Single-Point Nail Sampling to Diagnose Onychomycosis Caused by Non-Dermatophyte Molds: Utility of Polymerase Chain Reaction (PCR) and Histopathology

Abstract

The three most commonly used methods for diagnosing non-dermatophyte mold (NDM) onychomycosis are culture, polymerase chain reaction (PCR), and histopathology. Toenail samples from 512 patients (1 sample/patient) with suspected onychomycosis were examined using all three diagnostic tests. A statistically significant association was found between PCR and histopathology results, as well as between fungal culture and histopathology results. All PCR-positive and culture-positive dermatophyte samples were confirmed by histopathology. However, 15/116 (12.9%) of culture-positive NDM samples had negative histopathology results, while all PCR-positive NDM samples were confirmed by histopathology. The overall rate of dermatophyte detection was higher using PCR compared to culture (38.9% vs. 11.7%); the lower rate of NDM detection by PCR (11.7% vs. 38.9%) could be attributed to the restriction of the assay design to seven pre-selected targets. When repeat sampling in the clinic is not possible, a combination of NDM detection by PCR and positive histopathology of hyphae may be a proxy for NDM infection, particularly where the NDM occurs without a concomitant dermatophyte. There was a high degree of correlation between negative PCR and negative histopathology. A negative PCR result with negative histopathology findings may be a reliable proxy for the diagnosis of non-fungal dystrophy.

A comprehensive review of nondermatophyte mould onychomycosis: Epidemiology, diagnosis and management

“…Gupta et al recommend that any identification method should ideally identify the same species using PCR (rather than culture) in repeated samples from unique timepoints without any intervening treatment, and in conjunction with positive visualization (preferably using histopathology rather than KOH), to suggest an NDM is a pathogenic agent in the onychomycosis. 88,130 In a study assessing the utility of PCR, culture and histopathology for NDM detection using a single toenail sample, Gupta et al found that all NDM-positive samples detected by PCR were also positive for hyphae by histopathology, while 12.9% of NDM-positive samples detected by culture were negative by histopathology. 130 Matching results by PCR and histopathology, in the absence of a dermatophyte, may serve as a reliable proxy for the true presence of NDM using a single sample.…”

“…88,130 In a study assessing the utility of PCR, culture and histopathology for NDM detection using a single toenail sample, Gupta et al found that all NDM-positive samples detected by PCR were also positive for hyphae by histopathology, while 12.9% of NDM-positive samples detected by culture were negative by histopathology. 130 Matching results by PCR and histopathology, in the absence of a dermatophyte, may serve as a reliable proxy for the true presence of NDM using a single sample.…”

Clinical Diagnosis and Laboratory Testing of Abnormal Appearing Toenails: A Retrospective Assessment of Confirmatory Testing for Onychomycosis in the United States, 2022–2023

“…Confirmatory testing was conducted using fungal culture, histopathologic examination and/or multiplex real-time PCR; each test was conducted following a previously described protocol [24]. The choice of test was made according to physician's order, which includes the options of conducting a single test (e.g., histopathologic examination) or a combined test (e.g., histopathologic examination and PCR).…”

Treatment of onychomycosis in an era of antifungal resistance: Role for antifungal stewardship and topical antifungal agents

“…Most common and available among these are PCR‐based assays, in which fungal identification from clinical samples is enabled by amplification of species‐specific DNA, thus providing sensitivity that is similar to histopathology (e.g., periodic acid‐Schiff reaction) and greater than culture‐based or direct microscopy methods 94,96 . Utilizing PCR in conjunction with histopathology may provide indications of the fungal species present as well as confirming its pathogenic role; this may improve diagnostic confidence, particularly in cases of onychomycosis caused by non‐dermatophyte moulds 97 . Molecular identification of fungal species can also inform treatment selection, dosing and duration 94 .…”

“…94,96 Utilizing PCR in conjunction with histopathology may provide indications of the fungal species present as well as confirming its pathogenic role; this may improve diagnostic confidence, particularly in cases of onychomycosis caused by non-dermatophyte moulds. 97 Molecular identification of fungal species can also inform treatment selection, dosing and duration. 94 For example, PCR followed by comparative sequence analysis has been used to rapidly distinguish terbinafine-resistant from terbinafine-susceptible strains of T. interdigitale and T. rubrum via detection of single-point amino acid substitutions in the squalene epoxidase gene.…”