Optimising the diagnostic strategy for onychomycosis from sample collection to FUNGAL identification evaluation of a diagnostic kit for real‐time PCR

Abstract

Onychomycosis is a common nail disorder mainly due to dermatophytes for which the conventional diagnosis requires direct microscopic observation and culture of a biological sample. Nevertheless, antifungal treatments are commonly prescribed without a mycological examination having been performed, partly because of the slow growth of dermatophytes. Therefore, molecular biology has been applied to this pathology, to support a quick and accurate distinction between onychomycosis and other nail damage. Commercial kits are now available from several companies for improving traditional microbiological diagnosis. In this paper, we present the first evaluation of the real-time PCR kit marketed by Bio Evolution for the diagnosis of dermatophytosis. Secondly, we compare the efficacy of the kit on optimal and non-optimal samples. This study was conducted on 180 nails samples, processed by conventional methods and retrospectively analysed using this kit. According to our results, this molecular kit has shown high specificity and sensitivity in detecting dermatophytes, regardless of sample quality. On the other hand, and as expected, optimal samples allowed the identification of a higher number of dermatophytes by conventional mycological diagnosis, compared to non-optimal samples. Finally, we have suggested several strategies for the practical use of such a kit in a medical laboratory for quick pathogen detection.

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.…”

“…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.…”

Infection of Trichophyton verrucosum in cattle breeders, Poland: A 40‐year retrospective study on the genomic variability of strains

“…These methods should be handy, sensitive and time‐ and money‐saving screening tools . Molecular methods offer an opportunity to confront the lack of confidence in mycological diagnosis based on only phenotypic biological confirmation with a relatively high discriminatory power and good reproducibility of results . Nowadays, in studies conducted in some countries, for instance in France, T. verrucosum strains are identified without using any molecular techniques .…”

Comparison of fungal fluorescent staining and ITS rDNA PCR‐based sequencing with conventional methods for the diagnosis of onychomycosis

“…In this study, we compared microscopic results using two different methods: KOH and fluorescence. Although KOH is rapid and economical, it requires sufficient experience to identify the fungal elements and gives false‐negative results in 5–15% of cases . Fluorescence offers the advantages of high sensitivity and specificity because it observes fungal elements and budding patterns more easily, especially for the detection of rare hyphae and spores, and the percentage of false‐negative results is low compared with the culture method of diagnosis .…”

“…Although a false‐positive PCR result cannot be excluded, it is more likely that these represented false‐negative results of the conventional diagnostic methods . Divergences between PCR‐based sequencing and culture results may be related to the aliquots, which may not contain any further viable fungal elements for culture, whereas PCR may detect DNA contained in dead fungal elements . Using conventional methods as the gold standard, PCR demonstrated sensitivities up to 78%, higher than culture (72%), whereas the specificity was 90%.…”