title: Fast and reliable PCR/sequencing/RFLP assay for identification of fungi in onychomycoses.
abstract: Fusarium spp. and other non-dermatophyte fungi are repeatedly isolated from abnormal nails. To investigate whether these fungi are the aetiological agents of infection or simply transient contaminants, a PCR/sequencing/RFLP assay was developed for direct and routine identification of the infecting fungi in onychomycosis. Fungal DNA was readily extracted using a commercial kit after dissolving nail fragments in a Na2S solution. Amplification of part of the 28S rDNA by PCR was performed with universal primers and the fungal species were identified by sequencing. The PCR/sequencing results were comparable with microbiological identification from the same nail sample. In addition to dermatophytes, Fusarium spp. and other less frequently isolated non-dermatophyte fungi were identified as single fungal agents in onychomycosis. Moreover, mixed infections were clearly demonstrated in 10% of cases by RFLP analysis of PCR products. Identification of infectious agents could be obtained in 2 days, whilst results from fungal cultures take 1-3 weeks. Rapid and reliable molecular identification of the infectious fungus expedites the choice of appropriate antifungal therapy, thereby improving the cure rate of onychomycosis.
authors.affiliation: Department of Dermatology, Centre Hospitalier Universitaire Vaudois, CH-1011 Lausanne, Switzerland.
authors.forename: Michel
authors.initials: M
authors.lastname: Monod
authors.affiliation: Department of Dermatology, Centre Hospitalier Universitaire Vaudois, CH-1011 Lausanne, Switzerland.
authors.forename: Olympia
authors.initials: O
authors.lastname: Bontems
authors.affiliation: Department of Dermatology, Centre Hospitalier Universitaire Vaudois, CH-1011 Lausanne, Switzerland.
authors.forename: Christophe
authors.initials: C
authors.lastname: Zaugg
authors.affiliation: Department of Dermatology, Centre Hospitalier Universitaire Vaudois, CH-1011 Lausanne, Switzerland.
authors.forename: Barbara
authors.initials: B
authors.lastname: Léchenne
authors.affiliation: Department of Dermatology, Centre Hospitalier Universitaire Vaudois, CH-1011 Lausanne, Switzerland.
authors.forename: Marina
authors.initials: M
authors.lastname: Fratti
authors.affiliation: Department of Dermatology, Centre Hospitalier Universitaire Vaudois, CH-1011 Lausanne, Switzerland.
authors.forename: Renato
authors.initials: R
authors.lastname: Panizzon
mesh_terms: D004271:DNA, Fungal / Q000235:genetics*; D004275:DNA, Ribosomal / Q000235:genetics; D005658:Fungi / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D009262:Nails / Q000382:microbiology; D014009:Onychomycosis / Q000382:microbiology*; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D012339:RNA, Ribosomal, 28S / Q000235:genetics; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA
chemical_list: D004271:DNA, Fungal; D004275:DNA, Ribosomal; D012339:RNA, Ribosomal, 28S