Usefulness of techniques based on real time PCR for the identification of onychomycosis‐causing species

Abstract

Onychomycosis (OM) is a nail infection caused mainly by dermatophyte species but other species of yeast and moulds are frequently involved as well. Classical diagnosis has limitations thus empirical treatment is common. The usefulness of different real time PCR (RT-PCR) assays for identifying species causing OM was assessed in samples from seventy patients and fifteen controls. Conventional methods and four different RT-PCR assays were used: a panfungal, a pandermatophyte and two specific assays for detecting Candida spp. and Aspergillus spp. Fungal elements were visualised in 58% of the samples, and 54% of cultures were positive. Panfungal and pandermatophyte RT-PCR were positive in 28% and 60%, respectively, and the sensitivity relative to positive cultures was 47% and 90%. Candida spp. were detected in 76% of samples analysed and Aspergillus spp. in 60%. These species were also present in 80% of control cases. In conclusion, molecular techniques were useful but showed limitations. The panfungal assay showed a low sensitivity, the pandermatophyte assay was sensitive and specific but did not allow for differentiation among species of dermatophytes. Finally, the role of non-dermatophyte species detected by using specific RT-PCR techniques should be carefully analysed as these species were also present in healthy nails.

Development and evaluation of a pan‐dermatophyte polymerase chain reaction with species‐level identification using sloppy molecular beacon probes

“…Our data are in line with previous reports on RT‐PCR assays for the detection of dermatophytes; in general, an increase in the positivity rate of between 4% and 22% when compared with culture has been noted (in our case, it was 22·2%) . When PCR is compared with microscopy, reported data are inconsistent: in some studies microscopy was the method with the highest positivity rate, i.e.…”

“…In other studies, nondermatophytes were considered as commensal or contaminating fungi in most cases . Hafirassou and colleagues even found Candida and Aspergillus DNA in a majority of nails from healthy volunteers . Based on our results, we conclude for our assay that the detection of nondermatophytes currently has no benefit.…”

“…When PCR is compared with microscopy, reported data are inconsistent: in some studies microscopy was the method with the highest positivity rate, i.e. the most sensitive approach; in some studies PCR was the most sensitive; and in other studies – as in our case – the positivity rates for PCR and microscopy were similar . However, in many of these previous real‐time approaches, only dermatophytes in general or a single species (e.g.…”

“…However, one might criticize that in these studies, in approximately 30–40% of cases where the molecular assay revealed a single nondermatophyte, it was not the same species that was found in culture. In other studies, nondermatophytes were considered as commensal or contaminating fungi in most cases . Hafirassou and colleagues even found Candida and Aspergillus DNA in a majority of nails from healthy volunteers .…”

“…However, in many of these previous real‐time approaches, only dermatophytes in general or a single species (e.g. T. rubrum ) are detected . By combining several primers and probes in two reactions, some were able to identify up to six taxa .…”