Detection of infectious Cryptosporidium parvum oocysts in environmental water samples using an integrated cell culture-PCR (CC-PCR) system

Examination of naturally occurring C. parvum oocysts from environmental water samples has previously been hampered by the inability to determine the public health significance of detected organisms. As a result the safety of drinking water supplies was in question. These limitations have been resolved through the development and application of a method that incorporates immunomagnetic separation (IMS) and an infectivity determination using an integrated cell culture, polymerase chain reaction assay (CC-PCR). Briefly, the method concentrates water samples by filtration or centrifugation and isolates oocysts by IMS. An acidified Hank's balanced salt solution (HBSS) containing 1% trypsin was used for the dissociation of captured oocysts from the IMS beads. In vitro HCT-8 cell culture of purified oocysts was performed in 96-well microtiter plates and infected cells were detected using PCR primers specific for C. parvum. A total of 242 raw source waters or filter backwash water samples from twenty five sites in the U.S. were analyzed to validate the procedure. Oocyst seeded in raw and filter backwash water samples were used to evaluate recovery efficiencies and performance of the CC-PCR protocol with different water quality matrices. The CC-PCR detected infectious Cryptosporidium parvum in 6 of 121 (5.0%) raw and 9 of 122 (7.4%) filter backwash water samples. All CC-PCR positive samples were confirmed by cloning and DNA sequence analysis of the PCR products. Isolates were shown to originate from human and animal sources. Grouping of genotypes permitted evaluation of strain diversification and variation. Current studies are using this technique to examine oocysts in various watersheds and in the finished drinking water of over 80 surface water treatment plants.