Diagnosis of dermatophytoses: conventional methods and recent molecular biological methods

Abstract

Dermatophytoses such as tinea pedis and tinea unguium are very common diseases in the field of dermatology. The diagnosis of dermatophytoses is usually performed by direct microscopy and culture. The identification of species is based on morphological features of giant culture and slide culture. However, in some cases, it is difficult to identify the species clearly because the culture shows an atypical appearance or is false negative. Therefore, several molecular biological methods have been developed for precise identification of a species. The analysis of patterns of random amplification of polymorphic DNA (RAPD) and restriction fragment length polymorphisms (RFLP) of mitochondrial DNA is useful for identifying isolates which are not clearly identifiable by conventional biological techniques. The phylogenetic analysis of dermatophytes was made by using DNA direct sequencing of nuclear ribosomal internal transcribed spacer 1 (ITS1). Sequence analysis of chitin synthase 1 (CHS 1) is a rapid tool for species level identification. We attempted the identification and viability assessment of dermatophytes based on the quantitative measurement of dermatophyte actin (ACT) mRNA. An internal fragment of the ACT, 725 to 762 bp, was isolated by PCR from the genomic DNA of dermatophytes and sequenced. ACT intron based primers were dermatophyte species-specific and primer pairs crossing the intron were dermatophyte genus-specific. The results indicated that quantification of dermatophyte ACT mRNA correlated with the results of culture and KOH examination. It is important that the identification of dermatophyte be done by combining conventional methods with molecular biological methods. In some cases results of the two methods do not correspond, and is those the fungal species needs to be re-examined.