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- W4366182009 abstract "Approximately 5.5% of the world population is affected by onychomycosis, and infection rates continue to increase 21.2% annually. This costs Medicare alone over 27 million USD in 2016. Oral antifungals can lead to rare, but serious side effects including liver injury that can progress into complete hepatic failure requiring liver transplant. The recommendation from the American Academy of Dermatology is to perform confirmatory testing before oral therapy. However, current diagnostics are expensive, time consuming and require specialized skillsets and are, therefore, not widely used. Instead, presumptive treatment is still the standard of care. Our aim is to produce a viable, field-deployable, point of care, clustered regularly interspaced short palindromic repeat (CRISPR)-based assay for the diagnosis of onychomycosis at a fraction of current diagnostic costs. We developed a CRISPR Cas12a-based test using the highly conserved dermatophyte gene, Topoisomerase II, used to identify dermatophytes at the species level. Currently 8 patients are enrolled. Nail scrapings are divided for KOH testing, fungal culture and direct DNA extraction. Following the amplification of the DNA from dermatophytes species T. violaceum, T. verrucosum, and T. interdigitale, the amplicon was introduced to CRISPR Cas12a pre-incubated with the target-specific guide RNA. This resulted in double stranded DNA cleavage by Cas12a nuclease and subsequent cutting of FAM-biotin labeled single stranded DNA reporters. This reporter was then detected by a secondary antibody-based lateral flow strip. Gel electrophoresis confirmed highly specific target cutting without human genome off-targets. In this assay, all reactions were performed at room temperature without specialized equipment other than a thermocycler. Consequently, it has been estimated that costs for implementation of CRISPR assays could be less than 1USD/test. The success of this method can be expanded to other diagnostic applications in dermatology." @default.
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- W4366182009 date "2023-05-01" @default.
- W4366182009 modified "2023-09-28" @default.
- W4366182009 title "1582 Development of CRISPR point of care test for identification of dermatophytes in onychomycosis" @default.
- W4366182009 doi "https://doi.org/10.1016/j.jid.2023.03.1600" @default.
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