Evaluation of a New Commercial TaqMan PCR Assay for Direct Detection of the Clostridium difficile Toxin B Gene in Clinical Stool Specimens

Abstract

The ProGastro Cd assay (Prodesse, Inc., Waukesha, WI) is a new commercial TaqMan PCR assay that detects tcdB. The ProGastro Cd assay was compared to the Wampole Clostridium difficile toxin B test (TOX-B test; TechLab, Blacksburg, VA), a cell culture cytotoxicity neutralization assay (CCCNA), and to anaerobic toxigenic bacterial culture, as the "gold standard," for 285 clinical stool specimens. Assays were independently performed according to manufacturers' directions. A 1.0-ml sample was removed from the stool specimen, of which 20 l was used for extraction on the NucliSENS easyMAG platform (bioMérieux, Inc., Durham, NC) for the Prodesse ProGastro Cd assay and 200 l of the stool filtrate was used for the TOX-B CCCNA. Anaerobic toxigenic culture was done by heating an additional 1.0 ml of the stool sample to 80°C for 10 min before inoculation onto modified cycloserine, cefoxitin, and fructose agar with horse blood (Remel, Lenexa, KS) and into a prereduced chopped meat glucose broth (BBL, BD Diagnostics, Sparks, MD). The prevalence of toxin-producing strains of C. difficile was 15.7% (n ‫؍‬ 44) as determined by anaerobic toxigenic culture. The sensitivity, specificity, and positive and negative predictive values of the Prodesse ProGastro Cd assay compared to the TOX-B test were 83.3%, 95.6%, 69.4%, and 98%, respectively. Compared to toxigenic culture, the sensitivity, specificity, and positive and negative predictive values of the Prodesse ProGastro Cd assay were 77.3%, 99.2%, 94.4%, and 95.9%, respectively, and those of the TOX-B test were 63.6%, 99.2%, 93.3%, and 93.6%, respectively. Although no statistical difference (Fisher's exact test) was detected (P ‫؍‬ 0.242) between the sensitivities of the Prodesse ProGastro Cd assay and a standard CCCNA compared to anaerobic culture for the detection of toxigenic C. difficile, the Prodesse ProGastro Cd assay did detect more toxigenic C. difficile isolates than the CCCNA.

Comparison of two commercial molecular tests for the detection of Clostridium difficile in the routine diagnostic laboratory

“…A low percentage of invalid results was reported by Stamper et al (2009) and Terhes et al (2009). In our study the Cepheid test had a higher proportion of invalid results and they were rarely resolved after repeating the test.…”

“…Currently several realtime PCR assays that detect toxigenic C. difficile directly from stool are commercially available. Published reports cover only three of them: BD GeneOhm Cdiff assay (BD Diagnostics), ProGastro Cd assay (Prodesse) and Cepheid Xpert C. difficile, which detect the toxin B gene (tcdB) (Barbut et al, 2009;Huang et al, 2009;Stamper et al, 2009;Terhes et al, 2009;Babady et al, 2010;Doing et al, 2010;Knetsch et al, 2011;Kvach et al, 2010;NovakWeekley et al, 2010;Swindells et al, 2010;Tenover et al, 2010). Cepheid Xpert C. difficile also gives information about the binary toxin gene and 117 bp tcdC deletion, both features of the NAP1/BI/027 strain, a causative agent for epidemics of CDI in countries worldwide.…”

Loop-Mediated Isothermal Amplification Compared to Real-Time PCR and Enzyme Immunoassay for Toxigenic Clostridium difficile Detection

“…Molecular assays directed at one or more of five genes residing within the pathogenicity locus (PaLoc) have fostered much interest. Commercial kit-based real-time PCR tests targeting the tcdB gene have recently become available-the BD GeneOhm Cdiff assay (Becton Dickinson), proGastro Cd (Gen-Probe/Prodesse), and Xpert C. difficile (Cepheid)-with exceptional performance and relative ease of use documented (5,8,9,13,(28)(29)(30)(31). FDA clearance was recently granted for the Meridian illumigene C. difficile assay, which uses loop-mediated isothermal amplification (LAMP) to detect the toxin A gene (tcdA) within the PaLoc.…”

Clostridium difficile Infection: Epidemiology, Risk Factors, Pathogenesis, Clinical Features, Diagnosis and Therapy

“…Progastro CD yönteminde, DNA izolasyonu bioMerieux NucliSENS easyMAG otomatik sistemi ile, amplifikasyon işlemi ise Cepheid SmartCycler II cihazında yapılmak-ta; sonuçlar yaklaşık 4 saatte alınmaktadır. Çalışmalarda duyarlılığı %77.3-95.7, özgüllü-ğü ise %99-100 olarak bildirilmiştir 31,32 . Cepheid GeneExpert yöntemi GeneExpert cihazında gerçekleştirilmektedir.…”