PCR detection and prevalence of enterotoxin (cpe) gene in Clostridium perfringens isolated from diarrhea patients.

Clostridium perfringens isolated from patients with diarrhea (n=233) were analysed by a duplex PCR assay, in order to determine the prevalence of enterotoxin (cpe) gene and various factors involved in patients with cpe-positive isolates. This duplex PCR uses two sets of primers which amplify in the same reaction two different gene fragments: the phospholipase C (plc, alpha-toxin) and the enterotoxin (cpe) genes in C. perfringens. PCR analysis of 477 colonies of fecal spore isolates, from 159 patients who had a spore count > or = 10(3) cfu/g, gave positive plc gene detection in 436 colonies. The results were consistent with those obtained by using the standard method of C. perfringens species identification. 21 of 436 colonies gave positive results for both plc and cpe genes, indicating a prevalence of 4.8 per cent of C. perfringens that carried the cpe gene in cases of diarrhea. The majority of cases with cpe-positive isolates were women over 50 years of age (71.4%). These patients had diarrhea more than 6 times per day (71.4%) with a duration of 1-3 days (100%). Furthermore, 85.7 per cent of cases developed diarrhea after food consumption, 28.6 per cent had high spore counts of more than 106/g in their feces, and 71.4 per cent were co-infected with other enteric pathogens. The spore count should be interpreted with caution because not all isolates of C. perfringens from diarrhea patients with high fecal spore count carried the cpe gene, which encodes a sporulation-associated enterotoxin.

CONCLUSIONS

The duplex PCR assay can thus become a tool for C. perfringens species identification together with the detection of enterotoxin gene. This PCR assay is faster, less expensive and more suitable for large-scale use in epidemiological studies than conventional procedures. The authors recommend this assay to screen for enterotoxigenic C. perfringens isolates from primary fecal spore isolation cultures, particularly in elderly patients with food-borne diarrhea and non-food related diarrhea.