Molecular characterization of selected dermatophytes and their identification by electrophoretic mutation scanning

Abstract

Dermatophytes are fungi that can be contagious and cause infections in the keratinized skin of mammals, including humans. The etiological diagnosis of dermatophytosis relies on a combination of in vitro-culture and microscopic methods. Effective molecular tools could overcome the limitations of conventional methods of identification. In the present study, following phenetic identification as M. canis, M. fulvum, M. gypseum, T. mentagrophytes and T. terrestre, we genetically characterized key dermatophytes, employing the sequences of the first and second internal transcribed spacers of nuclear ribosomal DNA as well as part of the chitin synthase-1 gene, and assessed the utility of these DNA regions (based on levels of nucleotide variation within and among species/taxa) as markers for the classification of species and genotypes. Employing partial chitin synthase-1 gene as the marker, we also established a PCR-coupled SSCP approach as a diagnostic/analytical mutation-scanning tool. This tool should facilitate fundamental investigations of the ecology, epidemiology and population genetics of dermatophytes and, importantly, should assist in allowing a more rapid diagnosis of dermatophytoses in humans and other animals, thus overcoming the significant delays in targeted chemotherapy following diagnosis using conventional methods. (Nucleotide sequence data reported in this paper are available in the EMBL, GenBank and DDJB datadases under accession numbers FJ897707-FJ897713 (ITS-1), FJ897714-FJ897720 (ITS-2) and FJ897700-FJ897706 (pchs-1)).

Use of Restriction Fragment Length Polymorphism to Rapidly Identify Dermatophyte Species Related to Dermatophytosis

“…Recently, several PCR based techniques like arbitrarily primed PCR (AP-PCR) ( 15 ), random amplified polymorphic DNA (RAPD) ( 16 , 17 ), repetitive sequence PCR (rep-PCR) ( 18 ), restriction analysis of the mitochondrial DNA ( 19 , 20 ), semi-nested PCR ( 21 ), nested PCR ( 22 ), multiplex PCR ( 23 ) and single-strand conformation polymorphism (SSCP) analysis ( 24 ) are the available techniques for identification of dermatophytes. However, few methods reported a low sensitivity and specificity in identification of dermatophyte species.…”

An Over View of Canine Dermatophytosis

“…The first and second internal transcribed spacers (ITS-1 and ITS-2, respectively) of nuclear ribosomal DNA and a part of the chitin synthase gene (designated p chs-1) have showed particular promise as markers for the specific identification of dermatophytes [52]. Specific primer pairs were designed for the selective amplification of ITS _ ( _ ITS-1 _ 5.8S rRNA gene _ ITS-2) or p chs-1 of dermatophyte species (i.e., M. canis, M. gypseum, T. terrestre and T. mentagrophytes ) which are frequently isolated from the hair of animals , but not of other fungi associated with the coats [53]. One-step and nested PCRs used for identification of canine dermatophytes gave positive result for dermatophytes culture but were negative for other fungi isolates as shown in (Fig.…”

Non-culture based assays for the detection of fungal pathogens

“…These include fingerprinting methods, conventional PCR (employing selected genetic markers e.g. internal transcribed spacers (ITS), chitin synthase 1 gene (chs1), topoisomerase II gene, small and large ribosomal RNA subunit and non-transcribed spacer/NTS region), ITS restriction fragment length polymorphism (RFLP) and total mitochondrial DNA (mtDNA) analyses which change the view to the taxonomy of dermatophytes [29][30][31][32]. Molecular diagnosis provides specieslevel identification of dermatophytes more accurately regarding their development in human, soil or animals.…”