Ex vivo Cytopathic Effect Study of Human Acanthamoeba keratitis and Use of E. coli in Parasitic Culture

Abstract

Acanthamoeba is free living amoeba and can cause Acanthamoeba keratitis, a serious disease which affects the cornea. It is widely existing in fresh water and soil. Acanthamoeba has three phases during the life cycle; trophozoite and a resistant double walled cyst and protocysts. The pathogenesis starts when the trophozoites bind to the surface of cornea by expressing mannose-binding protein which can bind to mannose glycoproteins on the corneal surface. The contaminated contact lenses can increase the infection by increasing the binding ability of the trophozoites. This project aimed to study the cytopathic effect of different stages Acanthamoeba keratitis. Study strains of Acanthamoeba castellani (ATCC 50370) was cultured and seeded in Hep2 cell lines. E. coli (strain M12) was used to isolate the trophozoites. The third stage, protocysts was made using Neff's medium. The cytopathic effect was checked trophozoites invasion to monolayer cells cultured in a small flask and in 24 well tissue culture plate. The time of destroying Hep2 cells by the trophozoites is less than that of other Acanthamoeba stages. The double ring-shaped cell wall of the cysts may increase the time-kill of Hep2 due to their resistant which was very slow to hatch. Reactivating the trophozoites from cysts then seeding in Hep2 destroyed the cells at less time compared fresh trophozoites as the virulence of the reactivated trophozoites being higher. The result could suggest that the infection of corneas with reactivated trophozoites may be more complex. Finally, the study found that the number of the trophozoites can limit the cytopathic effect of Acanthamoeba.