Evaluation of Clostridium difficile Fecal Load and Limit of Detection during a Prospective Comparison of Two Molecular Tests, the illumigene C. difficile and Xpert C. difficile/Epi Tests
In a large prospective comparison, the illumigene test detected Clostridium difficile in 98% of toxin-positive and 58% of toxin-negative samples confirmed positive by other methods. The Xpert was uniformly sensitive. Most samples with discrepant results had C. difficile concentrations below the illumigene limit of detection. The significance of low-level C. difficile detection needs investigation.
The incidence and severity of Clostridium difficile infection (CDI) have increased dramatically in North America and Europe over the last decade (1). In the United States, rates of CDI are now at an all-time high (2). With this change in incidence, the performance of C. difficile testing has been reexamined and there has been a movement toward tests that detect C. difficile antigens or DNA directly versus tests that detect C. difficile toxins (3). As a group, C. difficile detection tests (e.g., PCR, loop-mediated amplification [LAMP], glutamate dehydrogenase immunoassay) are more sensitive than toxin tests, but the reported sensitivities still vary for unclear reasons. For example, the published sensitivity of the illumigene C. difficile assay ranges from 81 to 98% (4–11), leaving laboratories with uncertainty about which test to use and what performance to expect at their own institution.
To address this issue, we evaluated the fecal C. difficile DNA load of positive samples as part of a large, prospective comparison of two FDA-approved nucleic acid amplification tests (NAATs) for C. difficile, the illumigene C. difficile test and the Xpert C. difficile/Epi test, with toxigenic culture. When the clinical results and fecal C. difficile load comparisons suggested a genuine difference in the sensitivity of these two NAATs, we evaluated the inoculum size and analytical limit of detection (LOD) of both assays to further investigate the causes and significance of discrepant results.
Study population and test methods. Consecutive diarrheal stool samples submitted for C. difficile testing from adult inpatients 72 h after admission between January and October 2011 were included in the study; nonconforming stool samples were rejected. Each sample was tested for toxigenic C. difficile by three tests, (i) an illumigene C. difficile LAMP assay (Meridian Bioscience), (ii) an Xpert C. difficile/Epi real-time PCR assay (Cepheid), and (iii) a toxigenic culture. In addition, the fecal toxin status of each sample was determined by a toxin immunoassay (Premier C. difficile ToxinsA&B; Meridian Bioscience) performed on all samples and a cytotoxicity assay (Wampole C. difficile Tox-B [TechLab]; MHRF cells [Diagnostic Hybrids]) performed on immunoassay- negative, C. difficile-positive samples from frozen aliquots when available (55/61; 90.2%) (12). All tests except cytotoxicity were performed daily on fresh stool by following the manufacturer’s instructions. For toxigenic culture, 0.5 ml stool was mixed 1:1 with 95% EtOH for 10 min and a swab was used to inoculate a pre-reduced agar medium (cycloserine cefoxitin fructose agar supplemented with taurocholate [CCFA-ST]; Remel). Cultures were incubated anaerobically at 37°C for 3 days, and suspicious colonies were identified by colony and Gramstain morphology, odor, production of L-proline aminopeptidase, and fluorescence under long-range UV light. In vitro toxin production of isolates was confirmed by cytotoxicity testing of isolates grown in chopped meat broth (Remel) at 48 h (13). Samples that were negative with the initial EtOH shock culture but positive by either of the NAATs were cultured in enrichment broth (cycloserine cefoxitin mannitol broth with taurocholate lysozyme cysteine [CCMB-TAL]; Anaerobe Systems) from a frozen aliquot (n10) (14). The ability of the illumigene assay to detect toxigenic C. difficile isolates recovered from culture of illumigene-negative samples was tested directly using a 100-l volume of a 4 McFarland suspension from fresh subculture.