The Effect of Anti-Amoebic Agents and Ce6-PDT on Acanthamoeba castellanii Trophozoites and Cysts, In Vitro

PURPOSE: The purpose of this study was to analyze the concentration-dependent effects of biguanides (polyhexamethylene biguanide [PHMB], chlorhexidine [CH]); diamidines (hexamidine-diisethionate [HD], propamidine-isethionate [PD], dibromopropamidine-diisethionate [DD]); natamycin (NM); miltefosine (MF); povidone iodine (PVPI), and chlorin e6 PDT on Acanthamoeba trophozoites and cysts, in vitro. METHODS: Strain 1BU was cultured in peptone-yeast extract-glucose medium. Trophozoites or cysts were cultured in PYG medium containing each agent at 100%, 50%, and 25% of maximum concentration for 2 hours. The percentage of dead trophozoites was determined using a non-radioactive cytotoxicity assay and trypan blue staining. Treated cysts were also maintained on non-nutrient agar Escherichia coli (E. coli) plates and observed for 3 weeks. RESULTS: All tested drugs displayed significant cytotoxic effects on 1BU cells based on the biochemical and staining-based viability assays tested. On non-nutrient agar E. coli plates, neither trophozoites nor freshly formed cysts were observed after PHMB, PD, NM, and PVPI treatment, respectively, within 3 weeks. However, CH-, HD-, DD-, and MF-treated cysts could excyst, multiply, and encyst again. CONCLUSIONS: The off-label drugs PHMB, PD, NM, and PVPI are under in vitro conditions more effective against strain 1BU than CH, HD, DD, and MF. Our findings also suggest that the non-nutrient agar E. coli plate assay should be considered as method of choice for the in vitro analysis of the treatment efficacy of anti-amoebic agents. TRANSLATIONAL RELEVANCE: Ophthalmologists may optimize the treatment regime against Acanthamoeba keratitis by pre-testing the in vitro susceptibilities of the Acanthamoeba strain against drugs of interest with the non-nutrient E. coli agar plate assay.