Background: Conventional diagnostic methods for onychomycosis possess disadvantages related to limited sensitivity, extended turnaround times and strong reliance on operator expertise.
Objectives: To develop a molecular assay with high sensitivity and specificity for rapid detection of multiple fungal pathogens in nail specimens.
Methods: A multi-tube recombinase polymerase amplification (RPA) assay with fluorescent signal detection was developed, targeting pan-fungal, Trichophyton rubrum, T. interdigitale, pan-Candida, Candida albicans, pan-Trichosporon and pan-Aspergillus. The analytical sensitivity and specificity of the assay were evaluated using fungal strains, and its performance was assessed in 81 clinical nail specimens in parallel with calcofluor white fluorescence microscopy and fungal culture.
Results: The multi-tube RPA assay demonstrated a detection limit of 1 pg of genomic DNA for all targets, with a turnaround time of < 70 min. In the evaluation of 81 clinical nail specimens, Bayesian latent class analysis estimated sensitivities of 86.0% for multi-tube RPA, 67.0% for fungal culture and 77.0% for microscopy, with corresponding specificities of 85.0%, 89.0% and 87.0%. The positive and negative predictive values were 92.0% and 72.0% for multi-tube RPA, 93.0% and 55.0% for fungal culture, and 93.0% and 66.0% for microscopy.
Conclusion: Compared with fungal culture and microscopy, the multi-tube RPA assay demonstrated superior sensitivity while maintaining good specificity, facilitating the identification of fungal pathogens at the species level in nail specimens. Its operational simplicity, enhanced diagnostic performance and reduced processing times make it a promising alternative for the mycological diagnosis of onychomycosis.
Keywords: Bayesian latent class analysis; dermatophytes; isothermal amplification; onychomycosis; rapid detection; recombinase polymerase amplification.
© 2026 The Author(s). Mycoses published by Wiley‐VCH GmbH. Published by John Wiley & Sons Ltd.