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DETECTION OF CRYPTOSPORIDIUM OOCYSTS IN SOURCE AND FINISHED WATERS
Citation:
Schaefer, F W. DETECTION OF CRYPTOSPORIDIUM OOCYSTS IN SOURCE AND FINISHED WATERS. Presented at Cryptosporidium: From Molecules to Disease, Fremantle, Western Australia, Australia, October 7-12, 2001.
Impact/Purpose:
1) Refine new molecular and antibody labeling method for the detection of Giardia and Cryptosporidium in water.
2) Determine the occurrence and distribution of Giardia and Cryptosporidium in water supplies
Description:
Numerous waterborne outbreaks of cryptosporidiosis have occurred with the most notable being the 1993 episode in Milwaukee. As a result, the past decade has seen a massive effort expended on the development of methods to detect Cryptosporidium parvum oocysts in source and finished water. Initial analyses for C. parvum oocysts were based on methods originally developed for detecting Giardia cysts in water. This procedure involved sampling either 100 liters of source water or 1,000 liters or finished water using a fiber wound filter with a nominal porosity of 1.0 um. After washing the filter fibers with buffer, the particulates containing the parasites were concentrated by centrifugation. Further purification of the parasites was done by buoyant density centrifugation. Because buoyant density centrifugation is not a selective purification procedure, the parasites still were associated with and masked by particulates. To overcome this problem, the parasites were selectively stained with fluorescently labeled monoclonal antibodies. The stained samples were then microscopically read using epifluoresdcence microscopy to detect the presumptive parasites and differential interference contrast microscopy to confirm the identity of the parasites by demonstration of internal morphological characteristics. In the case of Cryptosporidium, recovery rates ranged from 0 to 11% in seeded samples. The poor oocyst recovery was based on the fact that up to 60% of the seeded oocysts passed through the fiber filter.
Method 1622 for Cryptosporidium was developed using off the shelf reagents and equipment in an attempt to improve on oocyst recovery. Instead of sampling large volumes of water, 10 liter samples are concentrated by passing the water through a 1.0 um absolute porosity filter. Immunomagnetic separation is a selective procedure employing monoclonal antibodies. To separate the parasites from the concentrated particulates, immunomagnetic separation now is used in place of buoyant density centrifugation. Like the previous method, oocysts are detected by staining with fluorescently labeled monoclonal antibodies. However, in addition to differential interference contrast microscopy for confirmation of internal morphological characteristics, the samples are counter stained with 4',6-diamidino-2-phenylindole (DAPI) which helps demonstrate sporozoite nuclei. Even with these improvements, the recovery results for the Method 1622 is only on average around 38%. Besides undefined parameters in water matrices, a number of other factors, such as laboatory proficiency and commercial reagent and equipment quality, are now known to compromise Cryptosporidium oocyst recovery. A modified version of the Information Collection Rule method, the ChemScan RDI solid phase cytometry method, and the AusFlow method were compared against Method 1622. The oocyst recovery results for Method 1622 in this compartive study were not as good as expected. Modification to immunomagnetic bead dissociation and DAPI staining are required, when these techniques are used in conjunction with any method in the future. Fluorescent in situ hybridization probes have veen suggested as assay additions for speciation of detected oocysts and for providing an indication of viability. Cryptosporidium cell culture has also been suggested as a mechanism for determining viability and infectivity of detected oocysts. Sensitivity, specificity, and the correlation with animal infectivity must be established for these new methods. At this point these new approaches are just research methods and remain to be multi-laboratory validated.