Clostridium difficile PCR Cycle Threshold Predicts Free Toxin

Abstract

There is no stand-alone <i>Clostridium difficile</i> diagnostic that can sensitively and rapidly detect fecal free toxins. We investigated the performance of the <i>C. difficile</i> PCR cycle threshold (<i>C_T</i> ) for predicting free toxin status. Consecutive stool samples (<i>n</i> = 312) positive for toxigenic <i>C. difficile</i> by the GeneXpert <i>C. difficile</i>/Epi <i>tcdB</i> 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 <i>C_T</i> was measured at different <i>C_T</i> cutoffs. Using RMEIA as the reference method, a <i>C_T</i> 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 <i>C_T</i> specificity to 78.0% (95% CI, 70.7% to 84.2%). Intercartridge lot <i>C_T</i> 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 <i>C_T</i> toxin specificity. The median <i>C_T</i> 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 <i>C. difficile</i> 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.