Identification of Dermatophyte and Non-Dermatophyte Agents in Onychomycosis by PCR and DNA Sequencing—A Retrospective Comparison of Diagnostic Tools

Abstract

Rapid and reliable fungal identification is crucial to delineate infectious diseases, and to establish appropriate treatment for onychomycosis. Compared to conventional diagnostic methods, molecular techniques are faster and feature higher accuracy in fungal identification. However, in current clinical practice, molecular mycology is not widely available, and its practical applicability is still under discussion. This study summarizes the results of 16,094 consecutive nail specimens with clinical suspicion of onychomycosis. We performed PCR/sequencing on all primary nail specimens for which conventional mycological diagnostics remained inconclusive. In specimens with a positive direct microscopy but negative or contaminated culture, molecular mycology proved superior and specified a fungal agent in 65% (587/898). In 75% (443/587), the identified pathogen was a dermatophyte. Positive cultures for dermatophytes, yeasts and non-dermatophyte molds (NDMs) were concordant with primary-specimen-DNA PCR/sequencing in 83% (10/12), 34% (22/65) and 45% (76/169), respectively. Among NDMs, agreement was high for Fusarium spp. (32/40; 80%), but low for Penicillium spp. (5/25; 20%) and Alternaria spp. (1/20; 5%). This study underlines the improvement in diagnostic yield by fungal primary-specimen-DNA PCR/sequencing in the event of a negative or contaminated culture, as well as its significance for the diagnosis of dermatophyte and non-dermatophyte onychomycosis. Molecular mycology methods like PCR and DNA sequencing should complement conventional diagnostics in cases of equivocal findings, suspected NDM onychomycosis or treatment-resistant nail pathologies.

Survey on Dermatophytes Isolated from Animals in Switzerland in the Context of the Prevention of Zoonotic Dermatophytosis

“…The 28S rRNA gene and the internal transcribed spacer (ITS) were amplified using the oligonucleotide pairs 5′-GATAGCGMACAAGTAGAGTG-3′/5′-GTCCGTGTTTCAAGACGGG-3′ (LSU1/LSU2) and 5′-GGTTGGTTTCTTTTCCT-3′/5′-AAGTAAAAGTCGTAACAAGG-3′ (ITS1/ITS2 or LR1/SR6R), respectively [ 17 , 18 , 21 , 22 ]. Species identification was performed using BLAST analysis of PCR product sequences, similar to the mycological analysis of human dermatophytosis [ 23 ].…”

Microbiological culture combined with PCR for the diagnosis of onychomycosis: Descriptive analysis of 121 patients

“…5 However, it is important to note that PCR cannot determine fungal viability, detects only dermatophytes, depends on the primers and cycles, and is more expensive than other techniques. 12 The objective of this study was threefold: to observe the prevalence of onychomycosis, determine the most suitable diagnostic test, and analyse the distribution of pathogens based on factors such as age, sex, quarter of the year, duration of symptoms and prior treatment history.…”

“…Finally, molecular methods such as PCR offers rapid identification of fungi, typically within 1 day, with a sensitivity of 87% based on previous studies 5 . However, it is important to note that PCR cannot determine fungal viability, detects only dermatophytes, depends on the primers and cycles, and is more expensive than other techniques 12 …”

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

“…The current and upcoming methods for diagnosing NDM onychomycosis are summarized in Table 4 41,88,110–129 …”

“…T A B L E 4 Diagnostic techniques for NDM onychomycosis. 41,88,[110][111][112][113][114][115][116][117][118][119][120][121][122][123][124][125][126][127][128][129] b Sample from fungal culture is required prior to the start of the assay, hence only viable isolates will be analysed. Aspergillus showed favourable MC and EC rates following treatment with terbinafine and itraconazole, with up to 88.2% (30/34) of patients achieving clinical improvement with mycological cure using pulsed terbinafine (Table 5).…”