Determination of the performance of a novel diagnostic test for Clostridioides difficile toxins A and B using latent class analysis

The diagnosis of Clostridioides difficile infection (CDI) remains challenging. Nucleic acid amplification tests (NAAT) targeting the C. difficile (CD) toxin B gene suffer from suboptimal specificity for CDI due to CD asymptomatic colonization. Enzyme immunoassays (EIAs) that detect the presence of CD toxins are more specific for CDI but suffer from low sensitivity. To address this challenge, assays detecting CD toxins were developed using single-molecule array (SIMOA) technology, which have much lower limits of toxin detection than conventional EIAs. In this study, stool specimens from 708 symptomatic patients were aliquoted for testing by cell cytotoxicity neutralization assay (CCNA), toxigenic culture, NAAT, conventional CD toxin EIA, and SIMOA CD toxin EIAs. Using latent class analysis, we calculated the sensitivity and specificity of each of these diagnostic tests for detecting, separately, the presence of CD bacterium, CD toxin gene, and CD toxin. We estimated that the prevalence of CDI in our cohort was 14% (95% credible interval [CI]: 0.11-0.17). While the specificity of NAAT for detecting the presence of CD toxin was 95% (95% CI: 0.94-0.97), its positive predictive value was poor due to the low prevalence of CDI. The specificity of the conventional CD toxin EIA for CDI was excellent, but the sensitivity was only 48% (95% CI: 0.41-0.55). In comparison, the sensitivities of the SIMOA toxins A and B EIAs were 76% (95% CI: 0.67-0.84) and 77% (95% CI: 0.67-0.84), respectively, while maintaining excellent specificity. We conclude that SIMOA CD toxin EIAs are significantly more sensitive than conventional CD toxin EIAs.IMPORTANCEClostridioides difficile infection (CDI) is the most important infectious cause of hospital-associated diarrhea worldwide, but its diagnosis remains challenging. Nucleic acid amplification tests (NAATs) targeting the C. difficile (CD) toxin B gene have suboptimal specificity due to the presence of CD asymptomatic colonization, while enzyme immunoassays (EIAs) that detect the toxin itself are much more specific but are limited by low sensitivity. New assays for detecting CD toxins were developed using single-molecule array (SIMOA) technology, which have much lower limits of toxin detection than conventional EIAs, potentially improving the sensitivity of these conventional EIAs while remaining highly specific. In this study, we use latent class analysis to evaluate the sensitivity and specificity of different diagnostic tests for CD, including the novel SIMOA toxin assays, in detecting the different CD targets: the presence of CD bacterium, the presence of CD toxin gene, and the presence of CD toxin.