Species Identification and Strain Differentiation of Dermatophyte Fungi by Analysis of Ribosomal-DNA Intergenic Spacer Regions

Abstract

Restriction fragment length polymorphisms (RFLPs) identified in the ribosomal-DNA (rDNA) repeat were used for molecular strain differentiation of the dermatophyte fungus Trichophyton rubrum. The polymorphisms were detected by hybridization ofEcoRI-digested T. rubrum genomic DNAs with a probe amplified from the small-subunit (18S) rDNA and adjacent internal transcribed spacer (ITS) regions. The rDNA RFLPs mapped to the nontranscribed spacer (NTS) region of the rDNA repeat and appeared similar to those caused by short repetitive sequences in the intergenic spacers of other fungi. Fourteen individual RFLP patterns (DNA types A to N) were recognized among 50 random clinical isolates ofT. rubrum. A majority of strains (19 of 50 [38%]) were characterized by one RFLP pattern (DNA type A), and four types (DNA types A to D) accounted for 78% (39 of 50) of all strains. The remaining types (DNA types E to N) were represented by one or two isolates only. A rapid and simple method was also developed for molecular species identification of dermatophyte fungi. The contiguous ITS and 5.8S rDNA regions were amplified from 17 common dermatophyte species by using the universal primers ITS 1 and ITS 4. Digestion of the amplified ITS products with the restriction endonucleaseMvaI produced unique and easily identifiable fragment patterns for a majority of species. However, some closely related taxon pairs, such as T. rubrum-T. soudanense andT. quinkeanum-T. schoenlenii could not be distinguished. We conclude that RFLP analysis of the NTS and ITS intergenic regions of the rDNA repeat is a valuable technique both for molecular strain differentiation of T. rubrum and for species identification of common dermatophyte fungi.

Evaluation of a single-tube real-time PCR for detection and identification of 11 dermatophyte species in clinical material

“…Previously described PCR assays for the detection of dermatophytes in clinical material are only moderately sensitive or cannot identify dermatophytes to the species level [16][17][18][19][20][21][22][23]. With other PCR-based assays, only one or a few dermatophyte species can be identified, the assays are not used directly on clinical material, or the assays require post-PCR analysis [2,16,[24][25][26][27][28][29]. One report described the detection and identification of dermatophytes in clinical material by 18S rRNA PCR and sequence analysis, which is relatively expensive for routine laboratories [30].…”

Fast and sensitive detection of Trichophyton rubrum DNA from the nail samples of patients with onychomycosis by a double-round polymerase chain reaction-based assay

“…The high variability and species-specific nature of the ITS1 target region allows it to be the tool of choice for PCR-based detection and differentiation of dermatophytes isolated from clinical samples relative to other markers that are less studied, such as the chitin synthase 1 region. [24][25][26][27][28][29][30] Therefore, we compared the actin gene-based PCR assay for detection of T. rubrum from the infected nails with the well-documented ITS1 region-based PCR assay. The PCR assay using actin primers was found to exhibit a significance level of P < 0AE01 relative to the ITS1 primer-based assay.…”

Trichophyton rubrumDNA strain switching increases in patients with onychomycosis failing antifungal treatments

“…Trichophyton rubrum DNA strain type characterization in a subpopulation of patients with onychomycosis T. rubrum DNA strain types were determined by RFLPs in rDNA according to the number of repeat units in the intergenic nontranscribed spacer. 13 Common T. rubrum DNA strain types have three rDNA fragments:~3 kb,~9 kb and a variable~4Á7-5Á8-kb fragment. Potential novel T. rubrum DNA strains produce banding patterns consisting of the common fragments, but with the addition of one or two fragments sized~4Á7-9Á7 kb.…”

“…1). 13 Gr€ aser et al 12 observed that genotypes of foreign origin seemingly disappear in new environments, to be replaced by local presumably fitter genotypes. Overall, taking into account the clonal reproduction of T. rubrum populations evidenced by their genomic stability, 12,[23][24][25] the similarity in prominent DNA strain types between this North American population and the European population 13 suggests that we have likely detected local variations within T. rubrum of the same origin with a history of conditions increasing in virulence across continents.…”

“…1 Genetic strain typing methods have revolutionized epidemiological studies of dermatophyte infections as they are sensitive enough to distinguish a high number of DNA strain types left uncharacterized by morphological identification, which is limited by the cultural stability and differentiation of strains. 2 T. rubrum DNA strain types have been characterized using a number of methods, [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] but no correlation between DNA strain type and culture morphology has been detected, 10,14,19 even after multiple passages over the course of a year. 20 Geographical trends have been found where isolates from Europe have been distinguished from Japanese isolates, 16 African isolates distinguished from non-African isolates, 10,12 and Japanese isolates distinguished from Chinese isolates.…”