Identification of Dermatophyte Species by 28S Ribosomal DNA Sequencing with a Commercial Kit

Abstract

We have shown that dermatophyte species can be easily identified on the basis of a DNA sequence encoding a part of the large-subunit (LSU) rRNA (28S rRNA) by using the MicroSeq D2 LSU rRNA Fungal Sequencing Kit. Two taxa causing distinct dermatophytoses were clearly distinguished among isolates of the Trichophyton mentagrophytes species complex.Dermatophytes are the main cause of superficial mycoses (9,15,16). These fungi have the capacity to invade keratinized tissue of humans or animals to produce infections that are generally restricted to the corneocytes of the skin, hair, and nails. Among the approximately 10 species isolated in Europe, Trichophyton rubrum and T. mentagrophytes are the most commonly observed, with frequencies varying from 27 to 74% and from 17 to 41%, respectively (13).Dermatophytes are usually identified on the basis of macroscopic appearance, together with microscopic examination of cultures. Important characteristics are the rate of growth, the shape and texture of the culture on solid media, color, diffusion of pigments into agar, and sporulation. However, identification of dermatophytes often remains difficult or uncertain because there are variations from one isolate to another. Recent advances in molecular biology and progress in technology have allowed the development of new techniques for species determination and strain typing in microbiology. The molecular approach used to identify fungi is often based on sequence analysis of the ribosomal DNA (rDNA) and in particular on the internal transcribed spacer (ITS). The polymorphism of the ITS1 and ITS2 regions flanking the DNA sequence encoding the 5.8S rRNA was previously shown to be suitable for the identification of clinically important yeasts (1), Aspergillus sp. (8),and dermatophyte species (3-5). In contrast, the gene coding for the small-subunit rRNA (18S rRNA) did not discriminate sufficiently between dermatophyte species (7).The MicroSeq D2 large-subunit (LSU) rRNA Fungal Sequencing Kit (Applied Biosystems, Rotkreuz, Switzerland) was recently developed to identify fungal species after amplification of a partial sequence of the DNA encoding the LSU rRNA (28S rDNA). The sequence of a given fungus can then be compared for identification with the rDNA sequences of the MicroSeq D2 Fungal database, which includes more than 500 validated sequences from different fungal species but not from dermatophytes. In the present study, we tested the MicroSeq D2 LSU rRNA Fungal Sequencing Kit by using it to identify dermatophyte species from patients referred to the mycological laboratory of the Department of Dermatology at the University Hospital in Lausanne, Switzerland (Table 1). Neotypes of different species and other reference strains (Table 2) were used for comparison of DNA sequences. This study allows the extension of the MicroSeq D2 Fungal database to the determination of dermatophytes.Isolates. Skin and nail scrapings and hair fragments were collected from patients with suspected mycoses. Routinely, one part of each sample was examine...

Phylogenetic analysis of Trichophyton mentagrophytes human and animal isolates based on MnSOD and ITS sequence comparison

“…They were identified on the basis of microscopy and colony characteristics and were classified using the following criteria: (i) clinical criteria (body localization, associated or not with clinical signs or symptoms of dermatophytosis), (ii) cultural aspects (texture, front and reverse colour of the colonies), and (iii) microscopic features (subspherical or clavate microconidia, macroconidia, spiral hyphae, and dense branchlets at right angles) (Graser et al, 1999b;Mochizuki et al, 1996Mochizuki et al, , 2003Ninet et al, 2003). Morphological data for the isolates, and their origins, are listed in under a laminar-airflow hood to avoid contamination.…”

“…In some cases, morphological identification can be difficult or uncertain because there is considerable variation and pleomorphism among isolates of the same species. In the last decade, genotyping approaches have proven to be useful for solving problems of dermatophyte taxonomy, as well as enhancing the reliability and speed of dermatophytosis diagnosis (De Hoog et al, 1998;Faggi et al, 2001Faggi et al, , 2002Graser et al, 2006;Jousson et al, 2004;Kano et al, 2003;Kardjeva et al, 2006;Liu et al, 2000;Mochizuki et al, 1990Mochizuki et al, , 1999Ninet et al, 2003;Weitzman & Summerbell, 1995).…”

Identification of Dermatophytes by an Oligonucleotide Array

“…These methods include restriction fragment length polymorphism analysis (4,17,22), sequencing of the large-submit rRNA gene (34) and protein-encoding genes (19), gene-specific PCR (16,17), random amplification of polymorphic DNA (20,33), PCR fingerprinting and amplified fragment length polymorphism analysis (12,13), and dot blot hybridization (6). Recently, several molecular studies have focused on the internal transcribed spacer (ITS) region of the rRNA gene.…”

The dermatophyte species Arthroderma benhamiae: intraspecies variability and mating behaviour

“…A. benhamiae isolates are generally recognized by the production of numerous pyriform or round microconidia, a characteristic of species of the T. mentagrophytes complex, and by considering the probable source of the infection, which is generally a guinea pig. They can be identified further by DNA sequence analysis of a 318 bp fragment of the 28S rRNA gene and/or of the internal transcribed spacer (ITS) region (Ninet et al, 2003;Fumeaux et al, 2004). Like other dermatophyte species, A. benhamiae is heterothallic and is able to produce cleistothecia (sexual fructifications) containing asci and ascospores when strains from two different mating types, mating type + (mt+) and mating type 2 (mt2), meet.…”