Recent studies have shown that Cryptosporidium can be found in finished reclaimedwater at levels higher than previously reported, which was prior to implementation of a revisedmethod for detecting the pathogens. In addition, recent work shows that infectiousCryptosporidium was detected in finished reclaimed water using a tissue culture assay to assesspotential human infectivity (Quintero-Betancourt, et al., 2003).The current detection method (USEPA Method 1623) allows for the direct enumerationof Cryptosporidium; however, it cannot assess the potential viability or ability of these organismsto infect humans. The polymerase chain reaction (PCR) is a novel method suggested foridentifying Cryptosporidium in water samples processed by Method 1623 (Xiao, et al., 2000).PCR detects the DNA of the pathogen and can be species and genotype specific. Thus, thepurpose of this study was to: evaluate and compare the use of Method 1623 alone or combinedwith PCR for detecting Cryptosporidium in reclaimed water; determine the levels ofCryptosporidium in finished water in reclaimed water in central Florida; and, ascertain whetherthe organisms detected were viable and potentially infectious to humans.USEPA Method 1623 was performed by concentrating 20 to 50 L of water onto anEnvirochek HV filter (Pall Gelman, Ann Arbor, MI). Target organisms were eluted off the filter,concentrated by centrifugation, and isolated using immunomagnetic separation (IMS) followedby an immunofluorescence staining procedure (IFA) for identification (USEPA Method 1623,2001). Isolation and preparation of Cryptosporidium DNA for PCR was performed usingDNazol Reagent (Invitrogen, Carlsbad, CA). Primers specific for a small subunit rRNA fragmentcommon to all Cryptosporidium species (Xiao, et al., 2000) were used to amplify the DNA. Includes 4 references, tables.
