title: Development and Validation of Digital Enzyme-Linked Immunosorbent Assays for Ultrasensitive Detection and Quantification of Clostridium difficile Toxins in Stool.
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 enzyme-linked 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.
authors.affiliation: Quanterix Corporation, Lexington, Massachusetts, USA.
authors.forename: Linan
authors.initials: L
authors.lastname: Song
authors.affiliation: Quanterix Corporation, Lexington, Massachusetts, USA.
authors.forename: Mingwei
authors.initials: M
authors.lastname: Zhao
authors.affiliation: Quanterix Corporation, Lexington, Massachusetts, USA.
authors.forename: David C
authors.initials: DC
authors.lastname: Duffy
authors.affiliation: Division of Gastroenterology, Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA.
authors.forename: Joshua
authors.initials: J
authors.lastname: Hansen
authors.affiliation: Division of Gastroenterology, Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA.
authors.forename: Kelsey
authors.initials: K
authors.lastname: Shields
authors.affiliation: Division of Gastroenterology, Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA.
authors.forename: Manida
authors.initials: M
authors.lastname: Wungjiranirun
authors.affiliation: Division of Gastroenterology, Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA.
authors.forename: Xinhua
authors.initials: X
authors.lastname: Chen
authors.affiliation: Division of Gastroenterology, Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA.
authors.forename: Hua
authors.initials: H
authors.lastname: Xu
authors.affiliation: Division of Gastroenterology, Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA.
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Leffler
authors.affiliation: Hines VA Medical Center, Hines, Illinois, USA Loyola University Chicago Stritch School of Medicine, Maywood, Illinois, USA.
authors.forename: Susan P
authors.initials: SP
authors.lastname: Sambol
authors.affiliation: Hines VA Medical Center, Hines, Illinois, USA Loyola University Chicago Stritch School of Medicine, Maywood, Illinois, USA.
authors.forename: Dale N
authors.initials: DN
authors.lastname: Gerding
authors.affiliation: Division of Gastroenterology, Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA.
authors.forename: Ciarán P
authors.initials: CP
authors.lastname: Kelly
authors.affiliation: Division of Infectious Diseases, Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA Department of Laboratory Medicine, Boston Children's Hospital, Boston, Massachusetts, USA nira.pollock@childrens.harvard.edu.
authors.forename: Nira R
authors.initials: NR
authors.lastname: Pollock
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001426:Bacterial Proteins / Q000032:analysis*; D001427:Bacterial Toxins / Q000032:analysis*; D019411:Clinical Laboratory Techniques / Q000379:methods*; D003015:Clostridium Infections / Q000175:diagnosis*; D003967:Diarrhea / Q000175:diagnosis*; D004768:Enterotoxins / Q000032:analysis*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005243:Feces / Q000737:chemistry*; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D012680:Sensitivity and Specificity; D055815:Young Adult
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004768:Enterotoxins; C041348:tcdA protein, Clostridium difficile; C031815:toxB protein, Clostridium difficile