Identification of infectious agents in onychomycoses by Polymerase Chain Reaction-Terminal Restriction Fragment Length Polymorphism.
Department of Dermatology, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland.
The need of a fast and reliable assay for the identification of dermatophyte and non-dermatophyte fungi (NDF) in onychomycosis is essential since NDF are especially difficult to cure using standard treatment. Diagnosis is usually based on both direct microscopic examination of nail scrapings, macroscopic and microscopic identification of the infectious fungus in culture assays. In the last decade, PCR assays have been developed for the direct detection of fungi in nail samples. In this study, we describe a Polymerase Chain Reaction – Terminal Restriction Fragment Length Polymorphism (PCR-TRFLP) assay to directly and routinely identify the infecting fungi in nails. Fungal DNA was easily extracted using a commercial kit after dissolving nail fragments in a Na(2)S solution. Trichophyton spp. as well as 12-non dermatophyte fungi (NDF) could be unambiguously identified by the specific restriction fragment size of 5′ -end labelled amplified 28S DNA. This assay enables the distinction of different fungal infectious agents and their identification in mixed infections. Infectious agents could be identified in 74% (162/219) of cases in which the culture results were negative. The PCR-TRFLP assay described herein is simple and reliable. Furthermore it has the possibility to be automated and thus routinely applied for the rapid diagnosis of a large number of clinical specimens in dermatology laboratories.
- PMID: 22170903 [PubMed - as supplied by publisher]