Free-living amoebas (FLAs) can cause neurological and ocular complications in humans. Water supplies play a critical role in transmitting FLAs to humans. The aim of the present study was to investigate the presence of FLAs in various aquatic sources including drinking water, stagnant water, and surface water in Alborz province, northern Iran, using morphological and molecular techniques. A total of 70 water samples were collected from 34 drinking waters, 23 surface waters, and 13 stagnant waters. Filtration and cultivation were employed to isolate FLAs. PCR assay was applied by using the genus-specific primers on positive samples. Pathogenicity tests (osmo- and thermo-tolerance properties) were performed for Acanthamoeba spp., positive sample. Considering the morphological criteria, four positive samples of Acanthamoeba sp., three Vermamoeba sp., two mixed Vermamoeba sp. with Vahlkamfiids, and one mixed Acanthamoeba sp. with Vahlkamfiids were isolated. Five Acanthamoeba sp. isolates were amplified using the JDP primer pairs. Among them, two genotypes, T4 (three isolates) and T5 (two isolates) corresponding to A. lenticulata, were identified. Four V. vermiformis samples were confirmed using the sequencing. This study highlighted the occurrence of potentially pathogenic waterborne FLAs in water habitats associated with high human activity. The results of such research on the prevalence of FLAs, as a human hazard, should be communicated to health policymakers.

自由生活的变形虫 (FLA) 可导致人类神经和眼部并发症。供水在将 FLA 传播给人类方面发挥着关键作用。本研究的目的是利用形态学和分子技术调查伊朗北部厄尔布尔士省各种水源中 FLA 的存在，包括饮用水、死水和地表水。从 34 个饮用水、23 个地表水和 13 个死水共采集了 70 个水样。过滤和培养用于分离FLAs。通过在阳性样品上使用属特异性引物应用PCR测定。对棘阿米巴属阳性样本进行了致病性测试（渗透和耐热特性） 。考虑到形态学标准，四个阳性样本Acanthamoeba sp.、三个Vermamoeba sp.、两个混合Vermamoeba sp. 与 Vahlkamfiids 和一种混合棘阿米巴sp。与 Vahlkamfiids 分离。五棘阿米巴 使用 JDP 引物对扩增分离株。其中，鉴定出与A. lenticulata相对应的两种基因型，T4（三个分离株）和T5（两个分离株） 。使用测序确认了四个V. vermiformis样品。该研究强调了在与人类高活动相关的水生境中存在潜在致病性水传播 FLA。此类关于作为人类危害的 FLA 流行率的研究结果应传达给卫生政策制定者。