Quintero-Betancourt Walter, Peele Emily R, Rose Joan B
Water Pollution Microbiology, College of Marine Science, University of South Florida, 140 7th Avenue South, St. Petersburg, FL 33701, USA.
J Microbiol Methods. 2002 May;49(3):209-24. doi: 10.1016/s0167-7012(02)00007-6.
Cryptosporidium and Cyclospora are obligate, intracellular, coccidian protozoan parasites that infest the gastrointestinal tract of humans and animals causing severe diarrhea illness. In this paper, we present an overview of the conventional and more novel techniques that are currently available to detect Cryptosporidium and Cyclospora in water. Conventional techniques and new immunological and genetic/molecular methods make it possible to assess the occurrence, prevalence, virulence (to a lesser extent), viability, levels, and sources of waterborne protozoa. Concentration, purification, and detection are the three key steps in all methods that have been approved for routine monitoring of waterborne oocysts. These steps have been optimized to such an extent that low levels of naturally occurring Cryptosporidium oocysts can be efficiently recovered from water. The filtration systems developed in the US and Europe trap oocysts more effectively and are part of the standard methodologies for environmental monitoring of Cryptosporidium oocysts in source and treated water. Purification techniques such as immunomagnetic separation and flow cytometry with fluorescent activated cell sorting impart high capture efficiency and selective separation of oocysts from sample debris. Monoclonal antibodies with higher avidity and specificity to oocysts in water concentrates have significantly improved the detection and enumeration steps. To date, PCR-based detection methods allow us to differentiate the human pathogenic Cryptosporidium parasites from those that do not infect humans, and to track the source of oocyst contamination in the environment. Cell culture techniques are now used to examine oocyst viability. While fewer studies have focused on Cyclospora cayetanensis, the parasite has been successfully detected in drinking water and wastewater using current methods to recover Cryptosporidium oocysts. More research is needed for monitoring of Cyclospora in the environment. Meanwhile, molecular methods (e.g. molecular markers such as intervening transcribed spacer regions), which can identify different genotypes of C. cayetanensis, show good promise for detection of this emerging coccidian parasite in water.
隐孢子虫和环孢子虫是专性细胞内球虫原生动物寄生虫，寄生于人和动物的胃肠道，可导致严重腹泻疾病。在本文中，我们概述了目前可用于检测水中隐孢子虫和环孢子虫的传统技术及更新颖的技术。传统技术以及新的免疫和基因/分子方法使得评估水源性原生动物的发生、流行、毒力（程度较低）、活力、水平和来源成为可能。浓缩、纯化和检测是所有已批准用于常规监测水源性卵囊的方法中的三个关键步骤。这些步骤已得到优化，以至于可以从水中有效地回收低水平自然存在的隐孢子虫卵囊。美国和欧洲开发的过滤系统能更有效地捕获卵囊，并且是水源水和处理后水中隐孢子虫卵囊环境监测标准方法的一部分。免疫磁分离和荧光激活细胞分选流式细胞术等纯化技术具有高捕获效率，能将卵囊与样品碎片选择性分离。对水浓缩物中卵囊具有更高亲和力和特异性的单克隆抗体显著改进了检测和计数步骤。迄今为止，基于聚合酶链反应（PCR）的检测方法使我们能够区分人类致病性隐孢子虫寄生虫和那些不感染人类的寄生虫，并追踪环境中卵囊污染的来源。现在细胞培养技术用于检测卵囊活力。虽然针对卡耶塔环孢子虫的研究较少，但使用目前回收隐孢子虫卵囊的方法已成功在饮用水和废水中检测到该寄生虫。环境中环孢子虫的监测还需要更多研究。同时，能够识别卡耶塔环孢子虫不同基因型的分子方法（例如间隔转录间隔区等分子标记），在检测水中这种新出现的球虫寄生虫方面显示出良好前景。
