Bukhari Zia, Holt David M, Ware Michael W, Schaefer Frank W
American Water, 1025 Laurel Oak Road, Voorhees, NJ 08043, USA.
Can J Microbiol. 2007 May;53(5):656-63. doi: 10.1139/W07-032.
An optimized cell culture immunofluorescence (IFA) procedure, using the HCT-8 cell line, was evaluated in blind trials to determine the sensitivity and reproducibility of measuring the infectivity of flow-cytometry-prepared inocula of Cryptosporidium parvum oocysts. In separate trials, suspensions consisting of between 0% and 100% viable oocysts were prepared at the US Environmental Protection Agency, shipped to the American Water Laboratory, and analyzed blindly by cell culture IFA. Data indicated the control (100% live) oocyst suspensions yielded statistically similar results to cell culture dose-response curve data developed previously at the American Water Laboratory. For test samples containing oocyst suspensions of unknown infectivity, cell culture IFA analyses indicated a high degree of correlation (r2 = 0.89; n = 26) with the values expected by the US Environmental Protection Agency. Cell culture infectivity correlates well with neonatal mouse infectivity assays, and these blind validation trials provide credibility for the cell culture IFA procedure as a cost-effective and expedient alternative to mouse infectivity assays for determining in vitro infectivity of C. parvum oocysts.
使用HCT - 8细胞系的优化细胞培养免疫荧光（IFA）程序，在盲法试验中进行了评估，以确定测量流式细胞术制备的微小隐孢子虫卵囊接种物感染性的灵敏度和可重复性。在单独的试验中，美国环境保护局制备了含有0%至100%活卵囊的悬浮液，运至美国水实验室，并通过细胞培养IFA进行盲法分析。数据表明，对照（100%活）卵囊悬浮液产生的结果与美国水实验室先前建立的细胞培养剂量反应曲线数据在统计学上相似。对于含有感染性未知的卵囊悬浮液的测试样品，细胞培养IFA分析表明与美国环境保护局预期的值具有高度相关性（r2 = 0.89；n = 26）。细胞培养感染性与新生小鼠感染性试验相关性良好，这些盲法验证试验为细胞培养IFA程序提供了可信度，该程序可作为一种经济高效且便捷的替代方法，用于替代小鼠感染性试验来确定微小隐孢子虫卵囊的体外感染性。
