Index > Vol. 86/2006 > Iss. 3/May > pp. 223-226 > Abstract
Investigative Report | Hair, Microbiology

18S rDNA Polymerase Chain Reaction and Sequencing in Onychomycosis Diagnostics

doi: 10.2340/00015555-0077

Abstract:

Diagnostic approaches to onychomycosis have traditionally been based on a combination of culture and microscopy. In the present study clinical specimens from 346 patients with suspected onychomycosis were analy­sed by 18S polymerase chain reaction (detection) followed by sequencing and subsequent database search (identification) in parallel with routine culture on agar (detection and identification). In 49 samples Trichophyton rubrum was identified by culture and sequencing. In 67 additional culture negative samples, a positive dermatophyte sequence was obtained (T. rubrum in 54, T. mentagrophytes in 5, and T. species in 8 samples). Fifteen samples cultured positive while no sequence was obtained. Two hundred and seven samples were negative by culture as well as by sequencing. Nails from 10 healthy controls were negative by culture and sequencing. In conclusion, the number of specimens that were positive by polymerase chain reaction was more than double the number that were positive by culture alone.

Authors:

Mette Walberg, Cato Mørk, Per Sandven, Anne Tomine Jorde, Magnar Bjørås and Peter Gaustad

Key words:

onychomycosis, Trichophyton rubrum, nails.

