Improved Risk Analysis by Dual Direct Detection of Total and Infectious Cryptosporidium Oocysts on Cell Culture in Combination with Immunofluorescence Assay

The inactivation of Cryptosporidium oocysts is a main driver in the selection of water treatment disinfection strategies, and microbial risk analysis provides a sound basis for optimizing water treatment processes. U. S. Environmental Protection Agency method 1622/23 provides an estimate of the total oocyst count; however, it cannot be used directly for risk assessment, as it does not determine the fraction of infectious oocysts. Improved assessment of the risk for designated sources or in treated water requires evaluation of the total number of oocysts and an estimate of their infectivity. We developed a dual direct detection method using differential immunofluorescent staining that allows detection of both oocysts and cell culture infection foci for each sample. Using Cryptosporidium parvum oocysts, various pH levels, proteases, and gastroenteric compounds and substrates were assessed to determine their abilities to enhance the number of infection foci. The results showed that the key trigger for oocyst stimulation was acidification. Addition of a low concentration of D-glucose (50 mM) to the infection media increased rates of infectivity, while a higher dose (300 mM) was inhibitory. The total number of oocysts in each sample was determined by counting the oocysts remaining on a cell monolayer and the oocysts recovered from cell monolayer washes during processing using a simple filtration technique. With the dual direct detection on cell culture with immunofluorescence assay method, it is now possible to determine the numbers of total and infectious oocysts for a given sample in a single analysis. Direct percentages of infectivity are then calculated, which allows more accurate assessments of risk.