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Journal of Clinical Microbiology, February 2009, p. 373-378, Vol. 47, No. 2
0095-1137/09/$08.00+0     doi:10.1128/JCM.01613-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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{triangledown}

Paul D. Stamper,1 Romina Alcabasa,2 Deborah Aird,2 Wisal Babiker,2 Jennifer Wehrlin,2 Ijeoma Ikpeama,2 and Karen C. Carroll1,2*

Division of Medical Microbiology, Department of Pathology, The Johns Hopkins University School of Medicine,1 The Johns Hopkins Hospital Microbiology Laboratory, Baltimore, Maryland2

Received 19 August 2008/ Returned for modification 23 September 2008/ Accepted 2 December 2008

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°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.

* Corresponding author. Mailing address: The Johns Hopkins Medical Institutions, Division of Medical Microbiology, Meyer B1-193, 600 N. Wolfe St., Baltimore, MD 21287. Phone: (410) 955-5077. Fax: (410) 614-8087. E-mail: kcarrol7{at}jhmi.edu

{triangledown} Published ahead of print on 10 December 2008.

Journal of Clinical Microbiology, February 2009, p. 373-378, Vol. 47, No. 2
0095-1137/09/$08.00+0     doi:10.1128/JCM.01613-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



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