Acanthamoeba, an opportunistic protozoan pathogen, is ubiquitous in nature, and therefore plays a predatory role and helps control microbial communities in the ecosystem. These Acanthamoeba species are recognized as opportunistic human pathogens that may cause blinding keratitis and rare but fatal granulomatous encephalitis. To date, there is not a single report demonstrating Acanthamoeba isolation and identification from environmental sources in Pakistan, and that is the aim of this study. Acanthamoeba were identified by morphological characteristics of their cysts on non-nutrient agar plates seeded with Escherichia coli. Additionally, the polymerase chain reaction (PCR) was performed with genus-specific primers followed by direct sequencing of the PCR product for molecular identification. Furthermore, our PCR and sequencing results confirmed seven different pathogenic and nonpathogenic genotypes, including T2-T10, T4, T5, T7, T15, T16, and T17. To the best of our knowledge, we have identified and isolated Acanthamoeba sp., for the first time, from water resources of Khyber Pakhtunkhwa, Pakistan. There is an urgent need to address (1) the pathogenic potential of the identified genotypes and (2) explore other environmental sources from the country to examine the water quality and the current status of Acanthamoeba species in Pakistan, which may be a potential threat for public health across the country.

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Abbreviations DNAAbstract. Naegleria is well recognized for primary amoebic meningoencephalitis which always results into death. To date there is not a single report demonstrating molecular identification of Naegleria from water resources of Pakistan thus the aim of the proposed study. Here, in total 135 various water samples (like domestic tap water, municipal water, sea water, well water, tube well water, canal water, boring water, sewage water, lake water and stream water) were collected across Pakistan and evaluated for Naegleria species. Naegleria australiensis (pathogenic) and Naegleria lovaniensis (non pathogenic) were isolated on non nutrient agar plates and were further identified by PCR and sequencing. To best of our knowledge we have described for the first time the isolation and molecular identification of thermotolerant pathogenic and non pathogenic Naegleria species from diverse water samples including drinking water across Pakistan. The presence of pathogenic Naegleria species in diverse water samples may add the health threat to the community.