title: 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 1,000 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.
authors.affiliation: Laboratoire de Parasitologie et Mycologie, Hôpital du Bocage, Dijon, France.
authors.forename: P
authors.initials: P
authors.lastname: Gobet
authors.forename: J C
authors.initials: JC
authors.lastname: Buisson
authors.forename: O
authors.initials: O
authors.lastname: Vagner
authors.forename: M
authors.initials: M
authors.lastname: Naciri
authors.forename: M
authors.initials: M
authors.lastname: Grappin
authors.forename: S
authors.initials: S
authors.lastname: Comparot
authors.forename: G
authors.initials: G
authors.lastname: Harly
authors.forename: D
authors.initials: D
authors.lastname: Aubert
authors.forename: I
authors.initials: I
authors.lastname: Varga
authors.forename: P
authors.initials: P
authors.lastname: Camerlynck
authors.forename: A
authors.initials: A
authors.lastname: Bonnin
mesh_terms: D000818:Animals; D016785:Cryptosporidium parvum / Q000302:isolation & purification*; D045647:DNA Glycosylases; D005243:Feces / Q000469:parasitology*; D006801:Humans; D009699:N-Glycosyl Hydrolases; D016133:Polymerase Chain Reaction / Q000379:methods*; D051981:Uracil-DNA Glycosidase
chemical_list: D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase