Ecole Polytechnique de Montréal, Chaire Industrielle CRSNG en Eau Potable, Département des Génies Civil, Géologique et des Mines, CP 6079, Succ. Centre-ville, Montréal, Québec, Canada.
Appl Environ Microbiol. 2010 Jan;76(2):566-77. doi: 10.1128/AEM.01496-09. Epub 2009 Nov 20.
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.
隐孢子虫卵囊的失活是选择水处理消毒策略的主要驱动因素，微生物风险分析为优化水处理过程提供了合理的依据。美国环保署方法 1622/23 提供了总卵囊数的估计值；然而，由于它不能确定感染性卵囊的比例，因此不能直接用于风险评估。要更好地评估指定来源或处理水中的风险，需要评估卵囊的总数及其感染力的估计值。我们开发了一种使用差异免疫荧光染色的双重直接检测方法，该方法允许对每个样本同时检测卵囊和细胞培养感染灶。使用微小隐孢子虫卵囊，评估了各种 pH 值、蛋白酶以及胃肠化合物和底物，以确定它们增强感染灶数量的能力。结果表明，卵囊刺激的关键触发因素是酸化。在感染培养基中添加低浓度的 D-葡萄糖（50mM）可提高感染率，而高浓度（300mM）则具有抑制作用。通过简单的过滤技术，在处理过程中通过计数留在细胞单层上的卵囊数量和从细胞单层洗涤中回收的卵囊数量来确定每个样品中总卵囊的数量。使用细胞培养的双重直接检测和免疫荧光分析方法，现在可以在单次分析中确定给定样品中的总卵囊和感染性卵囊的数量。然后计算直接感染率百分比，这可以更准确地评估风险。
