Dataset Information

Ultrasensitive Detection of Clostridioides difficile Toxins in Stool by Use of Single-Molecule Counting Technology: Comparison with Detection of Free Toxin by Cell Culture Cytotoxicity Neutralization Assay.

ABSTRACT: Laboratory tests for Clostridioides difficile infection (CDI) rely on the detection of free toxin or molecular detection of toxin genes. The Singulex Clarity C. diff toxins A/B assay is a rapid, automated, and ultrasensitive assay that detects C. difficile toxins A and B in stool. We compared CDI assays across two prospective multicenter studies to set a cutoff for the Clarity assay and to independently validate the performance compared with that of a cell culture cytotoxicity neutralization assay (CCCNA). The cutoff was set by two sites testing fresh samples from 897 subjects with suspected CDI and then validated at four sites testing fresh samples from 1,005 subjects with suspected CDI. CCCNA testing was performed at a centralized laboratory. Samples with discrepant results between the Clarity assay and CCCNA were retested with CCCNA when the Clarity result agreed with that of at least one comparator method; toxin enzyme immunoassays (EIA), glutamate dehydrogenase (GDH) detection, and PCR were performed on all samples. The cutoff for the Clarity assay was set at 12.0?pg/ml. Compared to results with CCCNA, the Clarity assay initially had 85.2% positive agreement and 92.4% negative agreement. However, when samples with discrepant results between the Clarity assay and CCCNA in the validation study were retested by CCCNA, 13/17 (76.5%) Clarity-negative but CCCNA-positive samples (Clarity+/CCCNA-) became CCCNA-, and 5/26 (19.2%) Clarity+/CCCNA- samples became CCCNA+, resulting in a 96.3% positive agreement and 93.0% negative agreement between Clarity and CCCNA results. The toxin EIA had 59.8% positive agreement with CCCNA. The Clarity assay was the most sensitive free-toxin immunoassay, capable of providing CDI diagnosis in a single-step solution. A different CCCNA result was reported for 42% of retested samples, increasing the positive agreement between Clarity and CCCNA from 85.2% to 96.3% and indicating the challenges of comparing free-toxin results to CCCNA results as a reference standard.

SUBMITTER: Hansen G

PROVIDER: S-EPMC6812992 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Publications

Ultrasensitive Detection of Clostridioides difficile Toxins in Stool by Use of Single-Molecule Counting Technology: Comparison with Detection of Free Toxin by Cell Culture Cytotoxicity Neutralization Assay.

Hansen Glen G Young Stephen S Wu Alan H B AHB Herding Emily E Nordberg Vickie V Mills Ray R Griego-Fullbright Christen C Wagner Aaron A Ong Chui Mei CM Lewis Shawna S Yoon Joseph J Estis Joel J Sandlund Johanna J Friedland Emily E Carroll Karen C KC

Journal of clinical microbiology 20191023 11

Laboratory tests for <i>Clostridioides difficile</i> infection (CDI) rely on the detection of free toxin or molecular detection of toxin genes. The Singulex Clarity C. diff toxins A/B assay is a rapid, automated, and ultrasensitive assay that detects <i>C. difficile</i> toxins A and B in stool. We compared CDI assays across two prospective multicenter studies to set a cutoff for the Clarity assay and to independently validate the performance compared with that of a cell culture cytotoxicity neut ...[more]

PMID: 31434724

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Project description:Ebselen, a reactive organoselenium compound, was shown to inhibit toxins TcdA and TcdB by covalently binding to their cysteine protease domains. It was suggested that ebselen lacked antimicrobial activity against Clostridioides difficile. However, this perception conflicts with C. difficile having essential cysteine-containing enzymes that could be potential targets and the reported antimicrobial activity of ebselen against other species. Hence, we reevaluated the anti-C. difficile properties of ebselen. Susceptibility testing revealed that its activity was either slightly reduced by pyruvate found in Wilkins-Chalgren agar or obliterated by blood in brucella agar. In brain heart infusion (BHI) agar, ebselen inhibited most C. difficile strains (MICs of 2 to 8 μg/ml), except for ribotype 078 that was intrinsically resistant (MIC = 32 to 128 μg/ml). Against C. difficile R20291, at concentrations below its minimal bactericidal concentration (MBC), 16 μg/ml, ebselen inhibited production of toxins and spores. Transcriptome analysis revealed that ebselen altered redox-associated processes and cysteine metabolism and enhanced expression of Stickland proline metabolism, likely to regenerate NAD+ from NADH. In cellular assays, ebselen induced uptake of cysteine, depleted nonprotein thiols, and disrupted the NAD+/NADH ratio. Taken together, killing of C. difficile cells by ebselen occurs by a multitarget action that includes disrupting intracellular redox, which is consistent with ebselen being a reactive molecule. However, the physiological relevance of these antimicrobial actions in treating acute C. difficile infection (CDI) is likely to be undermined by host factors, such as blood, which protect C. difficile from killing by ebselen. IMPORTANCE We show that ebselen kills pathogenic C. difficile by disrupting its redox homeostasis, changing the normal concentrations of NAD+ and NADH, which are critical for various metabolic functions in cells. However, this antimicrobial action is hampered by host components, namely, blood. Future discovery of ebselen analogues, or mechanistically similar compounds, that remain active in blood could be drug leads for CDI or probes to study C. difficile redox biology in vivo.

Dataset Information

Ultrasensitive Detection of Clostridioides difficile Toxins in Stool by Use of Single-Molecule Counting Technology: Comparison with Detection of Free Toxin by Cell Culture Cytotoxicity Neutralization Assay.

Publications

Ultrasensitive Detection of Clostridioides difficile Toxins in Stool by Use of Single-Molecule Counting Technology: Comparison with Detection of Free Toxin by Cell Culture Cytotoxicity Neutralization Assay.

Similar Datasets

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