Enhancing <i>Acanthamoeba</i> diagnostics: rapid detection of viable <i>Acanthamoeba</i> trophozoites and cysts using viability PCR assay

<i>Acanthamoeba</i> keratitis (AK) is a sight-threatening corneal infection that is challenging to diagnose and treat due to the resistance of <i>Acanthamoeba</i> 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 <i>Acanthamoeba</i> for rapid identification of viable <i>Acanthamoeba</i> trophozoites and cysts. Propidium monoazide (PMAxx) was used as a photoreactive dye. Mixtures containing decreasing percentages of viable <i>Acanthamoeba</i>, including reference strains <i>Acanthamoeba polyphaga</i> trophozoites and cysts, <i>Acanthamoeba castellanii</i> trophozoites, and <i>Acanthamoeba castellanii</i> trophozoites from a clinical sample, were prepared. Disinfectant efficacy against <i>Acanthamoeba</i> 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 <i>Acanthamoeba</i> 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 <i>Acanthamoeba</i> trophozoites and cysts showed increasingly lower delta Ct values as the percentage of viable <i>Acanthamoeba</i> decreased, as expected. This relationship was observed across all tested samples. Menicon Progent effectively eliminated <i>A. polyphaga</i> trophozoites and cysts, while propamidine, chlorhexidine, or their combination resulted in approximately 2-log reductions in <i>A. polyphaga</i> trophozoites and cysts. In the current study, a rapid v-PCR assay was developed that can distinguish between viable and non-viable <i>Acanthamoeba</i>, for both trophozoites and cysts, across multiple species. The presence of viable <i>Acanthamoeba</i>, 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 <i>Acanthamoeba</i> 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 <i>Acanthamoeba</i> trophozoites and cysts, addressing a critical need in the field. By effectively distinguishing between viable and non-viable <i>Acanthamoeba</i>, 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 <i>Acanthamoeba</i> 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.