Demonstration of toxin A and B by polymerase chain reaction and McCoy cell assay in clinical isolates of Clostridium difficile from Denmark

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Abstract
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.