Molecular epidemiology of Acanthamoeba in human infections and environmental reservoirs in India

Background and objectives Acanthamoeba spp. are free-living amoebae causing rare but frequently fatal, granulomatous amoebic encephalitis (GAE). Understanding their genotypic distribution is essential for defining epidemiology and identifying environmental sources. This study investigated Acanthamoeba in clinically suspected cases of GAE and in environmental water sources across India. Methods A total of 125 clinical specimens from patients with suspected GAE cerebrospinal fluid (CSF), brain biopsy tissue, or pus and 14 environmental water samples (pond, well, and household water) were analyzed between April 2023 and August 2025. Microscopic examination of samples and culture on non-nutrient agar overlaid with Escherichia coli was done. Centrifuged water pellets were processed similarly. DNA extraction and polymerase chain reaction assay (PCR) targeting the Acanthamoeba spp. diagnostic fragment were performed, followed by sequencing for genotype identification. Results Eighteen (14.4%) clinical samples were Acanthamoeba- PCR positive, with seven yielding positive cultures. These included samples from Kerala (n=9), Karnataka (n=4), Himachal Pradesh (n=2), and one each from Delhi, West Bengal, and Uttar Pradesh. Seven (50%) water samples were Acanthamoeba- PCR positive (Kerala, n=4, and West Bengal, n=3), of which two were culture-positive. Genotyping of 12 clinical and four environmental samples revealed that all isolates belonged to genotype T4, except for one T3 CSF-derived isolate from Karnataka. Interpretation and conclusions The predominance of Acanthamoeba genotype T4 across clinical and environmental samples mirrors global trends and suggests enhanced pathogenic potential and ecological adaptability of this genotype. The detection of environmental genotypes underscores the importance of systematic environmental surveillance in elucidating transmission pathways more effectively.