title: Comparison of a commercial real-time PCR assay for tcdB detection to a cell culture cytotoxicity assay and toxigenic culture for direct detection of toxin-producing Clostridium difficile in clinical samples.
abstract: Rapid detection of toxin-producing strains of Clostridium difficile is essential for optimal management of patients with C. difficile infection. The BD GeneOhm (San Diego, CA) Cdiff assay, a real-time PCR assay that amplifies tcdB, was compared to a cell culture neutralization assay (Wampole C. difficile Toxin B [TOX-B] test; TechLab, Blacksburg, VA) and to toxigenic culture. Using liquid (n = 273) and soft (n = 131) stool specimens from 377 symptomatic patients, all testing was performed on the same day by independent laboratory staff according to the manufacturers' protocols. Toxigenic bacterial culture was performed as follows. A 0.5-ml aliquot of stool was heated to 80 degrees C for 10 min, followed by inoculation onto modified cycloserine cefoxitin fructose agar with and without horse blood (Remel, Lenexa, KS) and into prereduced chopped-meat broth. Of the 404 stool specimens tested, 340 were negative and 40 were positive (10.0% prevalence) both by PCR for tcdB and by cytotoxin production. The overall agreement between the BD GeneOhm Cdiff assay and the TOX-B test was 94.8% (380/401). When the TOX-B test was used as the reference method, the initial sensitivity, specificity, and positive and negative predictive values of the BD GeneOhm Cdiff assay were 90.9% (40/44), 95.2% (340/357), 70.2% (40/57), and 98.8% (340/344), respectively. When toxigenic culture was used as the "gold standard," the sensitivity, specificity, and positive and negative predictive values of the BD GeneOhm Cdiff assay were 83.6%, 98.2%, 89.5%, and 97.1%, respectively, and those of the TOX-B test were 67.2%, 99.1%, 93.2%, and 94.4%, respectively. PCRs for three samples were inhibited upon initial testing; one sample was resolved upon retesting. One sample produced nonspecific cytotoxin results. The BD GeneOhm Cdiff assay performed well compared to a standard cell culture neutralization assay and to toxigenic culture for the detection of toxigenic C. difficile directly from fecal specimens.
authors.affiliation: Department of Pathology, Division of Medical Microbiology, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
authors.forename: Paul D
authors.initials: PD
authors.lastname: Stamper
authors.forename: Romina
authors.initials: R
authors.lastname: Alcabasa
authors.forename: Deborah
authors.initials: D
authors.lastname: Aird
authors.forename: Wisal
authors.initials: W
authors.lastname: Babiker
authors.forename: Jennifer
authors.initials: J
authors.lastname: Wehrlin
authors.forename: Ijeoma
authors.initials: I
authors.lastname: Ikpeama
authors.forename: Karen C
authors.initials: KC
authors.lastname: Carroll
mesh_terms: D000328:Adult; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics* / Q000633:toxicity*; D001427:Bacterial Toxins / Q000235:genetics* / Q000633:toxicity*; D018929:Cell Culture Techniques / Q000379:methods; D016360:Clostridioides difficile / Q000235:genetics / Q000302:isolation & purification* / Q000472:pathogenicity; D003470:Culture Media / Q000737:chemistry; D004760:Enterocolitis / Q000382:microbiology*; D005243:Feces / Q000382:microbiology; D006801:Humans; D009500:Neutralization Tests / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D003470:Culture Media; C031815:toxB protein, Clostridium difficile