Clostridium difficilePCR Cycle Threshold Predicts Free Toxin

ABSTRACTThere is no stand-alone Clostridium difficilediagnostic that can sensitively and rapidly detect fecal free toxins. We investigated the performance of the C. difficilePCR cycle threshold (CT) for predicting free toxin status. Consecutive stool samples (n= 312) positive for toxigenic C. difficileby the GeneXpert C. difficile/Epi tcdBPCR 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 CTwas measured at different CTcutoffs. Using RMEIA as the reference method, a CTcutoff 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 CTspecificity to 78.0% (95% CI, 70.7% to 84.2%). Intercartridge lot CTvariability 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 CTtoxin specificity. The median CTvalues 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. difficilein 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.