VAN DEN BERG R, BRUIJNESTEIJN LS, CLAAS EC, VAN DAM AP, KUIJPER EJ; Interscience Conference on Antimicrobial Agents and Chemotherapy (43rd: 2003: Chicago, Ill.).

Abstr Intersci Conf Antimicrob Agents Chemother Intersci Conf Antimicrob Agents Chemother. 2003 Sep 14-17; 43: abstract no. D-1872.

LUMC, Leiden, Netherlands.

BACKGROUND: Clostridium difficile-associated diarrhea (CD-AD) is diagnosed by the detection of specific enterotoxins in feces or by culture. The pathogenicity of CD is associated with the production of toxin A and B, although infections caused by toxin A negative, toxin B-positive (tox A-neg) strains are reported as well. The aim of this study was to develop a real-time PCR for rapid clinical diagnosis of CDAD from feces samples. METHODS: Primers for a real-time PCR were designed to amplify 177 bp of the nonrepeat region of the toxin B gene. A Taqman probe labeled with fluorophor FAM and quencher TAMRA was used as an internal probe specific for this amplicon. DNA isolation from feces samples was performed using polyvinylpolypyrrolydone (pvpp) pretreatment and Qiagen columns. Spiking experiments with a CD strain in 0,9% NaCl and in feces were used to determine the sensitivity of the real-time assay. Feces samples were obtained from 43 patients of which 28 had symptoms compatible with CDAD and 15 were asymptomatic. Cultures were performed on selective media before and after ethanol treatment. RESULTS: The sensitivity of the real-time PCR was 1 colony forming unit (CFU) in 0,9% NaCl and 10 CFU in feces. This correlates to a concentration of 2800 CFU/g feces. In 23 of 28 CDAD suspected patients and 4 of 15 asymptomatic patients toxinogenic CD was detected by culture. Using real-time PCR 25 of 28 CDAD suspected patients were tested positive. Three of these were culture negative, of which 2 had very high cycle tresholds and thus a low concentration of CD. The other was co-infected with E.coli O157. One patient was negative by real-time PCR and positive by culture, but did show a fragment of the right size on gel electrophoresis. All 4 culture-positive asymptomatic patients, had a positive signal in real-time PCR. CONCLUSION: Detection of CD toxin B gene in feces samples by real-time PCR is more sensitive than culture and can therefore be used as a rapid diagnostic method for CDAD and for the detection of asymptomatic carriership.

Publication Types:

• Meeting Abstracts

Keywords:

• Clostridium difficile
• DNA Primers
• Enterotoxins
• Feces
• Hematologic Tests
• Humans
• Polymerase Chain Reaction
• Sensitivity and Specificity
• analysis

Other ID:

• GWAIDS0025312

UI: 102264936

From Meeting Abstracts