, Volume 182, Issue 1–2, pp 57–65 | Cite as

Molecular Markers Useful for Intraspecies Subtyping and Strain Differentiation of Dermatophytes

  • Takashi Mochizuki
  • Kiminobu Takeda
  • Kazushi Anzawa


Dermatophytosis is a very common skin disorder and the most frequent infection encountered by practicing dermatologists. The identification, pathogenicity, biology, and epidemiology of dermatophytes, the causative agents of dermatophytosis, are of interest for both dermatologists and medical mycologists. Recent advances in molecular methods have provided new techniques for identifying dermatophytes, including intraspecies variations. Intraspecies subtyping and strain differentiation have made possible the tracking of infections, the identification of common sources of infections, recurrence or reinfection after treatment, and analysis of strain virulence and drug resistance. This review describes molecular methods of intraspecies subtyping and strain differentiation, including analyses of mitochondrial DNA and non-transcribed spacer regions of ribosomal RNA genes, random amplification of polymorphic DNA, and microsatellite markers, along with their advantages and limitations.


Trichophyton Epidemiology Dermatophytosis Molecular marker Molecular epidemiology 



This publication and cited research is partially supported by the research program on emerging and re-emerging infectious diseases from Japan Agency for Medical Research and Development, AMED.

Compliance with Ethical Standards

Conflict of interest

The authors declare there are no conflicts of interest including industrial links or affiliations.


