Development of a rapid LAMP assay toward point-of-care detection of dermatophyte infections and antifungal resistance

ABSTRACT Dermatophytosis is a common fungal infection affecting up to 25% of the global population. Increasing antifungal resistance and limited access to timely diagnostics hamper effective management, particularly in resource-limited settings. There is therefore an urgent need for reliable point-of-care diagnostics to guide treatment and support antifungal stewardship. Here, we report the development and validation of molecular assays based on loop-mediated isothermal amplification (LAMP) for rapid identification of dermatophytes and antifungal resistance-associated alleles. For taxonomic identification, we developed conventional LAMP assays targeting pan-dermatophytes, Microsporum spp., Trichophyton rubrum complex, and Trichophyton mentagrophytes complex. For detection of single-nucleotide polymorphisms (SNPs) associated with terbinafine resistance, we developed competitive SNP-based LAMP (sbLAMP) assays using unmodified self-stabilizing (USS) primers to differentiate specific from non-specific amplification. At 65°C, pan-dermatophyte, Microsporum spp., T. rubrum complex, and T. mentagrophytes complex samples were detected in less than 9 min of assay start, with Microsporum detection possible in less than 6 min. Limits of detection were 10 gene copies per reaction for T. mentagrophytes and 100–1,000 gene copies per reaction for the remaining assays. Using available clinical isolates, assays demonstrated sensitive and specific detection and were compatible with a simple colorimetric readout. For resistance detection, optimized primers validated wild-type allelic status of T. rubrum isolates, showing delayed amplification in mutant assays. Together, these assays show a means by which taxonomic identification and allele-specific resistance detection of dermatophyte infections might be possible within a point-of-care application. Such an application could support targeted treatment in remote or resource-limited settings that will also aid in the stewardship of emerging antifungal resistance. IMPORTANCE Antifungal resistance is an emerging global health threat, with the World Health Organization identifying inadequate fungal diagnostics as a major barrier to effective treatment and surveillance. Dermatophyte infections affect up to one-quarter of the global population, yet diagnosis remains slow, laboratory-dependent, and often inaccessible in the communities most affected. This diagnostic gap contributes to empirical antifungal use, delayed treatment, and the emergence of resistance to frontline therapies, such as terbinafine. Here, we present a proof-of-concept molecular diagnostic platform based on loop-mediated isothermal amplification (LAMP) for the rapid identification of dermatophyte infections and associated antifungal resistance markers. By integrating species-level detection with allele-specific resistance profiling in a rapid, low-cost, and portable format, these assays provide a foundation for future point-of-care diagnostics that could improve access to timely fungal diagnosis, enable targeted antifungal therapy, and strengthen global antifungal stewardship, particularly in resource-limited settings.