Toxinotyping of clostridium difficile in infected patients at AUBMC -

Abstract

Background: Pseudomembranous colitis by Clostridium difficile is directly associated with simultaneous prolonged hospitalization and antibiotic intake. The pathogenesis of C. difficile is due to two main virulence factors, toxins A and B encoded by toxin genes, tcdA and tcdB, on the PaLoc (pathogenicity locus) region. C. difficile strains with variations in tcdB and tcdA genes on PaLoc are defined as variant toxinotypes and currently 31 such groups are recognized (I to XXXI). Variations in PaLoc could result in production of toxins with altered properties. Toxinotyping in C. difficile is therefore important in determining the virulence potential of the strains. Due to the increase in the incidence of C. difficile associated diseases at the American University of Beirut Medical Center in Lebanon this study was undertaken to determine the prevalence of toxigenic C. difficile and their various toxinotypes.Methods: Eighty eight stool samples tested by the immunocard toxin A and B (Meridian Bioscience) were anonymously provided by the Clinical Microbiology Division. They were then cultured for C. difficile on CCFA agar for 48 hours in anaerobic media, and then stored at -80°C for further studies. The recovered isolates were preliminary identified-confirmed by API 20A anaerobic strips. PCR amplification on extracted DNA of the triose phosphate isomerase (tpi) gene were carried out on all isolates to confirm the presence of C. difficile in stool samples. PCR amplification of the toxin genes tcdA and tcdB followed by toxinotyping were also performed on recovered isolates.Results: Out of the 88 stool samples obtained, 46 were positive for C. difficile by the immunocard assay, but only 24 of the 88 stool samples were culture positive and tpi positive. Six stool samples did not show growth on CCFA agar but were positive for the tpi gene indicating presence of C. difficile. Of these 30 isolates, 4 were nontoxigenic (A-B-), and 26 were toxigenic out of which 4 were A+B+, 1 was A+B-, and 21 A-B+. Analysis of toxinotypi