  1. 1.
    Watanabe S, Harada T, Hiruma M, et al. Epidemiological survey of foot diseases in Japan: results of 30,000 foot checks by dermatologists. J Dermatol. 2010;37:397–406.CrossRefPubMedGoogle Scholar
  2. 2.
    Davison FD, Mackenzie DWR. DNA homology studies in the taxonomy of dermatophytes. Sabouraudia. 1984;22:117–23.CrossRefPubMedGoogle Scholar
  3. 3.
    de Bièvre C, Dauguet C, Nguyen VH, Ibrahim-Granet O. Polymorphism in mitochondrial DNA of several Trichophyton rubrum isolates from clinical specimens. Ann Inst Pasteur Microbiol. 1987;138:719–27.CrossRefPubMedGoogle Scholar
  4. 4.
    Kawasaki M, Ishizaki H, Aoki M, Watanabe S. Phylogeny of Nannizzia incurvata, N. gypsea, N. fulva and N. otae by restriction enzyme analysis of mitochondrial DNA analysis. Mycopathologia. 1990;112:173–7.CrossRefPubMedGoogle Scholar
  5. 5.
    Nishio K, Kawasaki M, Ishizaki H. Phylogeny of the genera Trichophyton using mitochondrial DNA analysis. Mycopathologia. 1992;117:127–32.CrossRefPubMedGoogle Scholar
  6. 6.
    Mochizuki T, Sugie N, Uehara M. Random amplification of polymorphic DNA is useful for the differentiation of several anthropophilic dermatophytes. Mycoses. 1997;40:405–9.CrossRefPubMedGoogle Scholar
  7. 7.
    Kim JA, Takahashi Y, Tanaka R, Fukushima K, Nishimura K, Miyaji M. Identification and subtyping of Trichophyton mentagrophytes by random amplification of polymorphic DNA. Mycoses. 2001;44:157–65.CrossRefPubMedGoogle Scholar
  8. 8.
    Makimura K, Mochizuki T, Hasegawa A, Uchida H, Saito H, Yamaguchi H. Phylogenetic classification of Trichophyton mentagrophytes complex strains based on DNA sequences of nuclear ribosomal internal transcribed spacer 1 regions. J Clin Microbiol. 1998;36:2629–33.PubMedPubMedCentralGoogle Scholar
  9. 9.
    Makimura K, Tamura Y, Mochizuki T, et al. Phylogenetic classification and species identification of dermatophyte strains based on DNA sequence of nuclear ribosomal internal transcribed spacer 1 regions. J Clin Microbiol. 1999;37:920–4.PubMedPubMedCentralGoogle Scholar
  10. 10.
    Kano R, Nakamura Y, Watari T, et al. Species-specific primers of chitin synthase 1 gene for the differentiation of the Trichophyton mentagrophytes complex. Mycoses. 1999;42:71–4.CrossRefPubMedGoogle Scholar
  11. 11.
    Kanbe T, Suzuki Y, Kamiya A, et al. Species-identification of dermatophytes Trichophyton, Microsporum and Epidermophyton by PCR and PCR-RFLP targeting of the DNA topoisomerase II genes. J Dermatol Sci. 2003;33:41–54.CrossRefPubMedGoogle Scholar
  12. 12.
    Sun PL, Hsieh HM, Ju YM, Jee SH. Molecular characterization of dermatophytes of the Trichophyton mentagrophytes complex found in Taiwan with emphasis on their correlation with clinical observations. Br J Dermatol. 2010;163:1312–8.CrossRefPubMedGoogle Scholar
  13. 13.
    Rezaei-Matehkolaei A, Mirhendi H, Makimura K, et al. Nucleotide sequence analysis of beta tubulin gene in a wide range of dermatophytes. Med Mycol. 2014;52:674–88.CrossRefPubMedGoogle Scholar
  14. 14.
    Gräser Y, Scott J, Summerbell R. The new species concept in dermatophytes—a polyphasic approach. Mycopathologia. 2008;166:239–56.CrossRefPubMedGoogle Scholar
  15. 15.
    Cafarchia C, Iatta R, Latrofa MS, Gräser Y, Otranto D. Molecular epidemiology, phylogeny and evolution of dermatophytes. Infect Genet Evol. 2013;20:336–51.CrossRefPubMedGoogle Scholar
  16. 16.
    Abdel-Rahman SM. Strain differentiation of dermatophytes. Mycopathologia. 2008;166:319–33.CrossRefPubMedGoogle Scholar
  17. 17.
    Kanbe T. Molecular approaches in the diagnosis of dermatophytosis. Mycopathologia. 2008;166:307–17.CrossRefPubMedGoogle Scholar
  18. 18.
    Gräser Y, Kuhnisch J, Presber W. Molecular markers reveal exclusively clonal reproduction in Trichophyton rubrum. J Clin Microbiol. 1999;37:3713–7.PubMedPubMedCentralGoogle Scholar
  19. 19.
    Kac G. Molecular approaches to the study of dermatophytes. Med Mycol. 2000;38:329–36.CrossRefPubMedGoogle Scholar
  20. 20.
    Wu Y, Yang J, Yang F, et al. Recent dermatophyte divergence revealed by comparative and phylogenetic analysis of mitochondrial genomes. BMC Genomics. 2009;10:238.CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Williams JGK, Kubelik AR, Livak KJ, Rafalski JA, Tingey SV. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acid Res. 1990;18:6531–5.CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Weising K, Nybom H, Wolff K, Meyer W. Chapter 4 Methodology, IV. PCR based DNA fingerprinting. DNA fingerprinting in plants and fungi. Boca Raton: CRC Press; 1995. p. 112–36.Google Scholar
