Instituto Universitario de Ingeniería del Agua y Medio Ambiente, Universitat Politècnica de València, Camino de Vera s/n, 46022 Valencia, Spain.
Estación Experimental de Zonas Áridas, Consejo Superior de Investigaciones Científicas (EEZA-CSIC), Carretera de Sacramento s/n, La Cañada, Almería, Spain.
Int J Hyg Environ Health. 2018 Jan;221(1):102-111. doi: 10.1016/j.ijheh.2017.10.008. Epub 2017 Oct 19.
Understanding waterborne protozoan parasites (WPPs) diversity has important implications in public health. In this study, we evaluated a NGS-based method as a detection approach to identify simultaneously most important WPPs using 18S rRNA high-throughput sequencing. A set of primers to target the V4 18S rRNA region of WPPs such as Cryptosporidium spp., Giardia sp., Blastocystis sp., Entamoeba spp, Toxoplasma sp. and free-living amoebae (FLA) was designed. In order to optimize PCR conditions before sequencing, both a mock community with a defined composition of representative WPPs and a real water sample inoculated with specific WPPs DNA were prepared. Using the method proposed in this study, we have detected the presence of Giardia intestinalis, Acanthamoeba castellanii, Toxoplasma gondii, Entamoeba histolytica and Blastocystis sp. at species level in real irrigation water samples. Our results showed that untreated surface irrigation water in open fields can provide an important source of WPPs. Therefore, the methodology proposed in this study can establish a basis for an accurate and effective diagnostic of WPPs to provide a better understanding of the risk associated to irrigation water.
了解水传播原生动物寄生虫（WPPs）的多样性对公共卫生具有重要意义。在这项研究中，我们评估了一种基于 NGS 的方法，该方法可通过 18S rRNA 高通量测序同时识别最重要的 WPPs，作为一种检测方法。设计了一组针对隐孢子虫属、贾第虫属、蓝氏贾第鞭毛虫属、内阿米巴属、刚地弓形虫和自由生活阿米巴（FLA）等 WPPs 的 V4 18S rRNA 区的引物。为了在测序前优化 PCR 条件，制备了具有代表性 WPPs 定义组成的模拟群落和用特定 WPPs DNA 接种的真实水样。使用本研究中提出的方法，我们在真实灌溉水样中检测到了肠道贾第虫、嗜热四膜虫、刚地弓形虫、溶组织内阿米巴和蓝氏贾第鞭毛虫属的存在。我们的研究结果表明，露天未处理的地表灌溉水可以成为 WPPs 的重要来源。因此，本研究中提出的方法可以为 WPPs 的准确和有效诊断奠定基础，从而更好地了解与灌溉水相关的风险。
