DEMONSTRATION OF TOXIN-A AND TOXIN-B BY POLYMERASE CHAIN-REACTION AND MCCOY CELL ASSAY IN CLINICAL ISOLATES OF CLOSTRIDIUM-DIFFICILE FROM DENMARK

A polymerase chain reaction (PCR) for demonstration of gene fragments of Clostridium difficile was established. One hundred and sixty-eight clinical isolates of C. difficile from three population groups were tested for production of cytotoxins by McCoy cell line assay (MCA) and for fragments of toxin A and B genes by PCR. The fragments for PCR amplification were at the 5' end of the toxin genes, which was found to be specific for C difficile. Full agreement between the PCR and MCA results was found with respect to toxicity. Fifty-eight of the 168 strains were cytotoxin positive. Isolates from 41 normal healthy children did not differ regarding cytotoxicity compared to isolates from hospitalized children. In adult hospitalized patients a much higher frequency of toxin-producing isolates was found. In two strains from two children, not of the same family, only the toxin A gene fragment was demonstrated, indicating that some strains of C difficile only harbour the gene for enterotoxin. When two isolates from different periods of time were tested from 36 of the healthy children, a variation in cytotoxicity was found: in seven children strains changed from non-toxic to toxic and in four vice versa. This may be explained by a fluctuating colonization of toxic and non-toxic C difficile strains, or it may indicate the need for examination of more than one strain from a positive faecal sample to demonstrate cytotoxicity.