<i>Clostridioides difficile</i> clinical diagnostic test methods and results are associated with recovery of <i>C. difficile</i> by stool culture

Accurate molecular epidemiology relies on recovering <i>Clostridioides difficile</i> 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 <i>C. difficile</i> 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 <i>C. difficile</i> 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. <i>C. difficile</i> was recovered by culture from 84.3% (1,527/1,811) of all specimens. In specimens tested with a reverse testing algorithm, <i>C. difficile</i> 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 <i>C. difficile</i> from stool cultures in PCR-only protocol cases was less likely when <i>C. difficile</i> 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 <i>C. difficile</i> surveillance programs, thereby enhancing the ability to generate robust molecular epidemiology data to inform prevention and control efforts.IMPORTANCEPublic health surveillance of <i>Clostridioides difficile</i> is essential for tracking strains, understanding transmission, and informing public health strategies. The foundation of the surveillance is the successful recovery of <i>C. difficile</i> from clinical specimens for molecular typing. This study reveals that the choice of diagnostic test significantly impacts the ability to recover <i>C. difficile</i> 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 <i>C. difficile</i> 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.