Evaluation of a complete‐kill assay for anti‐acanthamoeba solutions

Abstract

AbstractPurpose Acanthamoeba keratitis is a sight debilitating disease that requires effective topical drug therapy to eradicate the pathogenic agent.Methods An assay was created to determine whether 0.02% poly(hexamethylene biguanide) hydrochloride (PHMB), 0.02% chlorhexidinedigluconate (CHL), 0.1% c‐desomedine (DESO), and 1.0% c‐voriconazole (VOR) were effective in completely killing 15 different isolates of acanthamoeba at time points 24, 48, and 72 hours in comparison to a saline control. Each 0.5 ml volume of solution was inoculated with 0.1 ml of acanthamoeba cysts at a concentration of 1‐5 x 106 /ml determined with a hemacytometer and allowed to incubate at 30o C. At the time points listed, aliquots from each treatment group were inoculated onto non‐nutrient agar overlaid with Enterobacter aerogenes. The plates were microscopically examined for growth at time points 1 and 2 weeks. At 2 weeks, all plates were sub‐cultured onto fresh medium. At 7 days, growth in sub‐culture at each time point was graded 1 for growth and zero for no growth. The time points were combined for each drug with a possible grade from 0 to 3. The grades were non‐parametrically (Mann Whitney) compared to determine any significance in positive growth between the drugs.Results Complete–kill was determined more frequently with PHMB and DESO (p=1.0) than CHL (p=0.04). PHMB, DESO, and CHL demonstrated more complete‐kill than VOR (p=0.01) which was more effective than the saline control (p=0.0003).Conclusion The complete‐kill assay appears to provide separation in the effectiveness of different anti‐amoebic drug solutions, and may provide an alternative evaluation of possible new anti‐infectives in the treatment of acanthamoeba keratitis.