Natural does not always mean safe: first molecular detection of Cryptosporidium and Acanthamoeba in Iranian spring waters

Many people perceive spring water as a healthy alternative to tap water. However, unlike treated tap water, which undergoes rigorous filtration and disinfection, untreated or inadequately treated spring water may pose a higher risk of waterborne infections. Here, we analyzed spring water in three Iranian provinces for the presence of Cryptosporidium , Giardia , Acanthamoeba , and Microsporidia using molecular tools. Twenty-nine springs commonly used by locals, hikers, and mountaineers were selected across Hamedan ( n = 13), Kordestan ( n = 9), and Esfahan ( n = 7). From March to July 2023, water was collected and centrifuged to concentrate the sediment. Genomic DNA was extracted from the sediments and subjected to conventional PCR using genus-specific primers, followed by Sanger sequencing of positive samples. The results showed that two springs in Kordestan province were contaminated with C. parvum , and five springs in all examined provinces were contaminated with Acanthamoeba T4 ( n = 4) and T2 ( n = 1). Other parasite species were not detected. This is the first molecular report of Cryptosporidium and Acanthamoeba in spring water from Iran. Although only a limited number of springs tested positive, the detection of Cryptosporidium parvum and potentially pathogenic Acanthamoeba genotypes suggests that untreated spring water may represent a potential source of exposure to protozoan pathogens. These findings underscore the importance of routine monitoring of spring water used for human consumption and recreational purposes.