To develop a rat model of chronic Acanthamoeba polyphaga keratitis suitable for pharmacologic assessment of therapeutic agents.
An A. polyphaga isolate (ATCC #50495) was grown in peptone-yeast extract-glucose medium. Five-weeks-old, Sprague-Dawley male rats were injected with 103 or 104 trophozoites in the left cornea stromal layer. A subconjunctival injection of 0.14, 0.28, or 0.57 mg long-acting betamethasone was performed weekly. At the end of experiments, rats were killed; the superficial corneal epithelium gently scraped and cultured; and globes histologically examined. Topical polyhexamethylene biguanide (PHMB), hexamidine diisethionate, and miltefosine (hexadecylphosphocholine) were administered topically as eye drops 3 times a day at concentrations of 0.02%, 0.1%, and 0.01% respectively. In vitro minimal inhibitory concentration (MIC) and fractional inhibitory concentration values were measured in A. polyphaga cultures.
In infected eyes, lesions consisted of the sequential appearance within 2 weeks of edema, infiltrates, and/or abscesses. On day 35 postinfection, a combination of 104 parasites with a regimen of 0.28 mg/week betamethasone resulted in the highest ratio of rats with abscesses. Presence of A. polyphaga was confirmed histologically and inconsistently in cultures. In rats optimally prepared as said earlier, agents were administered on day 6 postinfection. A combination of PHMB and hexamidine diisethionate exerted a synergistic effect and was more effective than PHMB, hexamidine diisethionate, or miltefosine alone. In vitro, PHMB (MIC = 14.6 μM) and hexamidine diisethionate (MIC = 555 μM) exerted a synergistic effect (fractional inhibitory concentration = 0.06), and miltefosine exhibited antiamoebal activity (MIC = 27.4 μM).
In this study, a rat model of chronic A. polyphaga keratitis was obtained and found suitable for assessment of pharmacologic agents. It provides an in vivo approach of drug resistance, pathogenicity, and physiopathologic mechanisms of chronic amoebic keratitis.