Clostridium difficile Testing in the Clinical Laboratory by Use of Multiple Testing Algorithms

Abstract

The incidence of Clostridium difficile infection (CDI) has risen almost 3-fold in the United States over the past decade, emphasizing the need for rapid and accurate tests for CDI. The Cepheid Xpert C. difficile assay is an integrated, closed, nucleic acid amplification system that automates sample preparation and real-time PCR detection of the toxin B gene (tcdB). A total of 432 stool specimens from symptomatic patients were tested by a glutamate dehydrogenase (GDH) assay, a toxin A and B enzyme immunoassay (EIA), the Xpert C. difficile assay, and a cell culture cytotoxicity neutralization assay (CCCN). The results of these methods, used individually and in combination, were compared to those of toxigenic culture. Results for the Xpert C. difficile assay alone showed a sensitivity, specificity, positive predictive value, and negative predictive value (NPV) of 94.4, 96.3, 84.0, and 98.8%, while the EIA alone gave corresponding values of 58.3, 94.7, 68.9, and 91.9%, respectively. An algorithm using the GDH assay and the EIA (plus the CCCN if the EIA was negative) showed corresponding values of 83.1, 96.7, 83.1, and 96.1%. The Xpert C. difficile assay was statistically superior to the EIA (P, <0.001 by Fisher's exact test) and to the GDH-EIA-CCCN algorithm (P, 0.0363). Combining the GDH and Xpert C. difficile assays lowered both the sensitivity and the NPV of the Xpert assay. The GDH-EIA-CCCN procedure required, on average, 2 days to complete testing on GDH-positive results, while testing by the Xpert C. difficile assay was completed, on average, in less than 1 h. Xpert C. difficile testing yielded the highest sensitivity and NPV, in the least amount of time, of the individual-and multiple-test algorithms evaluated in this study.

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.…”

“…Since then, a variety of PCR assays targeting either tcdB or the tcdC regulator gene have been described. 28,34,56 All three FDA-cleared commercial PCR-based assays for C. difficile use tcdB as their major target. Evaluations of several commercial PCR assays 26,34,35 have appeared in the literature, and several investigators 8 -10 have proposed using PCR assays to confirm GDH screening tests in lieu of using EIA assays because of the low sensitivity of the EIA component of the assays.…”

“…28,34,56 All three FDA-cleared commercial PCR-based assays for C. difficile use tcdB as their major target. Evaluations of several commercial PCR assays 26,34,35 have appeared in the literature, and several investigators 8 -10 have proposed using PCR assays to confirm GDH screening tests in lieu of using EIA assays because of the low sensitivity of the EIA component of the assays. 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.…”

“…Sensitivity and specificity values are based on published literature 2,28,34,53,62 and represent midrange values. For the model, the sensitivity of the GDH portion of the GDH-EIA testing algorithm was set at 87%, 2,34,45 so the sensitivity of the testing algorithms that reflex to NAATs, CCCN, or toxigenic culture testing for GDH-positive/EIA-negative specimens cannot exceed this value, because specimens that are negative by GDH will be excluded from further testing. GDH and EIA testing may be either in parallel (ie, together in the same test) or sequential, if a stand-alone GDH assay is used, followed by an independent EIA toxin A/B test.…”

“…GDH and EIA testing may be either in parallel (ie, together in the same test) or sequential, if a stand-alone GDH assay is used, followed by an independent EIA toxin A/B test. 2,34 The model assumes that 32 specimens will be GDH positive and EIA negative and, thus, available for reflex testing. The sensitivities of the reflex tests are as follows: NAATs, 95% 26,33,34,56,57 ; toxigenic culture, 99% 42 ; and cytotoxin testing, 70%.…”