Clostridium difficile PCR Cycle Threshold Predicts Free Toxin

There is no stand-alone Clostridium difficile diagnostic that can sensitively and rapidly detect fecal free toxins. We investigated the performance of the C. difficile PCR cycle threshold (C-T) for predicting free toxin status. Consecutive stool samples (n = 312) positive for toxigenic C. difficile by the GeneXpert C. difficile/Epi tcdB PCR assay were tested with the rapid membrane C. Diff Quik Chek Complete immunoassay (RMEIA). RMEIA toxin-negative samples were tested with the cell cytotoxicity neutralization assay (CCNA) and tgcBIOMICS enzyme-linked immunosorbent assay (ELISA). Using RMEIA alone or in combination with CCNA and/or ELISA as the reference method, the accuracy of C-T was measured at different C-T cutoffs. Using RMEIA as the reference method, a C-T cutoff of 26.35 detected toxin-positive samples with a sensitivity, specificity, positive predictive value, and negative predictive value of 96.0% (95% confidence interval [CI], 90.2% to 98.9%), 65.9% (95% CI, 59.0% to 72.2%), 57.4% (95% CI, 52.7% to 62%), and 97.1% (95% CI, 92.8% to 98.9), respectively. Inclusion of CCNA in the reference method improved C-T specificity to 78.0% (95% CI, 70.7% to 84.2%). Intercartridge lot C-T variability measured as the average coefficient of variation was 2.8% (95% CI, 1.2% to 3.2%). Standardizing the input stool volume did not improve C-T toxin specificity. The median C-T values were not significantly different between stool samples with Bristol scores of 5, 6, and 7, between pediatric and adult samples, or between presumptive 027 and non-027 strains. In addition to sensitively detecting toxigenic C. difficile in stool, on-demand PCR may also be used to accurately predict toxin-negative stool samples, thus providing additional results in PCR-positive stool samples to guide therapy.