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Cryptosporidium Off-The-Slide Genotyping: Recovery of Oocysts Extracted from Slides Examined by Method 1623
Citation:
WARE, M. W. AND E. VILLEGAS. Cryptosporidium Off-The-Slide Genotyping: Recovery of Oocysts Extracted from Slides Examined by Method 1623. Presented at Water Quality Technology Conference, Cincinnati, OH, November 16 - 20, 2008.
Impact/Purpose:
The overall objective of this task is the development of improved occurrence detection methods for protozoan parasites and Microsporidia. Since this work is a primary focus of the Branch, this task supports several individual projects related to sample preparation and protozoan detection. Together these projects will lead to complete methods able to support the UCMR and the CCL2 and CCL3.
Description:
Cryptosporidium has been found world wide and is an important waterborne parasite causing a self-limiting diarrhea; however, in the immunocompromised it may become chronic and can even lead to death. To characterize the risk of exposure to Cryptosporidium in water the USEPA enacted Long Term 2 Enhanced Surface Water Treatment Rule (LT2) requiring United States surface water utilities to monitor for Cryptosporidium using USEPA Method 1623 (4, 5). This microscopic microscopy based method enumerates oocysts in a water sample,; but it can not determine oocyst species or identify the strain. Cryptosporidium speciation and genotyping can be achieved by molecular methods such as polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Studies of environmental water and wild life fecal samples reveal that several isolates and species from wildlife and livestock animals are more frequently identified than the two principle human pathogens C. hominis and C. parvum (6-8). Several approaches in the literature and as well as one commercial product have been developed to genotype oocysts detected on the well slide wells following examination ed by Method 1623 (1-3). These genotyping methods require extracting oocysts from the slide by removing the coverslip, scrapping the well to detach the oocysts, and transferring the oocysts to a tube for for molecular analysis. One of the procedures also includes the removales of the DABCO containing mounting media, which could inhibitmay inhibit PCR. These approaches have not been compared and validated in an independent study. If one or all of these approaches are robust enough to in which the oocysts are recovered efficiently recover oocysts, then this “off-the-slide” genotyping would greatly provide additional genotypinformation ing results tohat would improve the quality of the monitoring data gathered for assessing risk. However, if this is not the caseOn the other hand, the genotyping results may may have the potential to create false negatives by not detecting all of the species present or may indicate a false positive by Method 1623, creating data interpretation problems. This study focuses on recovery efficiencies of whole C. parvum oocysts using “off-the-slide” extraction techniques. The extraction approach was evaluated in two phases: 1) removal of the coverslip and mounting media, and 2) transfer of the oocysts from the slide to the genotyping tube. C. parvum oocysts were counted by flow cytometery to contain 200, 20 or 0 oocysts per well. The oocysts were either pre-stained or stained on the well. The and slides were counted by microscopy. then tThe coverslip was then removed and retained, and the well was washed. The coverslip and washed well were recounted. Results show that the number of oocysts remaining on the well was quite variable after removal of the coverslip and mounting media, ranging from nearly 5095% too nearly 9550%. Less than 2% of the oocysts on average were attached to the coverslip, with the remaining oocysts removed with the mounting media. These results show suggest that removal of the coverslip accounts for little oocysts loss; however, the mounting media retained on the slide must be transferred to the genotyping tube to accurately reflect the oocysts on contents of the slide. Phase 2 evaluated the transfer of oocysts from the slide to the genotyping tube. The methods used were similar to those used in phase 1 except that the surface of the well was scraped three times with a 10 l loop, as previously described prior to transfer (1-32) and the oocysts were transferred through a 0.8 m filter which was then restained and counted. The scrapped well and coverslip were also examined and only a few oocysts were observed on the scrapped well and coverslip, indicating the scrapping efficiently removes oocysts attached to the well slide. The overall oocyst average transfer rate and its implications for the utility of off-the-slide genotyping will be discussed.