PCR vs "PCAren't": limitations of PCR-based assays performed on formalin-fixed, paraffin-embedded mucosal biopsy specimens.

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 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