(‒)-Epicatechin enhances the anti-Acanthamoeba activity of chlorhexidine through synergistic action.

Acanthamoeba species are causative agents of the sight-threatening ocular disease, Acanthamoeba keratitis (AK). (‒)-Epicatechin (EC) has been reported to exhibit anti-amoebic activity against laboratory strains of A. castellanii and A. polyphaga, suggesting its potential as an AK drug alternative. However, its efficacy against clinical isolates of Acanthamoeba species and its feasibility as a treatment for AK have not yet been investigated. In this study, we analyzed the anti-amoebic activity of EC against Acanthamoeba clinical isolates and its synergistic effect with chlorhexidine (CHX) to validate its potential in developing or optimizing AK treatment regimes. The anti-amoebic activity of EC against trophozoites and cysts of 10 clinical isolates of Acanthamoeba species was analyzed. The synergistic anti-amoebic effect of EC and CHX was also investigated. EC exhibited anti-amoebic activity against clinical isolates of Acanthamoeba, but showed varying efficacy, with IC50 values ranging from 43.24 to 271.30 µM against trophozoites. EC induced apoptosis-like programmed cell death in the trophozoites of these clinical isolates, and also had partial cysticidal activity. Co-treatment with EC and CHX exhibited prominent in vitro synergistic anti-amoebic activity against Acanthamoeba trophozoites and cysts, and co-treatment with EC and a subclinical dose of CHX (0.001 and 0.002%) revealed a dose-dependent anti-amoebic effect on both trophozoites and cysts equivalent to or greater than a clinical dose of CHX (0.02%). The promising anti-amoebic activity of EC against Acanthamoeba clinical isolates, along with the observed synergistic effect with CHX, highlights its potential as a drug candidate or an adjunctive treatment for AK.