Impact of Strain Type on Detection of Toxigenic Clostridium difficile: Comparison of Molecular Diagnostic and Enzyme Immunoassay Approaches

Abstract

A multicenter clinical trial assessed the performance of the Cepheid Xpert C. difficile assay on stool specimens collected from patients suspected of having Clostridium difficile infection (CDI). A total of 2,296 unformed stool specimens, collected from seven study sites, were tested by Xpert C. difficile enrichment culture followed by cell culture cytotoxicity testing of the isolates (i.e., toxigenic culture with enrichment) and the study sites' standard C. difficile test methods. The methods included enzyme immunoassay (EIA), direct cytotoxin testing, and two-and three-step algorithms using glutamate dehydrogenase (GDH) screening followed by either EIA or EIA and an in-house PCR assay. All C. difficile strains were typed by PCR-ribotyping. Compared to results for toxigenic culture with enrichment, the sensitivity, specificity, and positive and negative predictive values of the Xpert assay were 93.5, 94.0, 73.0, and 98.8%, respectively. The overall sensitivity of the EIAs compared to that of enrichment culture was 60.0%, and the sensitivity of combined GDH algorithms was 72.9%; both were significantly lower than that of Xpert C. difficile (P < 0.001 and P ‫؍‬ 0.03, respectively). The sensitivity of the EIA was significantly lower than that of the Xpert C. difficile assay for detection of ribotypes 002, 027, and 106 (P < 0.0001, P < 0.0001, and P ‫؍‬ 0.004, respectively, Fisher's exact test), and the sensitivity of GDH algorithms for ribotypes other than 027 was lower than that for Xpert C. difficile (P < 0.001). The Xpert C. difficile assay is a simple, rapid, and accurate method for detection of toxigenic C. difficile in unformed stool specimens and is minimally affected by strain type compared to EIA and GDH-based methods.

Laboratory Diagnosis of Clostridium difficile Infection

“…Although GDH assays showed equivalent sensitivity to the PCR test for ribotype 027 strains when compared with the results of toxigenic culture, the sensitivity of GDH screening assays for non-027 strains decreased to 69.4% compared with toxigenic culture. 33 The sensitivities of toxins A and B EIA test results were significantly lower for ribotypes 002 (15.4%), 027 (78.4%), and 106 (18.8%) compared with sensitivities of 100%, 100%, and 75% for the PCR assay, respectively (P Ͻ 0.0001, P Ͻ 0.0001, and P Ͻ 0.0005, respectively). 33 This suggests that variations in the protein sequences, particularly within tcdB, directly affect the sensitivity of the antigen-based assays, whereas the PCR tests, which target conserved DNA regions, are less affected by sequence variation.…”

“…33 The sensitivities of toxins A and B EIA test results were significantly lower for ribotypes 002 (15.4%), 027 (78.4%), and 106 (18.8%) compared with sensitivities of 100%, 100%, and 75% for the PCR assay, respectively (P Ͻ 0.0001, P Ͻ 0.0001, and P Ͻ 0.0005, respectively). 33 This suggests that variations in the protein sequences, particularly within tcdB, directly affect the sensitivity of the antigen-based assays, whereas the PCR tests, which target conserved DNA regions, are less affected by sequence variation. To investigate this further, the amino acid sequences of toxin B from 16 isolates of C. difficile (based on DNA sequences available in GenBank) were aligned.…”

“…42 Direct plating of stool on selective agar media, which is common in many laboratories that perform anaerobic cultures for C. difficile, is 18% to 20% less sensitive than using broth enrichment to enhance recovery of organisms in culture; however, broth enrichment adds at least 24 hours to the turnaround time of testing, which is already slow. 33 …”

“…Indeed, a recent point-counterpoint article 6 on C. difficile laboratory methods cited the sensitivity of GDH assays as an unresolved issue. A recent report by Tenover and colleagues 33 suggests that some of the variability of the sensitivity of GDH assays reported in the literature may be because of reduced sensitivity of GDH assays for detecting PCR ribotypes other than type 027.…”

“…51 A third explanation for the disparity in results among antigen detection assays is related to the genetic diversity of the target proteins, particularly the antigenic variation of the toxins, which may decrease the sensitivity of antibody-based assays, depending on the strain mix present in a hospital or a community. For example, a recent study 33 of 350 human isolates of toxigenic C. difficile from North America reported that, although 18 different PCR ribotypes and seven different pulsed-field gel electrophoresis patterns were detected, approximately 30% of the isolates demonstrated unique undesignated ribotyping patterns and 46% were novel undesignated pulsed-field gel electrophoresis patterns, indicating a high degree of genetic diversity among the isolates. Of those organisms that were typable, a disparity in performance of both toxins A and B and GDH antigen tests compared with PCR was observed.…”