Recovery and viability ofCryptosporidium parvumoocysts andGiardia intestinaliscysts using the membrane dissolution procedure

Abstract

Previously, the cellulose acetate membrane filter dissolution method was reported to yield Cryptosporidium parvum oocyst recoveries of 70.5%, with recovered oocysts retaining their infectivity. In contrast, high spike doses (approximately 1 × 105Cryptosporidium parvum oocysts and Giardia intestinalis cysts) yielded recoveries ranging from 0.4% to 83.9%, and 3.2% to 90.3%, respectively, in this study. Recoveries with low spike doses (approximately 100 (oo)cysts) continued to demonstrate high variability also. Efforts to optimize the method included increased centrifugation speeds, suspension of the final concentrate in deionized water for organism detection on well slides, and analysis of the entire concentrate. A comparison of two monoclonal antibodies was also conducted to identify potential differences between antibodies in detection of organisms. Archived source and finished water samples were spiked, yielding variable recoveries of C. parvum oocysts (11.8% to 71.4%) and G. intestinalis cysts (7.4% to 42.3%). Effects of organic solvents used in the membrane dissolution procedure on the viability of recovered (oo)cysts was determined using a fluorogenic vital dyes assay in conjunction with (oo)cyst morphology, which indicated >99% inactivation. These data indicate that the membrane dissolution procedure yields poor and highly variable (oo)cyst recoveries, and also renders the majority of recovered organisms non-viable.Key words: Giardia, Cryptosporidium, membrane dissolution, viability.