Variables Affecting the Recovery of Acanthamoeba Trophozoites

While the results of Acanthamoeba testing have been extensively published, laboratories conducting such testing are left to develop their own methods in the absence of a standardized methodology. The wide disparity of methods has resulted in equally inconsistent reported results for contact lens care (CLC) products. This study's objective was to determine the source of these discrepancies by evaluating basic Acanthamoeba biology and their impact on antimicrobial efficacy testing, including the ability of a recovery method to stimulate a single trophozoite to proliferate. Antimicrobial efficacy testing was conducted using well-published Acanthamoeba strains, storage conditions, and growth-based recovery methods. To identify variables that influence results, test solutions with low Acanthamoeba disinfection rates were utilized to prevent differences from being masked by high log reductions. In addition, single-cell proliferation assays were executed to understand the growth requirements to stimulate trophozoite propagation in two recovery methods. These studies indicated that both nutrient density (>10⁶ CFU) and the length of plate incubation (at least 14 days) could significantly influence the accurate recovery of trophozoites. Together, this study emphasizes the need to understand how Acanthamoeba trophozoites biology can impact test methods to create divergent results.