Development and validation of digital enzyme-linked immunosorbent assays for ultrasensitive detection and quantification of clostridium difficile toxins in stool

Linan Song, Mingwei Zhao, David C. Duffy, Joshua Hansen, Kelsey Shields, Manida Wungjiranirun, Xinhua Chen, Hua Xu, Daniel A. Leffler, Susan P. Sambol, Dale N. Gerding, Ciarán P. Kelly, Nira R. Pollock

Research output: Contribution to journalArticlepeer-review

29 Scopus citations

Abstract

The 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 organisms (toxigenic culture [TC] and nucleic acid amplification testing [NAAT]) are confounded by asymptomatic colonization by toxigenic C. difficile. We developed ultrasensitive digital enzymelinked immunosorbent assays (ELISAs) for toxins A and B using single-molecule array technology and validated the assays using (i) culture filtrates from a panel of clinical C. difficile isolates and (ii) 149 adult stool specimens already tested routinely by NAAT. 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 that were negative by cytotoxicity assay/TC/NAAT, clinical cutoffs were set at 29.4 pg/ml (toxin A) and 23.3 pg/ml (toxin B); the resulting clinical specificities were 96% and 98%, respectively. The toxin B digital ELISA was 100% sensitive versus cytotoxicity assay. Twenty-five percent and 22% of the 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. The mean toxin levels by digital ELISA were 1.5-to 1.7-fold higher in five patients with CDI-attributable severe outcomes, versus 68 patients without, but this difference was not statistically significant. Ultrasensitive digital ELISAs for the detection and quantification of toxins A and B in stool can provide a rapid and simple tool for the diagnosis of CDI with both high analytical sensitivity and high clinical specificity.

Original languageEnglish (US)
Pages (from-to)3204-3212
Number of pages9
JournalJournal of Clinical Microbiology
Volume53
Issue number10
DOIs
StatePublished - Oct 1 2015
Externally publishedYes

ASJC Scopus subject areas

  • Microbiology (medical)

Fingerprint

Dive into the research topics of 'Development and validation of digital enzyme-linked immunosorbent assays for ultrasensitive detection and quantification of clostridium difficile toxins in stool'. Together they form a unique fingerprint.

Cite this