Characterization of Cryptosporidium parvum in human and animal feces by single-tube nested polymerase chain reaction and restriction analysis

A DNA isolation and purification method is described that produced DNA free of inhibitory substances in 148 of the 159 analyzed fecal samples. The pol ymerase chain reaction (PCR) product from a sensitive single-tube nested PC R that amplifies a part of an oocyst protein was used to characterize Crypt osporidium parvum genotypes by a simple restriction analysis. Genotype 1 wa s solely detected in human-derived oocysts, genotype 2 was present in both animal and human-derived oocysts. The ratio between both genotypes in human s in The Netherlands varied markedly between samples obtained during a peri od of augmented cases of cryptosporidiosis in the western part of the count ry and randomly selected samples from gastroenteritis patients. Sequence an alysis of a 581-bp fragment from the nested PCR product revealed 12 nucleot ide substitutions between the two genotypes. Sequences from isolates in eac h genotype group were identical.