Abstract：
Cryptosporidium poses significant public health risks as a cause of waterborne disease worldwide. Clinical surveillance of cryptosporidiosis is largely underreported due to the asymptomatic and mildly symptomatic infections, clinical misdiagnoses, and barriers to access testing. Wastewater surveillance overcomes these limitations and could serve as an effective tool for identifying cryptosporidiosis at the population level. Despite its potential, the lack of standardized wastewater surveillance methods for Cryptosporidium spp. challenges implementation design and the comparability between studies. Thus, this study compared and contrasted Cryptosporidium wastewater surveillance methods for concentrating wastewater oocysts, extracting oocyst DNA, and detecting Cryptosporidium genetic markers. The evaluated concentration methods included electronegative membrane filtration, Envirocheck HV capsule filtration, centrifugation, and Nanotrap Microbiome Particles, with and without additional immunomagnetic separation purification (except for the Nanotrap Microbiome Particles). Oocyst DNA extraction by either the DNeasy Powersoil Pro kit and the QIAamp DNA Mini kit were evaluated and the impact of bead beating and freeze-thaw pretreatments on DNA recoveries was assessed. Genetic detection via qPCR assays targeting either the Cryptosporidium 18S rRNA gene or the Cryptosporidium oocyst wall protein gene were tested. Oocyst recovery percentages were highest for centrifugation (39-77 %), followed by the Nanotrap Microbiome Particles (24 %), electronegative filtration with a PBST elution (22 %), and Envirocheck HV capsule filtration (13 %). Immunomagnetic separation purification was found to be unsuitable due to interference from the wastewater matrix. Bead-beating pretreatment enhanced DNA recoveries from both the DNeasy Powersoil Pro kit (314 gc/μL DNA) and the QIAamp DNA Mini kit (238 gc/μL DNA). In contrast, freeze-thaw pretreatment reduced DNA recoveries to under 92 gc/μL DNA, likely through DNA degradation. Finally, while both qPCR assays were specific to Cryptosporidium spp., the 18S rRNA assay had a 5-fold lower detection limit and could detect a wider range of Cryptosporidium spp. than the Cryptosporidium oocyst wall protein assay.
摘要：
隐孢子虫作为全球水传播疾病的一个原因，对公众健康构成重大风险。由于无症状和轻度症状感染，隐孢子虫病的临床监测在很大程度上被低估。临床误诊,以及进入测试的障碍。废水监测克服了这些局限性，可以作为在人群水平上识别隐孢子虫病的有效工具。尽管有潜力,缺乏对隐孢子虫的标准化废水监测方法。挑战实施设计和研究之间的可比性。因此，这项研究比较和对比了浓缩废水卵囊的隐孢子虫废水监测方法，提取卵囊DNA,并检测隐孢子虫遗传标记。评估的浓缩方法包括电负性膜过滤，Envirocheck高压胶囊过滤，离心,和纳米粒子微生物组，具有和不具有额外的免疫磁性分离纯化(除了Nanotrap微生物组颗粒)。评估了DNeasyPowersoilPro试剂盒和QIAampDNAMini试剂盒的卵囊DNA提取，并评估了珠打和冻融预处理对DNA回收率的影响。测试了通过针对隐孢子虫18SrRNA基因或隐孢子虫卵囊壁蛋白基因的qPCR测定的遗传检测。离心的卵囊回收率最高（39-77％），其次是纳米阱微生物组颗粒(24%)，电负性过滤与PBST洗脱（22％），和Envirocheck高压胶囊过滤(13%)。由于废水基质的干扰，发现免疫磁分离纯化不合适。打珠预处理可提高DNeasyPowersoilPro试剂盒（314gc/μLDNA）和QIAampDNAMini试剂盒（238gc/μLDNA）的DNA回收率。相比之下,冻融预处理将DNA回收率降低到92gc/μL以下，可能是通过DNA降解。最后,而两种qPCR检测都是隐孢子虫的特异性。,18SrRNA测定的检测限低5倍,可以检测更广泛的隐孢子虫.比隐孢子虫卵囊壁蛋白测定。
