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Can We Believe Our Results?

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Abstract:Numerous waterborne outbreaks of cryptosporidiosis have occurred recently with the most notable being the 1993 episode in Milwaukee. Due to these outbreaks and the concern for public health, 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 developed for detecting Giardia cysts in water. In short, the procedure involved sampling either 100 liters of source water or 1,000 liters of finished water using a fiber wound filter with a nominal porosity of 1.0um. After washing the filter fibers with buffer, the extracted particulates containing the parasites were further concentrated by centrifugation. Buoyant density centrifugation was then used to separate the parasites from the particulates. 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 results of the assay were microscopially determined using epifluorescence 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.

Method 1622 for Cryptosporidium was developed using off the shelf reagents and equipment in an attempt to improve on oocyst recovery rates. 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, a more selective procedure employing monoclonal antibodies, is used in place of buoyant density centrifugation to separate the parasites from the particulates. Like the prevoius method, staining is done with fluorescently labeled monoclonal antibodies for detection. However, in addition to differential interference contrast microscopy for confirmation, the samples are counter stained with 4',6=diamidino-2-phenyllindole (DAPI) which helps to demonstrate sporozoite nuclei. Even with these improvements, the recovery results for the Method 1622 only average around 38%.
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Citation:Schaefer III, F. W. Can We Believe Our Results? Presented at Royal Society of Chemistry Water Chemistry Forum. Cryptosporidium: The Analytical Challenge, Warwick, UK, October 25-26, 1999.
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Contact: Mary P. O'Bryant - (919)-541-4871 or obriant.mary@epa.gov
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Division: Microbiological & Chemical Exposure Assessment Division
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Branch: Biohazard Assessment Research Branch
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Product Type: Sympos/Conf
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Presented: 10/25/2001
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Last Updated on Monday, October 22, 2007
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