Detection of Infectious Cryptosporidium Oocysts by Cell Culture Immunofluorescence Assay: Applicability to Environmental Samples

Abstract

In the past few years many waterborne outbreaks related to Cryptosporidium have been described. Current methods for detection of Cryptosporidium in water for the most part rely on viability assays which are not informative concerning the infectivity of oocysts. However, for estimation of the risk of infection with Cryptosporidium this information is required. For environmental samples the oocyst counts are often low, and the oocysts have been exposed to unfavorable conditions. Therefore, determination of the infectivity of environmental oocysts requires an assay with a high level of sensitivity. We evaluated the applicability of in vitro cell culture immunofluorescence assays with HCT-8 and Caco-2 cells for determination of oocyst infectivity in naturally contaminated water samples. Cell culture assays were compared with other viability and infectivity assays. Experiments with Cryptosporidium oocysts from different sources revealed that there was considerable variability in infectivity, which was illustrated by variable 50% infective doses, which ranged from 40 to 614 oocysts, and the results indicated that not only relatively large numbers of fresh oocysts but also aged oocysts produced infection in cell cultures. Fifteen Dutch surface water samples were tested, and the cell culture immunofluorescence assays were not capable of determining the infectivity for the low numbers of naturally occurring Cryptosporidium oocysts present in the samples. A comparison with other viability assays, such as the vital dye exclusion assay, demonstrated that surrogate methods overestimate the number of infectious oocysts and therefore the risk of infection with Cryptosporidium. For accurate risk assessment, further improvement of the method for detection of Cryptosporidium in water is needed.Cryptosporidium is one of the important waterborne causative agents of gastrointestinal illness in humans (6, 10). More than 50 outbreaks of cryptosporidiosis associated with either contaminated drinking water or recreational water have been reported worldwide since 1983 (20). In immunocompetent individuals cryptosporidial diarrhea is usually self-limiting, lasting for about 3 weeks, but infected immunocompromised humans may develop severe and potentially fatal illness (5). At present, no consistently effective, approved therapeutic agent with anticryptosporidial activity is available.Cryptosporidium oocysts are ubiquitous in surface water used for recreation or drinking water production (9, 21). They are extremely resistant to chlorination at the concentrations commonly used for drinking water and swimming pool water disinfection (13). The number of oocysts in finished water is usually low but relevant for public health, and the concentrations are below the detection limit of the method used. Therefore, Dutch drinking water legislation requires drinking water companies to perform a quantitative risk assessment for Cryptosporidium, based on the concentration of oocysts in the source water and the efficiency of drinking water treatment proce...

Leaching of Cryptosporidium parvum Oocysts, Escherichia coli, and a Salmonella enterica Serovar Typhimurium Bacteriophage through Intact Soil Cores following Surface Application and Injection of Slurry

“…Cool temperatures (10°C) appear to increase viability and maintain infectivity of oocysts (61) as well as increasing the release of oocysts from manure and leaching through karst soil (13). The correlation between methods used to detect viable oocysts and infective oocysts has been questioned, and divergent results have been described in the literature (66,69). Application of vital dye staining or in vitro excystation provides information on oocyst viability, but the results do not always correlate with the outcome of in vitro and in vivo infectivity assays.…”

<b>Study of sequential disinfection for the inactivation of protozoa and indicator microorganisms in wastewater

“…Current cell culture-based methods are technically difficult to perform and the gold standard for determining the viability of cysts requires the use of in vivo animal infectivity studies which are complex, expensive, time demanding and involve ethical questions (SCHETS et al, 2005).…”

Aged HCT-8 Cell Monolayers Support Cryptosporidium parvum Infection

“…In previous Cryptosporidium cell culture studies, monolayers were typically allowed to develop for 24 or 48 h to reach 80 to 100% confluence (freshly confluent) just prior to inoculation (2,8,12,22,25,28,35). This can pose a challenge in coordinating water sample collection and processing with readiness of cell monolayers for inoculation, consequently making it difficult to perform Cryptosporidium cell culture assays routinely.…”

“…The human ileocecal adenocarcinoma HCT-8 cell line was previously found to support the greatest amount of Cryptosporidium parvum infection compared to several other cell lines (19,29) and is commonly used for Cryptosporidium infectivity assays (1,2,7,10,12,13,23,25,27,33). In the present study, we investigated the ability of freshly confluent (2-day-old) and aged (8-to 67-day-old) HCT-8 cell monolayers to support C. parvum infection.…”

“…For most Cryptosporidium cell culture assays, monolayers are allowed to develop for 24 or 48 h to reach 80 to 100% confluence prior to inoculation and then processed for detection of infection 48 to 72 h postinoculation (2,8,12,22,25,28,35). Since it is desirable to process water samples as soon as possible after collection and since most water samples are collected off-site and shipped to a processing laboratory, the required logistics can greatly impede the practical application of Cryptosporidium cell culture assays.…”