Comparison of Two Real Time PCR Assays to Culture for the Direct Detection of Toxogenic Clostridium difficile Strains from Stool Specimens

Background: C.difficile is a pathogen responsible for the majority of cases of infectious healthcare-associated diarrhea and colitis. Real time PCR is a new sensitive and rapid test for detection of C.difficile directly from stool specimens.

Objective: The objective of this study is to determine the sensitivity (SENS), specificity (SPEC), positive predictive value (PPV) and negative predictive value (NPV) of an in-house and the BD GeneOhm commercial PCR assays when compared to toxigenic culture for detection of toxin producing C .difficile directly from stool specimens.

Methods: 350 liquid/soft stool specimens from 323 patients were tested using convenience sampling. Anaerobic cultures were performed on all stool specimens by plating onto pre-reduced cycloserine-cefoxitin-fructose agar (CCFA-VA formulation) media. Plates were incubated anaerobically at 35°C for up to 5 days before a final negative interpretation. Yellow, spready colonies characteristic of C. difficile that were Gram variable rods, Pro Disc (Key Scientific Products) positive and did not grow aerobically were confirmed as C. difficile. All C. difficile isolates were grown in anaerobic chopped-meat (Remel) broth and supernatant was inoculated on MRC-5 cell lines and held for up to 48 hours to detect presence of toxin. A small aliquot of stool was also added to Cycloserine Cefoxitin Mannitol broth with Taurocholate Lysozyme Cysteine  and incubated up to 4 days to perform enriched cultures if primary culture was negative. A second culture method as described by Stamper et al (J. Clin. Microbiol 47:373, 2009) was also done on a subset of specimens to resolve discrepancies. For the in-house PCR method, stool specimens were diluted 1:10 in sterile saline and 100 µl was processed using the MagNA Pure LC DNA Isolation Kit III for Bacteria (Roche). 2 µl of extracted DNA was used in the PCR reaction run on the Roche LightCycler. For the BD PCR assay, a sterile swab was dipped in stool specimen and processed following package insert.

Results: Any positive culture was considered a gold standard for this evaluation. For 350 specimens the in-house PCR assay had a SENS, SPEC, PPV and NPV of 92.5%, 99%, 92.5% and 99% respectively. Three specimens could not be evaluated on the BD PCR and hence were excluded. For 347 specimens, the SENS, SPEC, PPV and NPV was 92.5%, 98.7%, 90.2% and 99%.

Conclusions: Both PCR assay performed equally well when compared to culture for detecting toxigenic C. difficile directly from stool specimens. PCR assays can be very useful for patient management and infection control.