Evaluation of tcdB Real-Time PCR in a Three-Step Diagnostic Algorithm for Detection of Toxigenic Clostridium difficile

Abstract

Clostridium difficile is the most common infectious cause of diarrhea in hospitalized patients. The optimal approach for the detection of toxigenic C. difficile remains controversial because no single test is sensitive, specific, and affordable. We have developed a real-time PCR method (direct stool PCR [DPCR]) to detect the tcdB gene encoding toxin B directly from stool specimens and have combined it with enzyme immunoassays (EIAs) in a three-step protocol. DPCR was performed on 699 specimens that were positive for C. difficile glutamate dehydrogenase (GDH) by Wampole C Diff Quik Chek EIA (GDH-Q) and negative for toxins A and B by Wampole Tox A/B Quik Chek EIA (AB-Q), performed sequentially. The performance of this three-step algorithm was compared with a modified "gold standard" that combined tissue culture cytotoxicity (CYT) and DPCR. A separate investigation was performed to evaluate the sensitivity of the GDH-Q as a screening test, and toxigenic C. difficile was found in 1.9% of 211 GDH-Q-negative specimens. The overall sensitivity, specificity, and positive and negative predictive values, respectively, were as follows for an algorithm combining GDH-Q, AB-Q, and DPCR: 83.8%, 99.7%, 97.1%, and 97.9%. Those for CYT alone were 58.8%, 100%, 100%, and 94.9%, respectively. In comparison, the sensitivity and specificity of DPCR were estimated to be 97.5% and 99.7%, respectively, using the same modified gold standard. Neither CYT nor toxin EIA was sufficiently sensitive to exclude toxigenic C. difficile, and combining EIAs with CYT in a three-step algorithm failed to substantially improve sensitivity. DPCR is a sensitive and specific method for the detection of toxigenic C. difficile that can provide same-day results at a cost-per-positive test comparable to those of other methods. A three-step algorithm in which DPCR is used to analyze GDH EIA-positive, toxin EIA-negative specimens provides a convenient and specific alternative with rapid results for 87.7% of specimens, although this approach is less sensitive than performing DPCR on all specimens.

Guidelines for Diagnosis, Treatment, and Prevention of Clostridium difficile Infections

“…Very occasionally, a patient with ileus and complicated disease will have a formed stool ( 3 ), in which case the laboratory should be made aware of this special clinical situation. Rectal swabs can be used for PCR 26. In patients with IBD, ongoing immunosuppression medications can be maintained in patients with CDI.…”

Laboratory Diagnosis of Clostridium difficile Infection

“…1 However, this approach often requires several days to complete, and neither assay is commonly available in clinical laboratories. The recent comprehensive study of C. difficile detection methods, 2 which reported the sensitivity of a commonly used GDH assay as 87.6% when compared with toxigenic culture, is consistent with concerns of falsely GDH-negative samples raised by the report of Larson et al 48 NovakWeekley et al 34 reported that initial GDH screening failed to identify approximately 15% of samples containing toxigenic C. difficile isolates. In addition, the mean sensitivity of membrane-type GDH assays in the ESCMID survey was only 60% when compared with toxigenic culture, 3 suggesting that GDH screening may not be as highly sensitive as previously assumed.…”

“…All four patients had subsequent stool samples that were positive by both PCR and toxigenic culture, and a review of the patients' medical records revealed that three of four patients already had been treated for CDI. 48 Thus, retesting of GDH-negative samples by PCR lowered the sensitivity of the GDH algorithm from 97.1% to 83.8%, and the negative predictive value also decreased from 99.7% to 97.9%.…”

“…The SHEA-IDSA guidelines suggest that PCR may be the most sensitive and specific diagnostic method for CDI detection, but additional studies in which toxigenic culture is used as the reference method are needed to validate this approach, particularly because PCR assays tend to be more expensive than antigen-based methods. 1 In their investigation comparing EIA, GDH, and PCR-based methods, Larson et al 48 retested GDH-negative stool samples by PCR and identified four additional positive samples among their cohort of patients. All four patients had subsequent stool samples that were positive by both PCR and toxigenic culture, and a review of the patients' medical records revealed that three of four patients already had been treated for CDI.…”

“…The report of Larson and colleagues 48 addresses the key controversy of using PCR methods for detection of CDI, which is whether the increased sensitivity and speed of direct PCR testing of liquid stool samples is worth the added expense of the method when compared with EIA testing. In their cost analysis, Larson et al 48 concluded that the increased cost of either direct PCR testing or the three-step algorithm (GDH testing, followed by EIA confirmation of positive samples, followed by PCR testing of GDH-positive EIA-negative samples) was justified by the earlier detection of CDI cases, which would prevent additional cases of nosocomial CDI and shorten the length of stay for patients with CDI.…”