We describe a nested PCR-restriction fragment length polymorphism (RFLP) method for detecting low densities of Cryptosporidium spp. oocysts in natural mineral waters and drinking waters. Oocysts were recovered from seeded 1-liter volumes of mineral water by filtration through polycarbonate membranes and from drinking waters by filtration, immunomagnetizable separation, and filter entrapment, followed by direct extraction of DNA. The DNA was released from polycarbonate filter-entrapped oocysts by disruption in lysis buffer by using 15 cycles of freeze-thawing (1 min in liquid nitrogen and 1 min at 65°C), followed by proteinase K digestion. Amplicons were readily detected from two to five intact oocysts on ethidium bromide-stained gels. DNA extracted from Cryptosporidium parvum oocysts, C. muris (RN 66), C. baileyi (Belgium strain, LB 19), human-derived C. meleagridis, C. felis (DNA from oocysts isolated from a cat), and C. andersoni was used to demonstrate species identity by PCR-RFLP after simultaneous digestion with the restriction enzymes DraI and VspI. Discrimination between C. andersoni and C. muris isolates was confirmed by a separate, subsequent digestion with DdeI. Of 14 drinking water samples tested, 12 were found to be positive by microscopy, 8 were found to be positive by direct PCR, and 14 were found to be positive by using a nested PCR. The Cryptosporidium species detected in these finished water samples was C. parvum genotype 1. This method consistently and routinely detected >5 oocysts per sample.
We determined the incidence of cryptosporidiosis in children aged <5 years presenting with diarrhoea in an urban and rural hospital-based setting in Malawi. Stools were collected over a 22-month period during both rainy and dry seasons. A range of microscopic methods were used to determine the presence of Cryptosporidium spp. oocysts. Species determination was by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of oocyst-extracted DNA using 18S rRNA and COWP gene loci. Cryptosporidium spp. oocysts were seen in 5.9% (50/848) of samples, of which 43 amplified by PCR-RFLP indicated the following species: C. hominis, C. parvum, C. hominis/C. parvum, C. meleagridis and C. andersoni. Seven samples could not be amplified by PCR. Wider species diversity was found in the rural setting, and may be a result of increased malnutrition and zoonotic exposure in this area. Improvements in water, sanitation, household hygiene and animal control are required to reduce the incidence of infection in this population.
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