(473) Non-toxigenic Clostridioides difficile: Implications for Diagnostic Stewardship

Objective : Clostridioides difficile (CD) is the causative pathogen for C. difficile infection (CDI), the most common healthcare-associated infection in the US. Previous studies have suggested that CD species lacking toxin genes (non-toxigenic C. difficile or NTCD) may confer protection against CDI. However, current diagnostic tests are limited to detecting either toxin proteins or toxin genes and cannot detect NTCD. This study aimed to develop a molecular testing method that identifies NTCD and to assess its prevalence in a clinical cohort.

Methods: A quantitative polymerase chain reaction (qPCR) assay was developed, with primers validation initially with PCR and reference strains. Analytic sensitivity of the NTCD qPCR assay was determined using DNA from reference strains and qPCR efficiency was assessed using the standard curves of DNA dilutions. A random selection of 100 clinical stool samples, tested using the glutamate dehydrogenase (GDH) enzyme and toxin enzyme immunoassay (EIA) at a local medical center, was used to evaluate the prevalence of NTCD in hospitalized patients tested for CDI.

Results: A qPCR NTCD assay was developed using reference strains and compared to toxigenic CD strains and related Clostridial strains. The qPCR efficiency was 97%, with the lowest detection limit of 30 copy number. Of 100 stool samples tested, 11 (11%) were detected with NTCD strains. Among these 11, the majority of samples (72.7%) were collected from patients with GDH+/Toxin– results, followed by 2 patients (18.2%) with GDH–/Toxin– results.

Conclusions: Using a newly developed qPCR NTCD assay, approximately 10% of hospitalized patients tested for CDI were found to be colonized with NTCD. This NTCD assay may have important implications for diagnostic and antimicrobial stewardship as colonization with NTCD may offer protection against CDI.