# Clostridioides difficile clinical diagnostic test methods and results are associated with recovery of C. difficile by stool culture

## TL;DR

This study shows that the type of diagnostic test used affects how well Clostridioides difficile can be recovered from stool samples, impacting public health surveillance accuracy.

## Contribution

The study identifies how diagnostic test choices influence C. difficile recovery and strain prevalence estimates in surveillance programs.

## Key findings

- C. difficile was recovered from 84.3% of test-positive stool specimens.
- Culture recovery was less likely with multiplex PCR compared to dedicated PCR.
- Toxin EIA-positive specimens had over eight times higher odds of successful culture recovery.

## Abstract

Accurate molecular epidemiology relies on recovering Clostridioides difficile from clinical specimens and is essential for informing public health responses, characterizing strains that cause disease, and tracking strain prevalence. We performed a cross-sectional study of the recovery of C. difficile isolates from test-positive stools received January 2020 through December 2022 from the Colorado and Georgia Emerging Infections Program sites. Multivariable logistic regression models determined the adjusted odds ratio (aOR) and 95% confidence intervals (CIs) for factors which may influence C. difficile recovery in specimens that tested positive by either a dedicated or multiplex polymerase chain reaction (PCR) as part of either a reverse testing algorithm (PCR-positive, arbitrated by toxin enzyme immunoassay [EIA]) or a PCR-only testing protocol. Whole-genome sequencing was performed to determine the multilocus sequence types. C. difficile was recovered by culture from 84.3% (1,527/1,811) of all specimens. In specimens tested with a reverse testing algorithm, C. difficile culture recovery was less likely with multiplex versus dedicated PCR (aOR: 0.53; 95% CI: 0.34–0.85) and more likely in toxin EIA-positive versus toxin EIA-negative specimens (aOR: 8.07; 95% CI: 4.10–18.31). Recovery of C. difficile from stool cultures in PCR-only protocol cases was less likely when C. difficile was detected by multiplex PCR (aOR: 0.16; 95% CI: 0.03–0.55). ST1 was associated with toxin EIA-positive specimens, whereas ST2/110 was associated with toxin EIA-negative specimens. These findings can be used to maximize C. difficile surveillance programs, thereby enhancing the ability to generate robust molecular epidemiology data to inform prevention and control efforts.

Public health surveillance of Clostridioides difficile is essential for tracking strains, understanding transmission, and informing public health strategies. The foundation of the surveillance is the successful recovery of C. difficile from clinical specimens for molecular typing. This study reveals that the choice of diagnostic test significantly impacts the ability to recover C. difficile by culture. These data reveal that culture recovery was lower from specimens that tested positive by syndromic multiplex polymerase chain reaction (PCR) panels compared to dedicated C. difficile PCR assays. Furthermore, recovery was more than eight times more likely from toxin enzyme immunoassay (EIA)-positive specimens than from toxin EIA-negative specimens, with high-virulence strains, such as PCR-ribotype 027, being associated with toxin EIA-positive results. These findings demonstrate that common diagnostic practices could introduce biases into surveillance data, potentially misrepresenting the true prevalence and epidemiology of clinically important strains. Understanding these factors is crucial for optimizing surveillance programs to generate accurate molecular epidemiologic data.