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

keratitis (AK) is a sight-threatening corneal infection that is challenging to diagnose and treat due to the resistance of 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 for rapid identification of viable trophozoites and cysts. Propidium monoazide (PMAxx) was used as a photoreactive dye. Mixtures containing decreasing percentages of viable , including reference strains trophozoites and cysts, trophozoites, and trophozoites from a clinical sample, were prepared. Disinfectant efficacy against 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 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 trophozoites and cysts showed increasingly lower delta Ct values as the percentage of viable decreased, as expected. This relationship was observed across all tested samples. Menicon Progent effectively eliminated trophozoites and cysts, while propamidine, chlorhexidine, or their combination resulted in approximately 2-log reductions in trophozoites and cysts. In the current study, a rapid v-PCR assay was developed that can distinguish between viable and non-viable , for both trophozoites and cysts, across multiple species. The presence of viable , as determined by v-PCR, allows monitoring of treatment response and efficacy in AK.IMPORTANCEThe development of a sensitive viability PCR (v-PCR) assay using propidium monoazide (PMAxx) as a photoreactive dye marks a significant advancement in the diagnosis and treatment of keratitis (AK), a severe corneal infection notorious for its resistance to conventional antimicrobials. This innovative assay offers a rapid and accurate method to distinguish viable from non-viable trophozoites and cysts, addressing a critical need in the field. By effectively distinguishing between viable and non-viable , this test enables monitoring of treatment response and efficacy, essential for guiding clinical interventions in AK cases. The successful validation of this v-PCR assay across various species and its ability to assess disinfectant efficacy further underline its potential as a valuable tool for improving diagnostic precision and therapeutic outcomes in the treatment of AK.