Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Thu, 25 Oct 2018 09:46:54 J. Med. Microbiol. - Vol. 38 (1993), 109-1 13 0 1993 The Pathological Society of Great Britain and Ireland Characterisation of Clostridhm difficile strains by polymerase chain reaction with toxin A- and B-specific primers B. W. WREN, S. R. HEARD, A. I. AL-SALEH and S. TABAQCHALI Department of Medical Microbiology, St Bartholome w 3 Hospital Medical College, West Smithfield, London EC1A 7BE Summary. A total of 218 Clostridium dzficile strains was examined for production of toxin A by ELISA, production of toxin B by a cytotoxin assay and the presence of toxin A and B gene- associated sequences by the polymerase chain reaction (PCR). After saturation amplification with toxin B-specific primers, the characteristic amplification product (59 1 bp) was detected in all 184 toxigenic strains examined. PCR with toxin A-specific primers gave positive results with all but one of the toxigenic strains. By contrast, PCR with toxin A- and toxin B-specific primers yielded negative results with all 34 non-toxigenic strains tested. This suggests that PCR detection of either the toxin A or B gene is a good indication of toxin production. PCR did not require DNA extraction or hybridisation and was convenient, sensitive and rapid. Toxigenic C. dificile could be detected in mixed cultures, suggesting a role for PCR in the identification of toxigenic C. dzficile in primary culture. Introduction Toxigenic strains of Clostridiurn dzficile are the major cause of pseudomembranous colitis and antibiotic-associated colitis and diarrhoea.’ Two toxins appear to be involved in disease: toxin A, an enterotoxin with cytotoxic activity; and toxin B, a potent cytotoxin.2 The diagnosis of C. dzficile- associated disease depends on the isolation and identification of the organism or the detection and neutralisation of cytotoxic activity from faecal specimens, or both. Results correlate well with clinical disease, but the cytotoxin assay is costly, the toxin cross-reacts with C. sordellii toxin, and some laboratories may lack the required facilities. Recently, a direct faecal ELISA (Meridian Diagnostics, Cincinnati, USA), which also cross-reacts with C. sordellii toxin, has been reported to have 84.1 YO sensitivity compared with cytotoxin assay.3 This is a rapid, sensitive but expensive test. Another less costly assay for the presence of toxin A is a latex test (Culturette Brand C. dzflcile test; Marion Laboratories, Kansas, USA) which has been shown to be non-specific for C. dificile and to give a large number of false positive results.*9 More rapid, specific and cost-effective tests for the detection of toxigenic C. dijicile are needed. The polymerase chain reaction (PCR) allows sen- sitive detection of a given DNA sequence even in a complex mixture of molecules and has been used in the . ~ ~~ Received 19 March 1992; accepted 17 July 1992. identification of a range of pathogenic organisms.‘ The decrease in cost of thermostable DNA poly- merases may make PCR a cost-effective alternative to traditional diagnostic procedures. A potential draw- back of PCR in diagnostic applications is that it detects genotypic rather than phenotypic character- istics, and “silent” genes may give false positive results. Recently, we described a rapid and sensitive assay for toxigenic C. dificile by PCR with boiled extracts.’.’ The toxin A-specific oligonucleotide primers used were tandem repeat nucleotide sequences from the toxin A gene, which resulted in a characteristic profile of amplified products that provided an easily inter- pretable alternative to conventional single-product PCR.’-’ Previous reports on PCR-based identification Fig. 1. Identification of toxigenic C. drfficile with toxin A-specific primers. Lanes B, D, E, W, X and Z, toxigenic C. dificile strains; A, C and Y, non-toxigenic C. dificile strains; 1, DNA size markers (Bsh 1 and Msp 1 digests of pHC314). 8 JMM 38 109