Rapid detection of Clostridium difficile in faeces by real-time PCR

Abstract number: P784

Egli K., Peter-Getzlaff S., Altwegg M.

Objectives: Traditional methods for the detection of C. difficile consist of immunoassays (sometimes believed to lack sufficient sensitivity), culture on selective medium, and traditional "gold standard" tissue culture cytotoxicity methods, which are difficult to perform and require several days to yield results. Our study was aimed at determining whether a commercial and a home-brew real-time PCR are suitable for the rapid detection of C. difficile in faecal specimens.

Methods: As part of an ongoing study for determining the performance of culture, cell culture cytotoxicity and three immunological toxin tests, a subset of 133 unformed stool specimens from patients with suspected CDAD was also analyzed by the BD GeneOhm™ Cdiff Assay (BD-PCR) consisting of a rapid, glass bead-based DNA preparation without further purification followed by real-time amplification of part of the toxin B gene on a SmartCycler II. The same extract was simultaneously analyzed with a home-brew duplex PCR detecting both toxin A and B genes using the 5' exonuclease format on a LightCycler 480 (BA-PCR). The performance of PCR was compared to the 'gold standard' (cell culture cytoxicity, with culture as reference for the resolution of discrepant results). 131 specimens were also analyzed by commercial and home-brew PCR after extraction/purification of DNA from specimens with the easyMAG system.

Results: DNA preparation according to the BD GeneOhm™ Cdiff Assay (N = 133) resulted in inhibition of 3 (2.3%) and 11 (8.3%) of specimens with BD- and BA-PCR, respectively. Sensitivity/specificity for not inhibited specimens was 95.7%/96.4% for BD-PCR, 95.1%/97.5% for BA-PCR, and 89.1%/98.8% for cell culture cytotoxicity. After easyMAG extraction (N = 131), 2 (1.5%) and 1 (0.8%) of specimens showed inhibition in BD- and BA-PCR, respectively. Sensitivity/specificity for not inhibited specimens was 100%/97.5% for BD-PCR, 97.6%/96.3% for BA-PCR, and 89.8%/100% for cell culture cytotoxicity.

Conclusions: Both PCR assays are more sensitive than and almost as specific as cell culture cytoxicity und thus provide simple and rapid stool tests that allow same-day identification of toxigenic C. difficile. DNA purification slightly increases the performance of the commercial assay and is a must for our home-brew PCR in order to avoid too many non valid results due to inhibition.