277.70 ng/L and was 105.94 ng/L
when excluding 1 outlier control. Finally, lesional tissue in
the block may simply be exhausted by the time it is subjected
to molecular analysis, and thus sampling error may cause a
negative result even when the patient truly has an infection.9
This study also emphasizes the critical importance of
correlating molecular data with all available histologic,
laboratory, and clinical information. Wiland et al8 give an
excellent example of this when they describe one of their
cases that was histologically categorized as C difficile colitis,
yet the patient tested negative by PCR-based stool assay.
Upon clinical review, the patient had multiple convincing
risk factors for ischemic colitis. One must bear in mind that
the PCR-based stool assays detect C difficile toxin genes but
do not necessarily indicate viability of the bacteria and toxin
production. A PCR assay also cannot gauge the severity of
disease in the same way that a colonoscopy or a biopsy can.
Moreover, molecular tests may detect C difficile in stools of
colonized patients, but it may not be the cause of diarrheal disease.10 Unfortunately, Wiland et al8 did not provide detailed
data regarding the colonoscopic appearance, risk factors for
C difficile, or results of molecular assays performed on stool
vs EIA for their study cases. Because previous studies10-12
have shown a troubling lack of correlation between clinical,
laboratory, and histologic data, the opportunity to see such a
correlation in these cases, particularly when combined with
the data from the PCR assay performed on the tissue blocks,
would no doubt have been interesting.
Robust clinicopathologic correlation is important for many
reasons in the practice of infectious disease pathology. Few
(if any) laboratory assays, including PCR performed on tissue
blocks, should be interpreted in a vacuum devoid of clinical
and histologic information. The result of the PCR assay must
be compared with the findings on the microscopic slide, as
well as available clinical and laboratory data. Obviously, the
negative PCR results from the tissue blocks in this study do
not mean that the patients with histologic and clinical evidence
of C difficile colitis were misdiagnosed. Yet, how many of us
have had a hallway conversation with a clinical colleague or
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medical student that started with, “How can you call it that?
The PCR was negative.” This is a particular problem in infections characterized by detection of few organisms. Some cases
of mycobacterial infection, for example, contain few bacilli on
acid-fast bacilli–stained slides. The PCR assay is often negative
in such situations due to lack of sufficient bacterial DNA.9
The PCR-based assays for C difficile detection are somewhat unusual because they are typically performed on stool
rather than on tissue. The ability to perform PCR assays for
bacterial DNA directly on gastrointestinal mucosal biopsy tissue blocks, however, is extremely important because patients
are often placed on empiric antibiotic therapy prior to either
mucosal biopsy or stool culture, thus limiting our ability to
detect a specific pathogen. Although their attempt met with
limited success, Wiland et al8 emphasize the importance of continuing to develop molecular assays for gastrointestinal pathogens that are suitable for use in fixed, paraffin-embedded tissue.
Part of our mission as pathologists, laboratorians, and
consultants is to educate our colleagues and the public as to the
limitations, as well as the advantages, of laboratory technology. This education includes the technical limitations of the
assays, as well as the importance of correlating all available
clinical and pathologic data when rendering a diagnosis. Many
of us are old enough to have witnessed the conflict between
technology and tradition with the advent and widespread use of
diagnostic immunohistochemistry, as well as heard our clinical
colleagues claim that “those brown stains” were a miraculous
tool that simply revealed the diagnosis by a color change and
had nothing to do with the interpretive abilities of the pathologist. New technology is available all the time, often at a high
cost to institutions and patients. Application of molecular technology, while often invaluable, must be done with the knowledge (by both pathologists and clinicians) of the limitations of
the test as well as the ability to interpret the test in light of the
tissue biopsy results and the clinical data.
The study by Wiland et al8 serves as a reminder that even
the most sophisticated technology has limitations and is best
interpreted in the context of the entire clinicopathologic picture. It is also a reaffirmation of pathologists’ ability to make
an accurate diagnosis of C difficile colitis. To paraphrase a
quote attributed to Hans Popper, the best diagnostic tool is still
a good-quality H&E slide connected to a brain.
From the Department of Pathology, University of Arkansas for
Medical Sciences, Little Rock.
References
1. Cohen S, Gerding D, Johnson S, et al. Clinical practice
guidelines for Clostridium difficile infection in adults: 2010
update by the Society for Healthcare Epidemiology of America
(SHEA) and the Infectious Diseases Society of America
(IDSA). Infect Control Hosp Epidemiol. 2010;31:431-455.
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AJCP / Editorial
2. Dumyati G, Stevens V, Hannett GE, et al. Communityassociated Clostridium difficile infections, Monroe County,
New York, USA. Emerg Infect Dis. 2012;18:392-400.
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7. Curry S. Clostridium difficile. Clin Lab Med. 2010;30:329-342.
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8. Wiland HO, Procop GW, Goldblum JR, et al. Interobserver
variability and feasibility of polymerase chain reaction–based
assay in distinguishing ischemic colitis from Clostridium
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PCR, culture & histopathology in the diagnosis of female
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10. Wilcox MH, Planche T, Fang FC, et al. What is the current
role of algorithmic approaches for diagnosis of Clostridium
difficile infection? J Clin Microbiol. 2010;48:4347-4353.
11. Guarner J, Bhatnagar J, Shane AL, et al. Correlation of
the detection of Clostridium difficile toxins in stools and
presence of the Clostridia in tissues of children. Hum Pathol.
2010;41:1586-1592.
12. Johal S, Hammond J, Solomon K, et al. Clostridium difficile
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community and hospital and role of flexible sigmoidoscopy.
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