A Rapid, Accurate, Single Molecule Counting Method Detects Clostridium difficile Toxin B in Stool Samples

Abstract

We describe a new rapid and accurate immunoassay-based technology capable of counting single target molecules using digital imaging without magnification. Using the technology, we developed a rapid test for Clostridium difficile toxin B, which is responsible for the pathology underlying potentially fatal C. difficile infections (CDI). There are currently no tests for CDI that are rapid, sensitive, and specific. The MultiPath C. difficile toxin B test images and counts complexes of target-specific magnetic and fluorescent particles that have been tethered together by toxin B molecules in minimally processed stool samples. The performance characteristics of the 30 minute test include a limit of detection of 45 pg/mL, dynamic range covering 4–5 orders of magnitude, and coefficient of variation of less than 10%. The MultiPath test detected all toxinotypes and ribotypes tested, including the one most commonly occurring in the US and EU; shows no cross reactivity with relevant bacterial species; and is robust to potential interferants commonly present in stool samples. On a training set of 320 clinical stool samples, the MultiPath C. difficile toxin B test showed 97.0% sensitivity (95% CI, 91.4–99.4%); 98.3% specificity (95% CI, 96.8–99.2%); and 98.2% accuracy (95% CI, 96.7–99.0%) compared to the cellular cytotoxicity neutralization assay (CCNA) reference method. Based on these compelling performance characteristics, we believe the MultiPath technology can address the lack of rapid, sensitive, specific, and easy-to-use diagnostic tests for C. difficile.

Advances in Optical Single‐Molecule Detection: En Route to Supersensitive Bioaffinity Assays

“…Gite et al [133] developed as andwich assay consisting of 1) amagnetic particle,2)ananti-Clostridium difficile capture antibody,3 )C. difficile, 4) an anti-C. difficile detection antibody,a nd 5) af luorescent microparticle.Amixture of avisible-light-absorbing dye-cushion reagent and the density agent iodixanol was dried on the bottom of each microwell (Figure 17). Thei mmunoreagents were added, and the magnetic beads were pulled to the bottom of the microplate with amagnet.…”

“…c) Fluorescent beads appear as bright pixels on the digital camera. d) Acomparison of aw ell with and without dye shows the efficiencyo f ad ye-cushion layer.R eprinted from ref [133]. with the permission of Creative Commons Attribution 4.0 International License.…”

Ultrasensitive Clostridioides difficile Toxin Testing for Higher Diagnostic Accuracy

“…Although a correlation between toxin concentrations and CDI severity has been observed (47) and high concentrations have been reported in individual patients with severe disease and ileus (32,36), the lack of such a correlation has also been reported (9,24,41,42). LoD (27) or cutoff (9,26) in that range may be too high and will lead to missed cases.…”

“…Indeed, the ultrasensitive assays detected toxins in 22.7% of NAAT+/toxin EIA-/CCNA- (25) and in 41.3% of GDH+/CCNA-/NAAT+ (26) samples, indicating that Simoa and Single Molecule Counting technologies are more sensitive than CCNA and that their specificity therefore may be underestimated in direct comparisons (21, 26). For less sensitive assays, such as the MultiPath assay that has an LoD in a similar range as CCNA, an accuracy comparison will look more favorable (27). In the prospective, multicenter study comparing Clarity with CCNA, samples with discrepant results were retested with CCNA when the ultrasensitive toxin result agreed with that of at least one other comparator method (GDH EIA, toxin EIA, or NAAT), and a different CCNA result was reported for as many as 42% of retested samples (21); CCNAs (in which results are read by microscopy) require experienced workers to optimize reproducibility.…”

Fortschritte in der optischen Einzelmoleküldetektion: Auf dem Weg zu höchstempfindlichen Bioaffinitätsassays

“…Gite et al [133] entwickelten einen Sandwich-Assay bestehend aus 1) einem magnetischen Partikel, 2) einem anti-Clostridium-difficile-Fängerantikçrper,3 ) C. difficile, 4) einem anti-C.…”