We compared two commercial PCR assays, the Prodesse ProGastro CD assay and the BD GeneOhm Cdiff assay, with a laboratory-developed Clostridium difficile toxin PCR assay with previously established performance characteristics. Results of all methods were in agreement for 333 (96%) of 346 stool specimens. No significant difference in performance among the assays was found (P values, >0.05).Clostridium difficile is the leading cause of antibiotic-associated diarrhea and pseudomembranous colitis in health care settings (6). Several laboratory-developed PCR assays for detection of C. difficile infection (CDI) have been described, and they have demonstrated favorable performances in comparative studies (3,7,10,14). We previously described a real-time PCR assay using a LightCycler (Roche Diagnostics, Indianapolis, IN), which we refer to herein as the LC-CDTX assay (11). We compared two commercial FDA-approved real-time PCR assays, the BD GeneOhm Cdiff assay (San Diego, CA) and the Prodesse ProGastro CD assay (Waukesha, WI), to our laboratory-developed assay.The study was approved by the Institutional Review Board, Mayo Clinic, Rochester, MN. Three hundred forty-six soft or liquid stool specimens submitted to the Clinical Microbiology Laboratory, Mayo Clinic, from different patients for C. difficile testing by PCR assay (LC-CDTX assay) were used to compare three molecular assays.For LC-CDTX assay extraction, swabs were inserted into the stool sample and transferred into 1 ml of sterile water. After the sediment settled, 200 l of supernatant was extracted using a total nucleic acid isolation kit (Roche Diagnostics) with a MagNA Pure system (Roche Diagnostics) and eluted into 100 l of elution buffer. For sample lysis with the BD GeneOhm Cdiff assay, swabs were dipped into the stool sample, broken into 1 ml of sample buffer, and vortexed. Ten microliters of sample was transferred into a lysis tube containing 40 l of sample buffer, vortexed for 5 min, centrifuged, heated at 95°C for 7 min, and placed on ice. For ProGastro CD assay extraction, approximately 100 l of stool was added to 400 l of stool transport and recovery buffer (S.T.A.R. buffer; Roche Diagnostics), vortexed, and centrifuged for 1 min at 13,000 ϫ g. Twenty microliters of the cleared stool supernatant and 10 l of the ProGastro CD assay internal control (IC) were extracted using bioMérieux easyMAG (bioMérieux, Durham, NC) and eluted into 110 l of elution buffer.The LC-CDTX assay detects tcdC, a downregulator of toxin production present in toxigenic C. difficile, and also, via melting curve analysis, detects 18-and 39-bp deletions in tcdC, which have previously been associated with increased toxin production. The assay was run as previously described (11), by using a LightCycler 2.0 real-time PCR system with software to detect the fluorescence resonance energy transfer (FRET) probes labeled with LC Red 640. The BD GeneOhm Cdiff assay targets tcdB of C. difficile and the IC, which was incorporated into the master mix. The assay was run with the SmartCycler instr...
