Development and validation of a real-time PCR assay for detection of toxigenic Clostridium difficile, as part of a Dutch study on the epidemiology of gastro-enteritis

Abstract number: P1773

de Boer R.F., Wijma J., Schuurman T., Luijt D.S., Dijk-Alberts B., Ott A., Kooistra-Smid A.M.D., van Duynhoven Y.T.H.P.

Objectives:Clostridium difficile is the aetiologic agent responsible for human diseases ranging from mild diarrhoea to severe pseudomembranous colitis, which are collectively referred to as C. difficile associated disease (CDAD). The cell cytotoxicity (CT) assay is considered the gold standard for detection of toxigenic C. difficile. However, this method is laborious and time-consuming (turn-around time: >48 hours). Rapid diagnosis of CDAD is important, since it may result in early treatment and help prevent nosocomial transmission. Therefore, a real-time PCR based assay was developed and validated. Also, this assay will be used as a screening tool for a gastro-enteritis study (GEops Study). This study will commence in May 2008 in 6 hospitals in The Netherlands. Its primary goal is to assess the incidence, aetiology and course of patients hospitalised for gastro-enteritis.

Methods: An assay targeting the tcdA and tcdB genes was developed. Stools were processed with the easyMAG specific A stool protocol (bioMérieux). The phocine herpes virus-1 was used as internal control. The selectivity of the assay was validated with a panel of well characterised C. difficile strains and clinical isolates (n = 50) and non-C. difficile strains (n = 43). The analytical sensitivity was assessed by dilution series (n = 3), spiked in a homogenous faecal matrix. Also, the assay was compared with the CT assay in a clinical validation performed on stool samples (n = 163) of patients suspected of CDAD.

Results: The screening assay proved to be specific for C. difficile as no cross-reaction was observed. The assay was capable of detecting approximately 1560 CFU/g of stool with a 100% hit rate. Sixteen PCR positive and 139 PCR negative samples were in complete concordance with the CT assay. The PCR was positive in 8 additional samples which remained negative in the CT assay. One of these 8 samples could be confirmed as truly positive by CT in a consecutive sample of the same patient. In comparison with the gold standard (CT) PCR showed 100% sensitivity, 95% specificity, 67% positive predictive value, and 100% negative predictive value. PCR inhibition was observed in less than 1% of all 163 screened stool samples.


1This in-house real-time PCR assay offers a rapid and sensitive method for the first screening for CDAD.

2The assay will be used as a rapid screening tool for the detection of C. difficile in the GEops Study that will commence in May 2008.

Session Details

Date: 19/04/2008
Time: 00:00-00:00
Session name: 18th European Congress of Clinical Microbiology and Infectious Diseases
Location: Barcelona, Spain
Presentation type:
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