Geneohm c diff assay

Testing was performed on stools in the clinical microbiology laboratory via an algorithm (Figure 1) using the C. DIFF QUIK CHEK COMPLETE^® test for C. difficile glutamate dehydrogenase (GDH) and toxins A or B (Techlab, Inc., Blacksburg, VA) by EIA. All GDH⁺/toxin⁻ stool tests were subjected to analysis for the tcdB gene by real-time PCR using the GeneOhm™ Cdiff Assay (BD, Franklin Lakes, NJ) run on a Cepheid SmartCycler^® System (Cepheid, Sunnyvale, CA). Confirmation of all positive C. difficile tests was attempted by anaerobic culture on taurocholate-cycloserine-cefoxitin-fructose agar at 37°C. Attempts were made to ribotype samples using high-throughput, fluorescent PCR-ribotyping as described elsewhere [21 (link), 22 ]. Blood culture collection and detection of positives was performed using the aerobic and anaerobic BacT/Alert system (BioMerieux, Durham, NC) and handled per the clinical microbiology laboratory protocol [23 ]. Briefly, all culture bottles are read by the automated system and positives reported to the laboratory staff every 15 minutes, followed by an initial Gram stain and subculturing / identification as appropriate.

IRB approval was obtained (HSR-IRB #13607). Sequential inpatients at our US academic hospital clinically diagnosed with CDI (May 2010–August 2011) were identified in the UVA Clinical Microbiology Laboratory once tested positive for via PCR for toxin B gene (BD GeneOhm™ Cdiff Assay, Becton, Dickinson and Company, Franklin Lakes, NJ or Xpert C. difficile, Cepheid, Sunnyvale, CA) based on manufactures instructions (20 (link)). Vital signs, laboratory values, and co-morbidities were obtained from the hospital electronic medical record. Most extreme value within 24 hours of CDI diagnosis was recorded. The Charlson comorbidity index was calculated for each subject from co-morbidities (21 (link)). Data was obtained by members of the research team and spot-checking and discrepancy resolution was done by the principle investigator. Delirium was identified retrospectively from medical record when acute cognitive change was cited by the clinical treatment team in medical record on the day of CDI diagnosis. Body-Mass index (BMI) used normative cut-offs for underweight (18.5 kg/m²) (22 ). Each subject was contacted via telephone for consent at 1month and, once consent was obtained, a semi-structured interview was performed with subject, surrogate, or facility staff to record outcomes. The medical record and state death registry was also reviewed for 30 day mortality.

Stool testing for CDI was requested based on clinical suspicion by the medical team. The hospital policy did not have specific criteria to limit or guide testing. Samples were tested in the clinical microbiology laboratory using polymerase chain reaction (PCR) for toxin B gene [either BD GeneOhm™ Cdiff Assay (Becton–Dickinson, Franklin Lakes, NJ, USA) or Xpert C. difficile (Cepheid, Sunnyvale, CA, USA)] according to manufacturers’ instructions. The clinical laboratory used both PCR methods during the time of the study, and choice of test was based on the time at which samples were received and batching protocols. PCR-positive samples were frozen and sent to TechLab, Inc. (Blacksburg, VA, USA) for stool toxin detection using the TOX-B cytotoxin test (TechLab), toxigenic culture, ribotyping, and measurement of intestinal inflammation (quantitative faecal lactoferrin, IBD-SCAN^®) using methods previously described.^{7 (link)} Fluoroquinolone resistance using E-test^® (bioMérieux, Durham, NC, USA) was performed according to the manufacturer’s instructions.