Metagenomic analysis as a support to clinical diagnosis of onychomycosis

Objective: Onychomycosis can be caused by yeasts and by dermatophytic and nondermatophytic moulds. These fungi lead to the gradual destruction of the nail plate. Antifungal therapies, both oral and topical, take a long time with very often disappointing results due to the lack both of accurate diagnosis and tests for sensitivity to antifungal drugs before therapy. Thus dermatologists increasingly require the analysis for the pathogens responsible of onychomycosis after a brief interruption of therapy; as a consequence culture dependent methods for onychomycosis diagnosis give frequently false-negative results. In order to overcome the above mentioned problems, the aim of the present work was to compare two different methods: the cultural methods and the sequence analysis method based on the metagenomic approach. Methods: 33 nail samples from 29 patients with clinically diagnosis of onychomycosis in feet and/or hands were investigated. Fungal cultivation was performed using Sabouraud dextrose agar (Oxoid) amended with antibiotics, with and without cycloheximide, to detect yeasts, dermatophyte and nondermatophytic moulds. Total DNA was obtained from nail powder. To obtain fungal amplicons, the ribosomal ITS1 region was targeted, by using primers BITS and B58S3 (Bokulich and Mills, 2013) linked to Illumina adapters. Sequencing libraries were finally constructed through the link of indexes (Nextera XT Index Kit, Illumina, San Diego, CA), quantified, normalized and pooled. Libraries were subjected to paired-end sequencing (2 × 250 bp, nano format). Data analysis was performed using the pipeline Qiime and high-quality reads were clustered into operational taxonomic units (OTUs) at 97%. OTUs were finally annotated using the UNITE fungal ITS reference data set within RDP classifier and the Worcup ITS reference as training dataset. Species assignation on selected OTUs was manually resolved through BLAST searching against mycobank, RDP and GeneBank. Relative abundances of microbial taxa in each sample were calculated and compared. Results: Candida spp. or Trichophyton spp were detected in 14 samples. In 1 sample both Candida and Trichophyton were detected. Filamentous nondermatophytic fungi having a recognized role as unique causative agent of onychomycosis or mixed infections, were recovered in 21 samples. It is difficult to establish when these moulds are etiologic agents of onychomycosis or contaminants. Only repeat testing may solve the problem. Comparing the results obtained by cultivation method and to those obtained by sequencing ITS1 amplicons we observed that 2 out of 33 samples did not provide concordant results. In fact, while from these samples any fungal growth was observed, the amplification of ITS region was achieved, thus suggesting the presence of fungi. Finally high-throughput deep sequencing let us to profile the biodiversity in the samples and by analysis of prevalence it was possible to identify and quantified the pathogen. Conclusion: The application of high-throughput sequence analysis of amplicons produced targeting ITS1 region allows us to detect the presence of fungal DNA even when in vitro growth of fungal colonies was not possible. Moreover the pathogen can be quantified by means the analysis of prevalence of the sequence data.