Comparative evaluation of conventional and nanopore approaches in onychomycosis diagnosis

Onychomycosis is a frequent and chronic nail infection caused by dermatophytes, yeast, and non-dermatophyte molds (NDM). Detection of the causative agent is relevant to overcoming recurrent infections and improving treatment. Combined microscopy and culture are the diagnostic gold standard; however, more sensitive tests are needed to speed detection. This work aimed to assess fungal nail diagnosis using conventional and molecular techniques. We explored the potential of nanopore sequencing for fungal detection.

A total of 104 nail samples from 51 patients with onychomycosis and 53 individuals with healthy nails were subjected to potassium hydroxide(KOH) microscopy, culture, PCR, and nanopore sequencing. Sensitivity was calculated using a composite reference standard based on microscopy and culture.

Distal subungual onychomycosis was the most frequent clinical presentation (43.1%). Infections were primarily caused by dermatophytes 60% (21), followed by yeast 22.9% (8) and NDM 11.4% (4), 5.7% (2) were mixed infections. The prevalent pathogens were T. rubrum (40%), T. mentagrophytes (14.3%), and C. albicans (14.3%). 90.2% (46/51) of patients were positive by KOH, 68.6% (35/51) by culture (58.8%, 30/51 positive in both), and 58.8%(30/51) were sequenced by nanopore. Fungal DNA sequences were identified in 65.7% (23/35) of culture-positive patients. Eight samples from healthy nails were positive for fungal DNA and were considered transient microbiota.

Dermatophytes are the predominant causative agent; however, at least 30% of nail infections are caused by yeast and NDM. Conventional diagnosis remains challenging; alternatively, nanopore sequencing represents a complementary diagnostic tool that should be further optimized for clinical application.