Detection of Cryptosporidium parvum DNA in formed human feces by a sensitive PCR-based assay including uracil-N-glycosylase inactivation

Abstract

We developed a PCR-based method that can be used to identify Cryptosporidium parvum in human feces. Fecal oocysts were concentrated by centrifugation on a sodium chloride gradient and filtration on a nitrocellulose filter prior to DNA extraction and PCR amplification of a 452- bp C. parvum-specific DNA sequence with a protocol including dUTP and uracil- N-glycosylase. All samples obtained from naturally infected humans (n = 10), calves (n = 4), and goats (n = 2) were positive. A 100% detection rate was achieved with both formed and solid stools (n = 10) seeded with 1,1100 C. parvum oocysts per g. Procedures based on stool concentration by a modified Ritchie method and subsequent oocyst identification by immunofluorescent labeling or acid-fast staining require concentrations of 50,000 to 500,000 oocysts per g to achieve a 100% detection rate with formed stools. The described PCR-based assay thus has a 50- to 500-fold increase in sensitivity compared to those of the methods commonly used to analyze formed feces.