Enhancing Acanthamoeba diagnostics: rapid detection of viable Acanthamoeba trophozoites and cysts using viability PCR assay

Acanthamoeba keratitis (AK) is a sight-threatening corneal infection that is challenging to diagnose and treat due to the resistance of Acanthamoeba to standard antimicrobial agents. Current detection methods have limitations. This study aimed to develop and validate a sensitive viability PCR (v-PCR) assay using a photoreactive dye to distinguish viable from non-viable Acanthamoeba for rapid identification of viable Acanthamoeba trophozoites and cysts. Propidium monoazide (PMAxx) was used as a photoreactive dye. Mixtures containing decreasing percentages of viable Acanthamoeba, including reference strains Acanthamoeba polyphaga trophozoites and cysts, Acanthamoeba castellanii trophozoites, and Acanthamoeba castellanii trophozoites from a clinical sample, were prepared. Disinfectant efficacy against Acanthamoeba was also assessed. Samples were divided into PMAxx-treated and non-PMAxx-treated parts, and v-PCR assay was applied to both. The difference in viable Acanthamoeba was determined by subtracting the cycle threshold (Ct) value of the PMAxx-treated sample from the non-PMAxx-treated sample. Mixtures with decreasing concentrations of viable Acanthamoeba trophozoites and cysts showed increasingly lower delta Ct values as the percentage of viable Acanthamoeba decreased, as expected. This relationship was observed across all tested samples. Menicon Progent effectively eliminated A. polyphaga trophozoites and cysts, while propamidine, chlorhexidine, or their combination resulted in approximately 2-log reductions in A. polyphaga trophozoites and cysts. In the current study, a rapid v-PCR assay was developed that can distinguish between viable and non-viable Acanthamoeba, for both trophozoites and cysts, across multiple species. The presence of viable Acanthamoeba, as determined by v-PCR, allows monitoring of treatment response and efficacy in AK.