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BLIND TRIALS EVALUATING IN VITRO INFECTIVITY OF CRYPTOSPORIDIUM PARVUM OOCYSTS USING CELL CULTURE IMMUNOFLUORESCENCE
BUKHARI, Z., D. M. HOLT, M. W. WARE, AND F. W. SCHAEFER. BLIND TRIALS EVALUATING IN VITRO INFECTIVITY OF CRYPTOSPORIDIUM PARVUM OOCYSTS USING CELL CULTURE IMMUNOFLUORESCENCE. CANADIAN JOURNAL OF MICROBIOLOGY. NRC Research Press, Ottawa, Canada, 53(5):656-663, (2007).
1) Refine new, practical methods for the detection of CCL-related and emerging waterborne human protozoa.
2) Perform field tests of devices or methods that have been developed under this task.
3) Evaluate these methods or devices in a variety of water matrices and parasite concentrations.
This work in this task supports CCL2 and 3 and is expected to be completed by 9/07.
An optimized cell culture-immunofluorescence (IFA) procedure, using the HCT-8 cell line, was evaluated in 'blind' trials to determine the sensitivity and reproducibility for measuring infectivity of flow cytometry prepared inocula of C. parvum oocysts. In separate trials, suspensions consisting of between 0-100% viable oocysts were prepared at the U.S. EPA, shipped to the American Water (AW) laboratory and analyzed 'blindly' by cell culture-IFA. Data indicated the control (100% live) oocyst suspensions yielded statistically similar results to a cell culture dose response curve data developed previously at AW. For test samples containing oocyst suspensions of unknown infectivity, cell culture-IFA analyses indicated a high degree of correlation (r2= 0.89; n=26) with the values expected by U.S. EPA. Cell culture infectivity correlates well with neonatal mouse infectivity assays and these 'blind' validation trials provide credibility for the cell culture-IFA procedure as a cost-effective and expedient alternative to mouse infectivity assays for determining in vitro infectivity of C. parvum oocysts.