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The aim of the study was to evaluate the effect of multipurpose contact lens (CL) solution (MPS) combined with autophagy inhibitors on the adhesion properties of Acanthamoeba castellanii (AC) trophozoites to silicone hydrogel CLs.AC trophozoites were inoculated onto discs trimmed from a silicone hydrogel CL with plasma surface treatment. After 18 hours, the number of adherent AC trophozoites on the CL treated with MPS was counted under phase-contrast microscopy. We analyzed the efficacy of MPS combined with autophagy inhibitors, 3-methyladenine (0.1, 1, and 5 mM) and chloroquine (10, 100 μM, and 1 mM), on Acanthamoeba adhesion by electron microscopy.Adhesion of AC trophozoites to the CL treated with MPS (average number of trophozoites adhered to the CL: 61.2 ± 8.1) was significantly lower compared with that of the CL treated without MPS (83.8 ± 10.2) (P = 0.027). In MPS application, the number of adhered AC trophozoites treated with 3-methyladenine [8.2 ± 2.5 for 5 mM (P = 0.008)] or chloroquine [19.4 ± 7.6 for 100 μM (P = 0.038) and 5.3 ± 1.9 for 1 mM (P = 0.001)] was significantly reduced compared with the sample without autophagy inhibitors. However, the number of adherent AC trophozoites was not significantly reduced in less than 5 mM in 3-methyladenine [50.4 ± 5.1 for 0.1 mM (P = 0.084) and 43.1 ± 5.0 for 1 mM (P = 0.079)] and 100 μM in chloroquine [40.6 ± 13.5 for 10 μM (P = 0.075)]. 3-methyladenine induced blebby structures or disrupted the membranes of AC trophozoites. AC trophozoites treated with chloroquine showed undigested organelles in the cytoplasm of Acanthamoeba cells.MPS combined with 3-methyladenine or chloroquine reduced the adhesion rate of AC trophozoites rather than MPS containing only polyhexamethylene biguanide. Appropriate concentrations of autophagy inhibitors, 3-methyladenine and chloroquine, added to commercial MPS should be considered to decrease the clinical rate of Acanthamoeba keratitis.