High Agreement Between an Ultrasensitive Clostridioides difficile Toxin Assay and a C. difficile Laboratory Algorithm Utilizing GDH-and-Toxin Enzyme Immunoassays and Cytotoxin Testing

Abstract

The Singulex Clarity C. diff toxins A/B (Clarity) assay is an automated, ultrasensitive immunoassay for the detection of Clostridioides difficile toxins in stool. In this study, the performance of the Clarity assay was compared to that of a multistep algorithm using an enzyme immunoassay (EIA) for detection of glutamate dehydrogenase (GDH) and toxins A and B arbitrated by a semiquantitative cell cytotoxicity neutralization assay (CCNA). The performance of the assay was evaluated using 211 residual deidentified stool samples tested with a GDH-and-toxin EIA (C. Diff Quik Chek Complete; Techlab), with GDH-and-toxin discordant samples tested with CCNA. The stool samples were stored at -80°C before being tested with the Clarity assay. For samples discordant between Clarity and the standard-of-care algorithm, the samples were tested with PCR (Xpert C. difficile; Cepheid), and chart review was performed. The testing algorithm resulted in 34 GDH⁺/toxin⁺, 53 GDH^-/toxin^-, and 124 GDH⁺/toxin^- samples, of which 39 were CCNA⁺ and 85 were CCNA^- Clarity had 96.2% negative agreement with GDH^-/toxin^- samples, 100% positive agreement with GDH⁺/toxin⁺ samples, and 95.3% agreement with GDH⁺/toxin^-/CCNA^- samples. The Clarity result was invalid for one sample. Clarity agreed with 61.5% of GDH⁺/toxin^-/CCNA⁺ samples, 90.0% of GDH⁺/toxin^-/CCNA⁺ (high-positive) samples, and 31.6% of GDH⁺/toxin^-/CCNA⁺ (low-positive) samples. The Singulex Clarity C. diff toxins A/B assay demonstrated high agreement with a testing algorithm utilizing a GDH-and-toxin EIA and CCNA. This novel automated assay may offer an accurate, stand-alone solution for C. difficile infection (CDI) diagnostics, and further prospective clinical studies are merited.

Keywords: C. difficile; C. difficile EIA; C. difficile PCR; CDI; cytotoxin; single-molecule counting; toxin; ultrasensitive.

FIG 1

The standard-of-care testing algorithm (YNHH) and Clarity results. One GDH⁺/toxin⁻/CCNA⁻ sample had invalid Clarity and NAAT results and was excluded from analysis. Abbreviations: GDH, glutamate dehydrogenase; CCNA, cell cytotoxicity neutralization assay.

FIG 2

C. difficile toxin concentration in samples in various result categories. Combined toxin A and B concentrations are shown for stool samples with different results by GDH-and-toxin EIA and a semiquantitative CCNA (dilution steps, 1:10, 1:100, 1:1,000, and 1:10,000). The dotted line represents the cutoff for the Clarity assay (12.0 pg/ml).