Quantitative sugar-flotation in a cover-slide system for quantifying Cryptosporidium parvum oocysts in feces: optimizing specific gravity, floating time, and counting strategies.

Cryptosporidium parvum is a zoonotic protozoan parasite that causes cryptosporidiosis, a disease characterized by intestinal tissue damage in animals and humans. The oocysts of Cryptosporidium parvum, which are the infective stage of this parasite in feces, are extremely small (4-6 μm in diameter), making microscopic detection and quantification difficult. In the present study, we developed a quantitative sugar-flotation in a cover-slide system (QUSIC), which is a novel, simple, sensitive, and cost-effective method for quantifying C. parvum oocysts in feces. Fecal samples from non-infected mice and purified oocyst stocks of C. parvum (HNJ-1 strain) were used in this study. To determine the optimal conditions for oocyst recovery, we evaluated sucrose solutions with different specific gravity (SG) values (1.306, 1.234, 1.216, and 1.207), incubation times (30-180 min), and oocyst numbers (102-106). Oocyst recovery was the highest (95.2%) when using a sucrose solution with SG 1.234, whereas lower recoveries were observed with SGs of 1.306, 1.216, and 1.207 (86.4%, 66.0%, and 22.7%, respectively). Furthermore, recovery peaked at 60 min (97.5%), but decreased with both shorter and longer incubation times. Across oocyst numbers ranging from 102 to 106, recovery remained consistently high (83.3-98.2%). These findings indicate that a sucrose solution with SG of 1.234 and a 60-min incubation time are optimal conditions for the QUSIC. By optimizing flotation conditions, the QUSIC offers a useful approach for quantifying C. parvum oocysts in fecal samples. These advantages make the QUSIC well-suited for routine diagnostics and research, particularly in resource-limited facilities.