  23. 23.
    Baeza LC, Matsumoto MT, Almeida AMF, Mendes-Giannini MJS. Strain differentiation of Trichophyton rubrum by randomly amplified polymorphic DNA and analysis of rDNA nontranscribed spacer. J Med Microbiol. 2006;55:429–36.CrossRefPubMedGoogle Scholar
  24. 24.
    Santos DA, Araujo RA, Hamdan JS, Cisalpino PS. Trichophyton rubrum and Trichophyton interdigitale: genetic diversity among species and strains by random amplified polymorphic DNA method. Mycopathologia. 2010;169:247–55.CrossRefPubMedGoogle Scholar
  25. 25.
    Liu D, Coloe S, Baird R, Pederson J. Application of PCR to the identification of dermatophyte fungi. J Med Microbiol. 2000;49:493–7.CrossRefPubMedGoogle Scholar
  26. 26.
    Irinyi L, Serena C, Garcia-Hermoso D, et al. International society of human and animal mycology (ISHAM)-ITS reference DNA barcoding database-the quality controlled standard tool for routine identification of human and animal pathogenic fungi. Med Mycol. 2015;53:313–37.CrossRefPubMedGoogle Scholar
  27. 27.
    Cafarchia C, Otranto D, Weigl S, et al. Molecular characterization of selected dermatophytes and their identification by electrophoretic mutation scanning. Electrophoresis. 2009;30:3555–64.CrossRefPubMedGoogle Scholar
  28. 28.
    Symoens F, Jousson O, Planard C, et al. Molecular analysis and mating behaviour of the Trichophyton mentagrophytes species complex. Int J Med Microbiol. 2011;301:260–6.CrossRefPubMedGoogle Scholar
  29. 29.
    Mochizuki T, Tanabe H, Kawasaki M, Ishizaki H, Jackson CJ. Rapid identification of Trichophyton tonsurans by PCR-RFLP analysis of ribosomal DNA regions. J Dermatol Sci. 2003;32:25–32.CrossRefPubMedGoogle Scholar
  30. 30.
    Jackson CJ, Barton RC, Evans EGV. Species identification and strain differentiation of dermatophyte fungi by analysis of ribosomal DNA spacer regions. J Clin Microbiol. 1999;37:931–6.PubMedPubMedCentralGoogle Scholar
  31. 31.
    Jackson CJ, Barton RC, Kelly SL, Evans EGV. Strain identification of Trichophyton rubrum by specific amplification of subrepeat elements in the ribosomal DNA nontranscribed spacer. J Clin Microbiol. 2000;38:4527–34.PubMedPubMedCentralGoogle Scholar
  32. 32.
    Gaedigk A, Gaedigk R, Adbel-Rahman SM. Genetic heterogeneity in the rRNA gene locus of Trichophyton tonsurans. J Clin Microbiol. 2003;41:5478–87.CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Mochizuki T, Ishizaki H, Barton RC, et al. Restriction fragment length polymorphism analysis of ribosomal DNA intergenic regions is useful for differentiating strains of Trichophyton mentagrophytes. J Clin Microbiol. 2003;41:4583–8.CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Mochizuki T, Kawasaki M, Ishizaki H, et al. Molecular epidemiology of Arthroderma benhamiae, an emerging pathogen of dermatophytoses in Japan, by polymorphisms of the non-transcribed spacer region of the ribosomal DNA. J Dermatol Sci. 2001;27:14–20.CrossRefPubMedGoogle Scholar
  35. 35.
    Mochizuki T, Watanabe S, Kawasaki M, Tanabe H, Ishizaki H. A Japanese case of tinea corporis caused by Arthroderma benhamiae. J Dermatol. 2002;29:221–5.CrossRefPubMedGoogle Scholar
  36. 36.
    Yazdanparast A, Jackson CJ, Barton RC, Evans EVG. Molecular typing of Trichophyton rubrum indicates multiple strain involvement in onychomycosis. Br J Dermatol. 2003;148:51–4.CrossRefPubMedGoogle Scholar
  37. 37.
    Gupta AK, Nakrieko KA. Trichophyton rubrum DNA strain switching increases in patients with onychomycosis failing antifungal treatments. Br J Dermatol. 2015;172:74–80.CrossRefPubMedGoogle Scholar
  38. 38.
    Takeda K, Mochizuki H, Izumi K, et al. Polyclonality of Trichophyton rubrum isolates in a dermatophytosis patient with multiple lesions. Med Mycol J. 2016;57E:E17–20.CrossRefGoogle Scholar
  39. 39.
    Jackson CJ, Mochizuki T, Barton RC. PCR fingerprinting of Trichophyton mentagrophytes var. interdigitale using polymorphic subrepeat loci in the rDNA nontranscribed spacer. J Med Microbiol. 2006;55:1349–55.CrossRefPubMedGoogle Scholar
  40. 40.
    Wakasa A, Anzawa K, Kawasaki M, Mochizuki T. Molecular typing of Trichophyton mentagrophytes var. interdigitale isolated in a university hospital in Japan based on the non-transcribed spacer region of the ribosomal RNA gene. J Dermatol. 2010;37:431–40.CrossRefPubMedGoogle Scholar
