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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.

Acanthamoeba Keratitis (AK) is a vision-threatening disease which can lead to blinding corneal tissue infection. Many patients who have been infected with Acanthamoeba in their eye do not respond to the current medical treatments involving polyhexamethylene biguanide or chlorhexidine despite the in vitro sensitivity of Acanthamoeba to these drugs. There is an urgent need for new therapeutic agents to eradicate the AK infection. This study focuses on the mechanism by which Acanthamoeba may distinguish between trophozoite, cyst and the newly identified lifecycle known as protocyst. The current study has tested 56 novel and existing therapeutic agents for their activity against Acanthamoeba spp. and their toxicity against a human epithelial cell line. The results of this research have revealed several compounds of interest for further study on their potential use in the treatment of AK. These compounds included, octenidine hydrochloride, alexidine, miltefosine and quaternary ammonium (didecyldimethylammonium chloride). The anti-amoebic effect of benzalkonium chloride, povidone iodine and tetracaine are superior to the current diamidines and slightly lower to the biguanides applied in the treatment for AK. The formulation of novel amidoamine compounds including myristoleyl-amidopropyl-dimethylamine (MOPD) and palmitoleyl-amidopropyl-dimethylamine (POPD) into contact lens solutions showed complete kill at a 4.5-log reduction against trophozoites compared with myristamidopropyl dimethylamine (MAPD) as an existing compound. The combination of biguanide compounds with lipid–based carriers has improved the antimicrobial activity from 1-fold to around 7-fold against cysts of Acanthamoeba spp. compared with the use of biguanides alone. The findings of encystment investigation (the transformation of trophozoites into cysts) showed that the agonists in particular the β ultra-long against indacaterol stimulated the encystment and the antagonists β1 metoprolol blocked the formation of cysts and protocysts. Two different herbicides including 2,6-dichlorobenzonitrile (DCB) and isoxaben were tested to target the biosynthesis of cellulose in the cyst form and also to evaluate their effects on the formation of protocyst of Acanthamoeba. The results of this study showed that the DCB at a high concentration of 500 μM, reduced encystment to 17.7% and protocyst production of Acanthamoeba at 24.6%, whereas isoxaben inhibited the transformation of trophozoites into cysts to only 45% and the percentage was decreased for protocyst formation by 37.2%. The test results for DCB and isoxaben individually at concentration of 100 uM showed 31.8% and 68.8% respectively for the conversion of trophozoites into cysts. In addition, a similar concentration of both DCB and isoxaben was evaluated for protocyst formation and the inhibition was observed at 36.9% for DCB and a much higher rate of protocysts production was recorded at 63 % for isoxaben. The combination of both isoxaben and DCB at a concentration of 100 μM caused a reduction in encystment to 49.1% and lowered the transformation of trophozoites into protocysts to 45.7%, these findings suggested that an antagonistic effect was occurred relative to the use of DCB alone. Finally, the data from LC/MS analysis for sugars suggested that the protocyst and cyst are different stages of Acanthamoeba, as the analysis of cyst walls indicated the presence of cellulose while the protocyst wall analysis showed the existing of cellulose and methylated sugar possibly corresponded to a methylated analogue of N-acetylglucosamine.

Purpose: To show the evolution of anterior chamber structures 6 years after cataract surgery in a case with Acanthamoeba keratitis (AK). Methods: A 37-year-old woman with AK receiving long-term treatment with chlorhexidine, propamidine isethionate and steroids developed a white cataract and iris atrophy. Penetrating keratoplasty and cataract surgery were performed with subsequent intraocular pressure elevation requiring Molteno shunt implantation. Two years after the last surgery, endothelial decompensation developed and another penetrating keratoplasty was performed. Intraoperatively, the anterior and posterior capsules were completely transparent. Results: Six years after cataract surgery, the intraocular lens was centered with clear anterior and posterior capsules without lens epithelial cells proliferation. No Soemmering’s ring formation or posterior capsule opacification was found. Also, no zonular damage or pseudophacodonesis was observed. Conclusions: This case suggests that AK infection and AK treatment not only cause white progressive cataract but also lens epithelial cell death. The capsules may be completely clear 6 years after cataract surgery, with a good quality of vision regardless of intraocular lens material or design

Acanthamoeba are ubiquitous protists that play an environmental role in regulating microbial diversity; they also occasionally cause infections of the eye (Acanthamoeba keratitis) and brain (granulomatous amoebic encephalitis). These organisms exhibit two distinct phenotypes. The trophozoite form dominates in favorable conditions, in which the Acanthamoeba move through the extension of pseudopodia, engulfing microbes and other particles. During stressful conditions, the Acanthamoeba undergo a process of encystment, in which they build a double cell wall and become relatively inactive. The cyst form can survive years until more favorable conditions arise, at which point they may excyst. For this study, multiple laboratory encystment methods were compared to determine the percent encystment and the different viabilities of laboratory-produced cysts. Furthermore, four different encystment genes were targeted for development of a primer library for reverse-transcription, polymerase chain reaction expression studies. The library was developed using sequences accessed from various databases, including NCBI and EMBL; primers were screened through polymerase chain reaction, and those primers producing positive results were used to further screen cellular RNA that was extracted from encysting cells over various time points during the encystment process, and using various encystment media. Using these methods, target gene involvement in the encystment process was compared between species and encystment methods. These studies lay the foundation for quantitative gene expression analysis, and provide the basis for comparison of various encystment methods.