Rapid discrimination among dermatophytes, Scytalidium spp., and other fungi with a PCR-restriction fragment length polymorphism ribotyping method

Dermatomycoses are very common infections caused mainly by dermatophytes. S cytalidiosis is a differential mycological diagnosis, especially in tropica l and subtropical areas. Since a culture-based diagnosis takes 2 to 3 weeks , we set up a PCR-restriction fragment length polymorphism (RFLP) method fo r rapid discrimination of these fungi in clinical samples. The hypervariabl e V4 domain of the small ribosomal subunit 18S gene was chosen as the targe t for PCR. The corresponding sequences from 19 fungal species (9 dermatophy tes, 2 Scytalidium species, 6 other filamentous fungi, and 2 yeasts),were o btained from databases or were determined in the laboratory. Sequences were aligned to design primers for dermatophyte-specific PCR and to identify di gestion sites for RFLP analysis, The reliability of PCR-RFLP for the diagno sis of dermatomycosis was assessed on fungal cultures and on specimens from patients with suspected dermatomycosis. Two sets of primers preferentially amplified fungal DNA from dermatophytes (DH1L and DH1R) or from Scytalidiu m spp. (DH2L and DH1R) relative to DNA from bacteria, yeasts, some other fi lamentous fungi, and humans. Digestion of PCR products with EaeI or BamHI d iscriminated between dermatophytes and Scytalidium species, as shown with c ultures of 31 different fungal species. When clinical samples were tested b y PCR-RFLP, blindly to mycological findings, the results of the two methods agreed for 74 of 75 samples. Dermatophytes and Scytalidium spp. can thus b e readily discriminated by PCR-RFLP within 24 h. This method can be applied to clinical samples and is suited to rapid etiologic diagnosis and treatme nt selection for patients with dermato mycosis.