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

Glen Hansen; Stephen Young; Alan H B Wu; Emily Herding; Vickie Nordberg; Ray Mills; Christen Griego-Fullbright; Aaron Wagner; Chui Mei Ong; Shawna Lewis; Joseph Yoon; Joel Estis; Johanna Sandlund; Emily Friedland; Karen C Carroll

doi:10.1128/JCM.00719-19

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

J Clin Microbiol. 2019 Oct 23;57(11):e00719-19. doi: 10.1128/JCM.00719-19. Print 2019 Nov.

Authors

Glen Hansen^{1

2}, Stephen Young^{3

4}, Alan H B Wu⁵, Emily Herding^{1

2}, Vickie Nordberg^{1

2}, Ray Mills³, Christen Griego-Fullbright³, Aaron Wagner³, Chui Mei Ong⁵, Shawna Lewis⁶, Joseph Yoon⁶, Joel Estis⁷, Johanna Sandlund⁷, Emily Friedland⁷, Karen C Carroll⁸

Affiliations

¹ Department of Pathology, Hennepin County Medical Center, Minneapolis, Minnesota, USA.
² Division of Infectious Diseases, Department of Pathology and Laboratory Medicine, University of Minnesota, Minneapolis, Minnesota, USA.
³ TriCore Reference Laboratories, Albuquerque, New Mexico, USA.
⁴ Department of Pathology, University of New Mexico HSC, Albuquerque, New Mexico, USA.
⁵ Department of Laboratory Medicine, University of California, Zuckerberg San Francisco General Hospital, San Francisco, California, USA.
⁶ Division of Medical Microbiology, Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
⁷ Singulex, Inc., Alameda, California, USA.
⁸ Division of Medical Microbiology, Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA kcarrol7@jhmi.edu.

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.

Keywords: C. difficile; single-molecule counting technology; toxins A and B; ultrasensitive; ultrasensitivity.

Publication types

Comparative Study
Multicenter Study
Validation Study

MeSH terms

Adolescent
Adult
Aged
Bacteriological Techniques
Child
Child, Preschool
Clostridioides difficile
Clostridium Infections / diagnosis*
Cytotoxicity Tests, Immunologic / methods
Enterotoxins / isolation & purification*
Feces / chemistry*
Female
Humans
Immunoenzyme Techniques
Male
Middle Aged
Prospective Studies
Sensitivity and Specificity
Single Molecule Imaging / methods*
Young Adult

Substances

Enterotoxins