Establishing an ex vivo model of Acanthamoeba keratitis and investigating the phenotypic similarities between the protozoan Acanthamoeba and human macrophages

Acanthamoeba is a small free-living amoeba found in tap water and soil with two life stages: the trophozoite and cyst. Acanthamoeba species are opportunistic pathogens of humans that cause two main diseases including a potentially blinding infection of the cornea called Acanthamoeba keratitis (AK) in immunocompetent individuals, and a fatal granulomatous encephalitis in the immunocompromised. In this study, an ex vivo model of AK was developed to better understand the pathophysiological processes that occur in this disease. The model has several applications such as studying the interaction of Acanthamoeba with cells of innate immunity, investigating the efficacy of different pharmaceutical products in stopping the progression of the disease beside using the model to correlate between in vivo and ex vivo confocal microscopy findings of various morphologies of Acanthamoeba cysts and trophozoites. Furthermore, the study evaluated the phenotypic similarities between Acanthamoeba and cells of the innate immune system mainly macrophages using flow cytometry analysis to enhance the understanding of how immune cells interact with Acanthamoeba Porcine corneas were used to establish a reproducible ex vivo model which was maintained for four weeks and optimised by the supplement of CO2 and the use of air - liquid interface rocking system that mimics natural eye blinking. Once the model was established, Acanthamoeba trophozoites and cysts were added to the corneal model to evaluate the pathogenesis of Acanthamoeba keratitis, and the study successfully demonstrated the development of the infection in the model. This study was also able to demonstrate that the addition of macrophages and neutrophils to the AK model did not limit the process of the infection as these cells were phagocytosed by Acanthamoeba. The model was also used to investigate the efficacy of doxycycline and polyhexamethylene biguanide (PHMB) in stopping the disease progression and the results demonstrated the ability of PHMB to inactivate Acanthamoeba trophozoites with minimal toxicity to the corneal epithelium. Doxycycline was not found to have any major antimicrobial effect on the viability of Acanthamoeba. The model was also utilized to study the ex vivo confocal microscopy (EVCM) features of various forms of Acanthamoeba and correlate these findings to in vivo confocal microscopy (IVCM) images from culture positive AK cases. The study demonstrated similarity in the morphological features of Acanthamoeba in both ex vivo and in vivo confocal microscopy images, which makes EVCM images a reliable reference to validate IVCM findings. Finally, this study evaluated the phenotypic similarities between Acanthamoeba and macrophages using flow cytometry analysis which identified various degrees of positive reactivity of amoebic cell surface to a limited number of anti-human monoclonal antibodies. This suggests some structural and functional similarities in protein surfaces between amoeba and macrophages which can potentially offer a future tool for screening.