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Research Article: Diagnostic accuracy of fluorescence microscopy of hematoxylin–eosin-stained nail sections for onychomycosis

Date Published: 2026-07-27

Abstract:
Onychomycosis requires laboratory confirmation before prolonged oral antifungal therapy is initiated. Periodic acid–Schiff (PAS) staining is highly sensitive for the histopathologic diagnosis of onychomycosis but increases cost and turnaround time. This study evaluated the diagnostic accuracy of fluorescence microscopy of routine hematoxylin–eosin (H&E)-stained nail sections compared with PAS-stained nail sections. A diagnostic accuracy study was performed using archived nail clipping slides from patients evaluated between 2019 and 2023. Fluorescence microscopy of H&E-stained nail clippings served as the index test, while PAS-stained nail clippings served as the reference standard. Slides were randomly selected from de-identified records and independently assessed. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated. A total of 84 slides were included, comprising 42 PAS-positive onychomycosis slides and 42 PAS-negative non-onychomycosis slides. Fluorescence microscopy showed a sensitivity of 80.95% (95% confidence interval [CI], 65.88–91.40%) and a specificity of 97.62% (95% CI, 87.43–99.94%). The PPV and NPV were 97.14% (95% CI, 85.08–99.93%) and 83.67% (95% CI, 70.34–92.68%), respectively. False-negative results were primarily associated with sparse fungal elements and yeast forms lacking pseudohyphae, while the single false-positive result was associated with fluorescent plasma globules and neutrophils. Fluorescence microscopy of H&E-stained nail sections exhibits high specificity and may serve as a rapid, lower-cost adjunctive diagnostic method for confirming onychomycosis, particularly in resource-limited settings where fluorescence microscopy is available.

Introduction:
Onychomycosis requires laboratory confirmation before prolonged oral antifungal therapy is initiated. Periodic acid–Schiff (PAS) staining is highly sensitive for the histopathologic diagnosis of onychomycosis but increases cost and turnaround time. This study evaluated the diagnostic accuracy of fluorescence microscopy of routine hematoxylin–eosin (H&E)-stained nail sections compared with PAS-stained nail sections.

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