Cytomorphological changes and susceptibility of clinical isolates of Acanthamoeba spp. to heterocyclic alkylphosphocholines.

The treatment of diseases caused by pathogenic strains of Acanthamoeba spp. is to date limited and frequently unsuccessful. Alkylphosphocholines (APCs) are promising agents with interesting results of antiparasitic activity in experimental and clinical conditions. In the present study susceptibilities of two clinical isolates of Acanthamoeba spp. to four heterocyclic APCs were investigated. The isolates showed high degrees of susceptibility to studied APCs and all the tested concentrations inhibited the growth with the highest concentrations of 500-1000µM causing 100% eradication of the trophozoites and cysts. The highest susceptibility was noted in IF16-P-4-Pip with EC50 values of 28.62-43.73µM, and EC90 values of 30.70-63.16µM after 48h of incubation. The cytomorphological changes of trophozoites after the exposure to APCs included rounding up of cells, resorption of acanthopodia and subsequent lysis. The remains of cells were typical with oval shape and identifiable nucleus. After the application of IF16-P-4-Pip, IF16-P-2-MetPip, and IF16-P-Azep, at concentrations of 62.5-125µM to trophozoite suspension, a formation of pseudocysts was detected. The single-layered coat covering the surface of pseudocyst stained positively with a fluorescence brightener, Rylux. Destroyed cysts were characteristic with shrinkage of the cytoplasm and separation of the cytoplasmic membrane from the endocyst. IF16-P-2-MetPip at the highest concentration formed large spherical vesicles which frequently enclosed inactivated cysts. Heterocyclic APCs used in the study demonstrated strong amoebicidal activity and the cytotoxic effect of IF16-P-4-Pip similar to that of miltefosine indicates its possible therapeutic potential.