Journal Of Clinical Microbiology | July 22, 2015

Linan Song et al.,
Journal of Clinical Microbiology
DOI: 10.1128/JCM.01334-15

Background: Currently available diagnostics for Clostridium difficile infection(CDI) have major limitations. Despite mounting evidence that toxin detection is paramount for diagnosis, conventional toxin immunoassays are insufficiently sensitive and cytotoxicity assays too complex; assays that detect toxigenic organism [toxigenic culture(TC) and nucleic acid amplification testing(NAAT)] are confounded by asymptomatic colonization by toxigenic C. difficile.

Methods: We developed ultrasensitive “digital ELISA” assays for toxins A and B using single molecule array technology and validated the assays using a) culture filtrates from a panel of clinical C. difficile isolates and b) 149 adult stool specimens already tested routinely by NAAT.

Results: The digital ELISAs detected toxins A and B in stool with limits of detection of 0.45 and 1.5 pg/mL respectively, quantified toxins across a 4-log range, and detected toxins from all clinical strains studied. Using specimens negative by cytotoxicity assay/TC/NAAT, clinical cutoffs were set at 29.4 pg/mL(A) and 23.3 pg/mL(B); resulting clinical specificities were 96% and 98%, respectively. The toxin B digital ELISA was 100% sensitive vs cytotoxicity assay. 25% and 22% of samples positive by NAAT and TC, respectively, were negative by the toxin B digital ELISA, consistent with the presence of organism but minimal or no toxin. Mean toxin levels by digital ELISA were 1.5-1.7-fold higher in five patients with CDI-attributable severe outcomes, vs. 68 patients without, but this difference was not statistically significant.

Conclusions: Ultrasensitive digital ELISAs for detection and quantification of toxins A and B in stool can provide a rapid, simple tool for diagnosis of CDI with both high analytical sensitivity and high clinical specificity.