SPECIFIC DETECTION OF CLOSTRIDIUM-DIFFICILE TOXIN-A GENE-SEQUENCES INCLINICAL ISOLATES

The polymerase chain reaction (PCR) was used to specifically detect to xin A gene sequences of Clostridium difficile in DNA isolated from hum an faeces. A set of oligonucleotide primers derived from the non-repet itive region of the toxin A gene was developed to amplify a 634-bp DNA fragment. AII 28 cytotoxic strains of C. difficile, previously charac terized by a toxin B-PCR assay, were positive for the presence of toxi n A gene sequences. No amplification products were obtained from DNAs extracted from non-toxigenic strains, strains of C. sordellii, or C. b ifermentans. In addition, amplification of DNA extracted from C. diffi cile 8864, a strain which does not produce toxin A, resulted in multip le bands which probed negative for toxin A gene sequences. DNAs extrac ted from nine stool specimens which were positive for toxin B by the c ytotoxicity assay and by the toxin B-PCR assay were also positive in t his assay. Toxin A gene sequences were detected in DNAs obtained from 4/11 stool specimens which were negative by the toxin B cytotoxicity a ssay. These four specimens were from patients who had a history of rel apses due to C. difficile-associated colitis, and whose stools had pre viously been found to be positive by the toxin B-PCR test despite no d etectable toxin B in the specimens. These data indicate a comparable d egree of clinical sensitivity between these two toxin-gene PCR-based a ssays. This rapid, sensitive and specific assay may be useful not on l y in the diagnosis of C. difficile infections, but also in molecular s tudies of the toxin A gene in C. difficile strains.