Detection of infectious Cryptosporidium oocysts by cell culture: applicability to environmental samples

Cryptosporidium is one of the important causative agents of gastrointestinal illness in humans. Cryptosporidium infections are often waterborne and can be transmitted through drinking water or recreational water. Estimation of the risk of infection with Cryptosporidium after exposure to drinking water requires information on oocyst infectivity. According to the Dutch drinking water regulations, the annual risk of infection should not exceed one infection per 10,000 persons. We evaluated the applicability of in vitro cell culture assays on HCT-8 and Caco-2 cells for determination of oocyst infectivity in naturally contaminated water samples. Experiments with Cryptosporidium oocysts showed considerable variability in infectivity. Dilution and survival experiments indicated that only relatively large numbers of fresh or aged oocysts produced infection in cell cultures. Naturally contaminated Dutch surface waters usually contain low numbers of Cryptosporidium oocysts. The cell culture assays are not sensitive enough to detect infectivity of such low numbers of oocysts. The assays can however successfully be used to study the effect of disinfection processes on oocyst infectivity. Surrogate methods, such as vital dye exclusion assays, used to determine oocyst viability as opposed to infectivity were found to overestimate the number of infectious oocysts. Using oocyst viability in risk assessment will therefore overestimate the risk of infection with Cryptosporidium. For accurate risk assessment, further improvement of the detection method for Cryptosporidium in water is required. PCR detection of infection in cell lines may improve sensitivity whereas enhanced recovery of the detection method may result in the detection of higher numbers of oocysts