PCR Detection and Prevalence of Enterotoxin (cpe)
Gene in Clostridium perfringens Isolated from Diarrhea
Patients
UNCHALEE TANSUPHASIRI, M.Sc. *,
GUMPHOL WONGSUVAN, M.Sc .. **,
BOONCHUAY EAMPOKALAP, M.Sc.***
Affiliation : * Department of Microbiology, Faculty of Public Health,
** Wellcome Tropical Disease Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400,
*** Bamrasnaradura Hospital, Ministry of Public Health, Nonthaburi 11000, Thailand.
AbstractClostridium 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 (pic, 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 ~103 cfu/g, gave positive pic 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 pic and cpe
genes, indicating a prevalence of 4.8 per cent of C. perjringens 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.
Conclusion : 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 expen
sive 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.
Keywords : Clostridium Perfringens, Diarrhea Patients, Duplex PCR, Enterotoxin
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