  41. 41.
    Takeda K, Nishibu A, Anzawa K, Mochizuki T. Molecular epidemiology of a major subgroup of Arthroderma benhamiae isolated in Japan by restriction fragment length polymorphism analysis of the non-transcribed spacer region of ribosomal RNA gene. Jpn J Infect Dis. 2012;65:233–9.CrossRefPubMedGoogle Scholar
  42. 42.
    Abliz P, Takizawa K, Nishimura K, Fukushima K. Molecular typing of Trichophyton tonsurans by PCR–RFLP of the ribosomal DNA nontranscribed spacer region. J Dermatol Sci. 2004;36:125–7.CrossRefPubMedGoogle Scholar
  43. 43.
    Mochizuki T, Kawasaki M, Tanabe H, Anzawa K, Ishizaki H, Choi JS. Molecular epidemiology of Trichophyton tonsurans isolated in Japan using RFLP analysis of non-transcribed spacer regions of ribosomal RNA genes. Jpn J Infect Dis. 2007;60:188–92.PubMedGoogle Scholar
  44. 44.
    Sugita T, Shiraki Y, Hiruma M. Genotype analysis of the variable internal repeat region in the rRNA gene of Trichophyton tonsurans isolated from Japanese judo practitioners. Microbiol Immunol. 2006;50:57–60.CrossRefPubMedGoogle Scholar
  45. 45.
    Anzawa K, Mochizuki T, Nishibu A, et al. Molecular epidemiology of Trichophyton tonsurans isolated in Japan between 2006 and 2010 and their susceptibility to oral antimycotics. Jpn J Infect Dis. 2011;64:458–62.PubMedGoogle Scholar
  46. 46.
    Adbel-Rahman SM, Preuett B, Gaedigk A. Multilocus genotyping identifies infections by multiple strains of Trichophyton tonsurans. J Clin Micobiol. 2007;45:1949–53.CrossRefGoogle Scholar
  47. 47.
    Adbel-Rahman SM, Sugita T, Gonzalez GM, et al. Divergence among an international population of Trichophyton tonsurans isolates. Mycopathologia. 2010;169:1–13.CrossRefGoogle Scholar
  48. 48.
    Adbel-Rahman SM, Simon S, Wright KJ, Ndjountche L, Gaedigk A. Tracking Trichophyton tonsurans through a large urban child care center: defining infection prevalence and transmission patterns by molecular strain typing. Pediatrics. 2006;118:2365–73.CrossRefGoogle Scholar
  49. 49.
    Pasquetti M, Peano A, Soglia D, et al. Development and validation of microsatellite marker-based method for tracing infections by Microsporum canis. J Dermatol Sci. 2013;70:123–9.CrossRefPubMedGoogle Scholar
  50. 50.
    Ohst T, de Hoog GS, Presber W, Stavrakieva V, Gräser Y. Origins of microsatellite diversity in the Trichophyton rubrum-T. violaceum clade (dermatophytes). J Clin Microbiol. 2004;42:4444–8.CrossRefPubMedPubMedCentralGoogle Scholar
  51. 51.
    Gräser Y, Fröhlich J, Presber W, de Hoog GS. Microsatellite markers reveal geographic population differentiation in Trichophyton rubrum. J Med Microbiol. 2007;56:1058–65.CrossRefPubMedGoogle Scholar
  52. 52.
    Sharma R, Presber W, Rajak RC, Gräser Y. Molecular detection of Microsporum persicolor in soil suggesting widespread dispersal in central India. Med Mycol. 2008;46:67–73.CrossRefPubMedGoogle Scholar
  53. 53.
    Sharma R, de Hoog GS, Presber W, Gräser Y. A virulent genotype of Microsporum canis is responsible for the majority of human infections. J Med Microbiol. 2007;56:1377–85.CrossRefPubMedGoogle Scholar
  54. 54.
    da Costa FV, Farias MR, Bier D, et al. Genetic variability in Microsporum canis isolated from cats, dogs and humans in Brazil. Mycoses. 2013;56:582–8.CrossRefPubMedGoogle Scholar
  55. 55.
    Spesso MF, Nuncira CT, Burstein VL, Masih DT, Did MD, Chiapello LS. Microsatellite-primed PCR and random primer amplification polymorphic DNA for the identification and epidemiology of dermatophytes. Eur J Clin Microbiol Infect Dis. 2013;32:1009–15.CrossRefPubMedGoogle Scholar
  56. 56.
    Dobrowolska A, Debska J, Kozlowska M, Staczek P. Strains differentiation of Microsporum canis by RAPD analysis using (GACA)4 and (ACA)5 primers. Pol J Microbiol. 2011;60:145–8.PubMedGoogle Scholar
  57. 57.
    Martinez DA, Oliver BG, Gräser Y, et al. Comparative genome analysis of Trichophyton rubrum and related dermatophytes reveals candidate genes involved in infection. MBio. 2012;3:e00259-12.CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2016

Authors and Affiliations

  • Takashi Mochizuki
    • 1
  • Kiminobu Takeda
    • 1
  • Kazushi Anzawa
    • 1
  1. 1.Department of DermatologyKanazawa Medical UniversityUchinadaJapan

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