| Summary: | <i>Acanthamoeba</i> spp. are ubiquitous and opportunistic free-living amoebae (FLA) that can cause <i>Acanthamoeba</i> keratitis and other infections in the human host. A quick and efficient diagnosis is often challenging. Our study aimed to establish a qPCR assay to detect and, at the same time, quantify the predominant <i>Acanthamoeba</i> genotype T4. DNA from clinical corneal scrapings and <i>Acanthamoeba</i> reference strains, including genotypes T3, T4, T5, T6, T10, T11, and T12, were used to develop the new T4 assay and it was compared to published protocols and one commercial kit for evaluation. The T4 assay showed no amplification with <i>Acanthamoeba</i> genotypes T3, T5, T6, T10, T11, and T12. The efficiencies ranged from 92.01 to 97.59% (R<sup>2</sup> of 0.9768 to 0.9951). The calculated LOD range was 3.63 to 33.27 cells/µL. The protocol published by Qvarnstrom and colleagues was more sensitive compared to the other assays, and an overall good agreement was observed between the new T4 and the Qvarnstrom assays. We successfully developed and validated a genotype T4 assay that could be run in duplex with the Qvarnstrom assay to reliably and simultaneously diagnose <i>Acanthamoeba</i> genotype T4 and other genotypes from clinical samples.
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