Acanthamoeba castellanii is the causative agent of blinding keratitis. Though reported in non-contact lens wearers, it is most frequently associated with improper use of contact lens. For contact lens wearers, amoebae attachment to the lens is a critical first step, followed by amoebae binding to the corneal epithelial cells during extended lens wear. Acanthamoeba attachment to surfaces (biological or inert) and migration is an active process and occurs during the trophozoite stage. Thus retaining amoebae in the cyst stage (dormant form) offers an added preventative measure in impeding parasite traversal from the contact lens onto the cornea. Here, we showed that as low as 3% DMSO, abolished A. castellanii excystation. Based on the findings, it is proposed that DMSO should be included in the contact lens disinfectants as an added preventative strategy against contracting Acanthamoeba keratitis.
For the past several decades, there has been little improvement in the morbidity and mortality associated with Acanthamoeba keratitis and Acanthamoeba encephalitis, respectively. The discovery of a plethora of antiacanthamoebic compounds has not yielded effective marketed chemotherapeutics. The rate of development of novel antiacanthamoebic chemotherapies of translational value and the lack of interest of the pharmaceutical industry in developing such chemotherapies have been disappointing. On the other hand, the market for contact lenses/contact lens disinfectants is a multi-billion-dollar industry and has been successful and profitable. A better understanding of drugs, their targets, and mechanisms of action will facilitate the development of more-effective chemotherapies. Here, we review the progress toward phenotypic drug discovery, emphasizing the shortcomings of useable therapies.
Acanthamoeba is an opportunistic protist pathogen that is responsible for serious human and animal infection. Being one of the most frequently isolated protists from the environment, it is likely that it readily encounters microaerophilic environments. For respiration under anaerobic or low oxygen conditions in several amitochondriate protists, decarboxylation of pyruvate is catalyzed by pyruvate ferredoxin oxidoreductase instead of pyruvate dehydrogenase. In support, Nitazoxanide, an inhibitor of pyruvate ferredoxin oxidoreductase, is effective and non-mutagenic clinically against a range of amitochondriate protists, Giardia intestinalis, Entamoeba histolytica and Trichomonas vaginalis. The overall aim of the present study was to determine in vitro efficacy of Nitazoxanide against Acanthamoeba castellanii. At micromolar concentrations, the findings revealed that Nitazoxanide neither affected A. castellanii growth or viability nor amoeba-mediated host cell monolayer damage in vitro or extracellular proteolytic activities. Similarly, microaerophilic conditions alone had no significant effects. In contrast, microaerophilic conditions together with Nitazoxanide showed amoebicidal effects and inhibited A. castellanii-mediated host cell monolayer damage as well as extracellular proteases. Using encystation assays, it was observed that Nitazoxanide inhibited trophozoite transformation into cysts both under aerophilic and microaerophilic conditions. Furthermore, pre-treatment of cysts with Nitazoxanide inhibited A. castellanii excystation. These findings are important in the identification of potential targets that could be useful against parasite-specific respiration as well as to understand the basic biology of the life cycle of Acanthamoeba.
Acanthamoeba keratitis is a serious infection with blinding consequences and often associated with contact lens wear. Early diagnosis, followed by aggressive topical application of drugs, is a prerequisite in successful treatment, but even then prognosis remains poor. Several drugs have shown promise, including chlorhexidine gluconate; however, host cell toxicity at physiologically relevant concentrations remains a challenge. Nanoparticles, subcolloidal structures ranging in size from 10 to 100 nm, are effective drug carriers for enhancing drug potency. The overall aim of the present study was to determine whether conjugation with gold nanoparticles enhances the antiacanthamoebic potential of chlorhexidine. Gold-conjugated chlorhexidine nanoparticles were synthesized. Briefly, gold solution was mixed with chlorhexidine and reduced by adding sodium borohydride, resulting in an intense deep red color, indicative of colloidal gold-conjugated chlorhexidine nanoparticles. The synthesis was confirmed using UV-visible spectrophotometry that shows a plasmon resonance peak of 500 to 550 nm, indicative of gold nanoparticles. Further characterization using matrix-assisted laser desorption ionization-mass spectrometry showed a gold-conjugated chlorhexidine complex at m/z 699 ranging in size from 20 to 100 nm, as determined using atomic force microscopy. To determine the amoebicidal and amoebistatic effects, amoebae were incubated with gold-conjugated chlorhexidine nanoparticles. For controls, amoebae also were incubated with gold and silver nanoparticles alone, chlorhexidine alone, neomycin-conjugated nanoparticles, and neomycin alone. The findings showed that gold-conjugated chlorhexidine nanoparticles exhibited significant amoebicidal and amoebistatic effects at 5 μM. Amoebicidal effects were observed by parasite viability testing using a Trypan blue exclusion assay and flow-cytometric analysis using propidium iodide, while amoebistatic effects were observed using growth assays. In contrast, chlorhexidine alone, at a similar concentration, showed limited effects. Notably, neomycin alone or conjugated with nanoparticles did not show amoebicidal or amoebistatic effects. Pretreatment of A. castellanii with gold-conjugated chlorhexidine nanoparticles reduced amoeba-mediated host cell cytotoxicity from 90% to 40% at 5 μM. In contrast, chlorhexidine alone, at similar concentrations, had no protective effects for the host cells. Similarly, amoebae treated with neomycin alone or neomycin-conjugated nanoparticles showed no protective effects. Overall, these findings suggest that gold-conjugated chlorhexidine nanoparticles hold promise in the improved treatment of A. castellanii keratitis.