A new keratitis rat model for evaluation of anti–Acanthamoeba polyphaga agents

Abstract

Abstract: : Purpose: Free–living Acanthamoeba sp. are a cause of potentially devastating human keratitis. Current lack of non–empirical, validated therapies and frequent sequellae prompt the development of experimental models suitable for evaluation of candidate anti–amoebal agents. The aim of this work was to obtain a rat model of chronic amoebic keratitis suitable for screening candidate therapeutic agents with documented anti–acanthamoebal activity in vitro. Methods: Sprague–Dawley rats were infected by intracorneal inoculation of 10³ or 10⁴ axenic Acanthamoeba polyphaga trophozoites associated with local corticosteroids. Controls consisted of non–infected steroid–treated or untreated rats. Corneal morphology was examined weekly and, 35 days after inoculation, animals were killed and local infection assessed by corneal microbiological and histocytological examination. In vivo and in vitro anti–amoebal activities of miltefosine were evaluated using this model and axenic cultures, and compared with those of the established anti–amoebal agents polyhexamethylene biguanide (PHMB) and hexamidine di–isethionate tested alone or in combination. Results: Size of inoculum and corticosteroid dose were found key factor for the maintenance of a chronic infection which was best obtained with 10⁴ Acanthamoeba polyphaga combined with a weekly subconjunctival injection of betamethasone (0.28mg) (p<0.05). In vivo, combination of PHMB and hexamidine diisethionate resulted in corneal healing twelve days after inoculation and was more effective than miltefosine, PHMB and hexamidine diisethionate alone. In vitro, miltefosine was found active (MIC: 27.4 µmol), while PHMB (MIC: 14.6 µM) and hexamidine diisethionate (MIC: 555µm) exhibited a synergistic effect (FIC: 0.006). Conclusions: The present chronic rat model of Acanthamoeba polyphaga keratitis was found suitable for pharmacological studies of candidate agents.

Keywords: Acanthamoeba • keratitis • antibiotics/antifungals/antiparasitics