Comparative study of PCR, microscopic examination and culture-based methods for detection and identification of fungi in onychomycosis

This work was supported by a grant from the Swiss Government Excellence Scholarships for Foreign Students to da Cunha KC. Onychomycosis is the most common disease of the nails and represents about a half of all nail abnormalities worldwide. It affects, in particular, males, the elderly, diabetics, and immune-compromised individuals, with toenails being affected to a greater extent than fingernails. The diagnosis of onychomycosis cannot be done only based on clinical presentation, due to the similarity with other onychopathies. Traditionally, fungi identification is based on direct microscopic examination of clinical samples as well as microscopic and macroscopic examination of cultures, and when needed in metabolism tests. However, it is time-consuming, laborious, costly, and requires a certain level of morphological and taxonomical expertise. Furthermore, in the case of onychomycosis the culture sensitivity is only about 50%. Moreover, the identification of cultured isolates is sometime challenging especially when strains do not produce typical characteristics or when Non Dermatophytes Molds (NDM) are the causative agent. With the growing number of organisms recognized as possible fungal pathogens in nails, it is becoming important to ensure the accurate identification of the causative organism. This will help clinicians in selecting the most appropriate therapy. Indeed antifungal agents, such as terbinafine, itraconazole, and fluconazole have a broad spectrum with activity against fungi but some NDM may be poorly responsive or unresponsive to systemic treatments. We have introduced the identification based on DNA extraction directly from human nail samples. We use PCR amplification of DNA coding nuclear ribosomal internal transcribed spacer (ITS). The clinical specimen consisted of 80 nails samples with 60 being positives under microscope using blankophor and 20 were negative. As expected PCR allows detection of fungi in all nail specimen that were positive by microscopy and culture. Moreover, PCR also allows detection of fungal DNA in nails with negative culture but with positive microscopy. In some samples, PCR lead to the amplification of more than one amplicon, which suggests mixed infection. Sequencing of purified amplicons permitted identification of the implicated fungi in the vast majority of case. The results of the present study indicated that PCR is highly advantageous as a diagnostic tool for detection and identification of fungi on direct application to nail specimens. Because during the last decade, cost for DNA sequencing have been dramatically reduced and given the poor sensitivity of culture in onychomycosis, it is becoming reasonable to use PCR/sequencing as a first line method for the diagnosis of onychomycosis. This will provide a quick identification of the implicated fungi and will help clinicians in making rational and effective therapeutic decisions. This will be a direct benefit to patients with dermatophytosis.