Intraspecies genetic diversity and host-associated distribution of Endolimax nana in Sumba Island, Indonesia.

BACKGROUND: Endolimax nana is a common intestinal archamoebid within Evosea (Amoebozoa), alongside genera such as Entamoeba, Iodamoeba, and Mastigamoeba. Despite its frequent colonization of humans, it has received limited attention as a presumed non-pathogenic commensal, and its genetic diversity and host-associated distribution remain poorly resolved. Here, we define the molecular diversity, subtype structure, and host-associated distribution of E. nana. METHODS: Cross-sectional surveys were conducted in Wainyapu Village, Sumba Island, Indonesia. A total of 315 stool samples were collected from humans (n = 144) and animals (n = 171), including rats, pigs, dogs, ducks, chickens, horses, buffaloes, and goats between 2015 and 2016. Samples were screened by PCR targeting the 18S rRNA gene, followed by direct sequencing and sub-cloning. Sub-cloning was performed for 26 samples with ambiguous chromatograms. Phylogenetic relationships were inferred using Bayesian inference, Neighbor-Joining, and Maximum Parsimony methods. In human, the association between E. nana colonization and diarrheal stool form was evaluated using logistic regression analysis. RESULTS: E. nana was detected in humans (42.4%, 61/144), rats (12.0%, 6/50), pigs (11.1%, 5/45), dogs (7.7%, 2/26), ducks (20.0%, 1/5), and chickens (3.6%, 1/28). Sequencing yielded 127 sequences (1252-1283 bp) comprising 118 unique 18S rRNA haplotypes. Phylogenetic analyses resolved two major subtypes (ST1 and ST2), each consistently recovered monophyletic clusters and further subdivided into distinct subclusters. Subtype distribution revealed clear host-associated structure: ST1-1 and ST2-2 were predominantly human-associated, ST1-2 was shared between humans and pigs, and ST2-1 was distributed across humans, pigs, and rats. Notably, no subtype was restricted to animal hosts, indicating that all subtype lineages include human-associated populations. No association was observed between E. nana colonization and diarrheal stool form. CONCLUSIONS: We provide the first molecular characterization of human-derived E. nana, defining its subtype structure and host-associated distribution. The absence of animal-restricted subtypes suggests that E. nana circulates primarily within human-associated transmission networks. No association was observed between E. nana colonization and diarrheal stool form in the present dataset. However, these findings should not be interpreted as definitive evidence regarding the commensal or pathogenic nature of the organism.