References

  1. Roberts DT. Prevalence of dermatophyte onychomycosis in the United Kingdom: results of an omnibus survey. Br J Dermatol 1992; 126 (suppl 39): 23–27. 
.
  2. Heikkila H, Stubb S. The prevalence of onychomycosis in Finland. Br J Dermatol 1995; 133: 699–703.
  3. Evans EG. Causative pathogens in onychomycosis and the possibility of treatment resistance: a review. J Am Acad Dermatol 1998; 38: 32–36.
  4. Verweij PE, Klont RR, Donnelly JP. Validating PCR for detecting invasive aspergillosis. Br J Haematol 2004; 125: 196–202. Link to article
  5. Kamiya A, Kikuchi A, Tomita Y, Kanbe T. PCR and PCR-RFLP techniques targeting the DNA topoisomerase II gene for rapid clinical diagnosis of the etiologic agent of dermatophytosis. J Dermatol Sci 2004; 34: 35–48. Link to article
  6. Kanbe T, Suzuki Y, Kamiya A, Mochizuki T, Fujihiro M, Kikuchi A. PCR-based identification of common dermatophyte species using primer sets specific for the DNA 
topoisomerase II gene. J Dermatol Sci 2003; 32: 151–161. Link to article
  7. Kanbe T, Suzuki Y, Kamiya A, Mochizuki T, Kawasaki M, Fujihiro M, Kikuchi A. 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. 
. Link to article
  8. Kano R, Hirai A, Muramatsu M, Watari T, Hasegawa A. Direct detection of dermatophytes in skin samples based on sequences of the chitin synthase 1 (CHS1) gene. J Vet Med Sci 2003; 65: 267–270. Link to article
  9. Makimura K, Tamura Y, Mochzuki T, Hasegawa A, Tajiri Y, Hanazaki R, et al. Phylogenetic classification and species identification of dermatophyte strains based on DNA sequences of nuclear ribosomal internal transcribed spacer 1 regions. J Clin Microbiol 1999; 37: 920–924.
  10. Harmsen D, Schwinn A, Brocker EB, Frosch M. Molecular differentiation of dermatophyte fungi. 1999; 42: 67–70.
  11. El Fari M, Tietz HJ, Presber W, Sterry W, Graser Y. Development of an oligonucleotide probe specific for Trichophyton rubrum. Br J Dermatol 1999; 141: 240–245. Link to article
  12. Graser Y, El Fari M, Vilgalys R, Kuijpers AF, De Hoog GS, Presber W, et al. Phylogeny and taxonomy of the family Arthrodermataceae (dermatophytes) using sequence analysis of the ribosomal ITS region. Med Mycol 1999; 37: 105–114. 
.
  13. Hirai A, Kano R, Nakamura Y, Watanabe S, Hasegawa A. Molecular taxonomy of dermatophytes and related fungi by chitin synthase 1 (CHS1) gene sequences. Antonie Van Leeuwenhoek 2003; 83: 11–20. Link to article
  14. Bock M, Maywald M, Kappe R, Nickel P, Naher H. Poly­merase chain reaction-based detection of dermatophyte DNA with a fungus-specific primer system. Mycoses 1994; 37: 79–84.
  15. Kappe R, Okeke CN, Fauser C, Maiwald M, Sonntag HG. Molecular probes for the detection of pathogenic fungi in the presence of human tissue. J Med Microbiol 1998; 47: 811–820.
  16. Baek SC, Chae HJ, Houh D, Byun DG, Cho BK. Detection and differentiation of causative fungi of onychomycosis using PCR amplification and restriction enzyme analysis. Int J Dermatol 1998; 37: 682–686.
  17. Turin L, Riva F, Galbiati G, Cainelli T. Fast, simple and highly sensitive double-rounded polymerase chain reaction assay to detect medically relevant fungi in dermatological specimens. Eur J Clin Invest 2000; 30: 511–518. 
. Link to article
  18. Arca E, Saracli MA, Akar A, Yildiran ST, Kurumlu Z, Gur AR. Polymerase chain reaction in the diagnosis of onychomycosis. Eur J Dermatol 2004; 14: 52–55. 
.
  19. Ninet B, Jan I, Bontems O, Lechenne B, Jousson O, 
Panizzon R, et al. Identification of dermatophyte species by 28S ribosomal DNA sequencing with a commercial kit. J Clin Microbiol 2003; 41 826–830.
  20. Graser Y, El Fari M, Presber W, Sterry W, Tietz HJ. Identification of common dermatophytes (Trichophyton, Microsporum, Epidermophyton) using polymerase chain reaction. Br J Dermatol 1998; 138 (4): 576–582.
  21. Elias Costa MR, Da Silva Lacaz C, Kawasaki M, De 
Camargo ZP. Conventional versus molecular diagnostic tests. Med Mycol 2000; 38 (suppl 1): 139–145.
  22. Faggi E, Pini G, Campsi E, Bertellini C, Difonzo E, Mancianti F. Application of PCR to distinguish common species of dermatophytes. J Clin Microbiol 2001; 39: 3382–3385. Link to article
  23. Jackson CJ. Molecular identification and strain typing of dermatophyte fungi. Nippon Ishinkin Gakkai Xasshi 2001; 42: 7–10.
  24. Shin JH, Sung JH, Park SJ, Kim JA, Lee JH, Lee DY, et al. Species identification and stain differentiation of dermatophyte fungi using polymerase chain reaction amplification and restriction enzyme analysis. J Am Acad Sci 2003; 48: 857–865.
  25. Okeke CN, Tsuboi R, Kawai M, Hiruma M, Ogawa H. Isolation of an intron-containing sequence of the gene encoding dermatophyte actin (ACT) and detection of a fragment of the transcript by reverse transcription-nested PCR as a means of assessing the viability of dermatophytes in skin scales. J Clin Microbiol 2001; 39: 101–106. Link to article
  26. Kawai M. Diagnosis of dermatophytoses: conventional methods and molecular biology methods. Nippon Ishinkin Gakkai Zasshi 2003; 44: 261–264. 
.
  27. Larone DH. Medically important fungi – a guide to identification 4th edn. Washington DC: American Society of Microbiology, 2002.
  28. Sambrook J, Fritsch E, Maniatis T. Molecular cloning: A laboratory manual 2nd edn. NY: Cold Spring Harbor, 1989.
  29. Kano R, Nakamura Y, Watari T, Watanabe S, Takahashi H, Tsujimoto H, et al. Phylogenetic analysis of 8 dermatophyte species using chitin synthase 1 gene sequences. Mycoses 1997; 40: 411–414. Link to article
  30. Lefler E, Haim S, Merzbach D. Evaluation of direct 
microscopic examination versus culture in the diagnosis of superficial fungal infection. Mykosen 1981; 24: 102–106.