Genotypic diversity and molecular epidemiology of Acanthamoeba keratitis in China: Identification of novel T4 variants and source attribution.

Acanthamoeba keratitis (AK) is a severe, vision-threatening protozoan infection with a rising global incidence linked to contact lens use, necessitating robust genotyping to clarify its epidemiology and pathogenesis. This study analyzed the genotype distribution of 102 Acanthamoeba isolates collected from clinical settings in China between 1991 and 2014, comprising 100 isolates from corneal scrapings of clinically diagnosed AK patients, one from soil, and one from a patient's contact lens solution. Genetic analysis was conducted via amplification and sequencing of the 18S rRNA hypervariable DF3 region, followed by phylogenetic reconstruction and sequence identity assessment against reference clades. Twenty-eight distinct DF3 sequence types were identified, with genotype T4 demonstrating overwhelming dominance (99.0%; 101/102). Three novel T4 variants (T4/42-T4/44) were discovered, expanding the known diversity of this pathogenic lineage. Notably, T4/31 and T4/41 together accounted for 36% of clinical isolates, suggesting regionally endemic, infection-enriched variants. Only one isolate belonged to the rarely reported T11 genotype in China. Molecular source tracing confirmed two transmission chains: one linking a patient's corneal infection to their contact lens solution (both T4/6 variant), and another linking infection to periresidential soil (both T4/25 variant). These findings confirm the near-exclusive dominance of T4 among Chinese AK cases, identify prevalent local T4 variants, and demonstrate the utility of high-resolution DF3 genotyping for transmission tracing and disease control.