A quantitative PCR to detect non-toxigenic Clostridioides difficile

Clostridioides difficile species lacking toxin genes (non-toxigenic C. difficile or NTCD) may confer protection against CDI. However, current diagnostic tests detect either toxin proteins or toxin genes and cannot detect NTCD. This study developed a molecular testing method that uniquely identified NTCD and assessed its prevalence in a clinical cohort. A quantitative PCR (qPCR) assay was developed and validated using reference strains. Analytic sensitivity was determined using DNA from reference NTCD strains, and qPCR efficiency was assessed using the slope of the standard curves of DNA dilutions. A random selection of 95 clinical stool samples, tested using the GDH enzyme and toxin enzyme immunoassay (EIA), was used to evaluate the prevalence of NTCD in hospitalized patients tested for CDI. The KB-1/KB-2 primers and probe designed were specific for NTCD strains and did not amplify with toxigenic C. difficile or other related strains. The NTCD qPCR assay analytical sensitivity was linear between 3 × 10¹ and 3 × 10⁶ gDNA (R² = 0.999; P < 0.0001). No NTCD was found in 25 GDH-EIA -/- samples compared to 5 of 25 (20%) GDH-EIA +/- samples and 2 of 23 (8.7%) GDH-EIA +/+ samples. Of samples detected with NTCD, median NTCD DNA was 33,039 (IQR: 22.449-45.688) in GDH-EIA +/- samples and 370 [IQR: 159-583] in GDH-EIA +/+ samples. The new qPCR NTCD assay identified NTCD colonization in 7% of hospitalized patients tested for CDI. This NTCD assay may have important implications for diagnostic and antimicrobial stewardship as colonization with NTCD strains may offer protection against CDI.IMPORTANCECurrent diagnostic strategies do not detect non-toxigenic Clostridioides difficile (NTCD) strains, which may provide protection against C. difficile infection (CDI). Detecting these strains is critical as it underscores the importance of avoiding unnecessary antibiotic treatment in patients colonized with NTCD. To better guide clinical decisions and enhance the understanding of NTCD epidemiology, molecular assays that specifically target non-coding regions unique to NTCD strains are needed. In this study, we developed and validated a qPCR assay capable of uniquely identifying NTCD strains. This innovative assay holds significant potential for applications in public health, infection control, diagnostic, and therapeutic strategies related to CDI.