Ann Dermatol.  2013 Feb;25(1):73-79. 10.5021/ad.2013.25.1.73.

Molecular Biological Identification of Malassezia Yeasts Using Pyrosequencing

• Kim JY
• Hahn HJ
• Choe YB
• Lee YW
• Ahn KJ
• Moon KC

• KMID: 2265974
• DOI: http://doi.org/10.5021/ad.2013.25.1.73

Abstract

BACKGROUND
A Pyrosequencing assay has been used in identification of fungal species such as Candida or Aspergillus and diagnosis of pathogenic bacteria such as Helicobacter pylori but there has been no report on successful isolation and identification of Malassezia yeasts using the pyrosequencing method.
OBJECTIVE
Examine the applicability and plausibility of the pyrosequencing method in identification of the Malassezia species.
METHODS
At internal transcribed spacer (ITS) sites 1 and 2, three primers were developed using Pyrosequencing Assay Design Software (Biotage AB). Pyrosequencing was performed on 11 standard strains and 83 genomic DNA samples obtained from 66 healthy controls aged from 1 to 80.
RESULTS
The eleven Malassezia standard species and 83 genomic DNA samples were successfully identified using the pyrosequencing assay.
CONCLUSION
The pyrosequencing method is a new tool for analysis of Malassezia yeasts, and its precision and rapidity suggests its clinical applicability.

Keyword

Malassezia yeasts; Pyrosequencing assay

MeSH Terms

Aged
Aspergillus
Bacteria
Candida
DNA
Helicobacter pylori
Humans
Malassezia
Yeasts
DNA

Figure

• 

  Fig. 1 Polymerase chain reaction (PCR) amplification with primer of internal transcribed spacer (ITS) 1~2 from 11 Malassezia standard strains. Lanes: M, molecular marker; 1. M. dermatis (JCM 11348); 2. M. furfur (KCTC 7743); 3. M. globosa (CBS 7966); 4. M. japonica (CBS 9432); 5. M. nana (JCM 12085); 6. M. obtusa (KCTC 7847); 7. M. pachydermatis (KCTC 17008); 8. M. restricta (KCTC 7848); 9. M. slooffiae (KCTC 17431); 10. M. sympodialis (KCTC 7985); 11. M. yamatoensis (CBS 9725).

Reference

1. Janik MP, Heffernan MP. Wolff K, Goldsmith LA, Katz SI, Gilchrest BA, Paller AS, Leffell DJ, editors. Yeast infections: Candidiasis, pityriasis (tinea) versicolor. Fitzpatrick's dermatology in general medicine. 2008. 7th ed. New York: McGraw-Hill;1822–1830.

2. Ahn KJ. Taxonomy of the genus malassezia. Korean J Med Mycol. 1998. 3:81–88.

3. Ljubojević S, Skerlev M, Lipozencić J, Basta-Juzbasić A. The role of Malassezia furfur in dermatology. Clin Dermatol. 2002. 20:179–182.

4. Kanda N, Tani K, Enomoto U, Nakai K, Watanabe S. The skin fungus-induced Th1- and Th2-related cytokine, chemokine and prostaglandin E2 production in peripheral blood mononuclear cells from patients with atopic dermatitis and psoriasis vulgaris. Clin Exp Allergy. 2002. 32:1243–1250.

5. Ginarte M, Fabeiro JM, Toribio J. Confluent and reticulated papillomatosis (Gougerot-Carteaud) successfully treated with tacalcitol. J Dermatolog Treat. 2002. 13:27–30.

6. Yesudian P, Kamalam S, Razack A. Confluent and reticulated papillomatosis (Gougerot-Carteaud). An abnormal host reaction to Malassezzia furfur. Acta Derm Venereol. 1973. 53:381–384.

7. Chowdhary A, Randhawa HS, Sharma S, Brandt ME, Kumar S. Malassezia furfur in a case of onychomycosis: colonizer or etiologic agent? Med Mycol. 2005. 43:87–90.

8. Gupta AK, Batra R, Bluhm R, Boekhout T, Dawson TL Jr. Skin diseases associated with Malassezia species. J Am Acad Dermatol. 2004. 51:785–798.

9. Devlin RK. Invasive fungal infections caused by Candida and Malassezia species in the neonatal intensive care unit. Adv Neonatal Care. 2006. 6:68–77.

10. Curvale-Fauchet N, Botterel F, Legrand P, Guillot J, Bretagne S. Frequency of intravascular catheter colonization by Malassezia spp. in adult patients. Mycoses. 2004. 47:491–494.

11. Senczek D, Siesenop U, Böhm KH. Characterization of Malassezia species by means of phenotypic characteristics and detection of electrophoretic karyotypes by pulsed-field gel electrophoresis (PFGE). Mycoses. 1999. 42:409–414.

12. Lee YW, Lim SH, Ahn KJ. The application of 26S rDNA PCR-RFLP in the identification and classification of Malassezia yeast. Korean J Med Mycol. 2006. 11:141–153.

13. Gharizadeh B, Norberg E, Löffler J, Jalal S, Tollemar J, Einsele H, et al. Identification of medically important fungi by the Pyrosequencing technology. Mycoses. 2004. 47:29–33.

14. Nilsson I, Shabo I, Svanvik J, Monstein HJ. Multiple displacement amplification of isolated DNA from human gallstones: molecular identification of Helicobacter DNA by means of 16S rDNA-based pyrosequencing analysis. Helicobacter. 2005. 10:592–600.

15. Gupta AK, Boekhout T, Theelen B, Summerbell R, Batra R. Identification and typing of Malassezia species by amplified fragment length polymorphism and sequence analyses of the internal transcribed spacer and largesubunit regions of ribosomal DNA. J Clin Microbiol. 2004. 42:4253–4260.

16. Theelen B, Silvestri M, Guého E, van Belkum A, Boekhout T. Identification and typing of Malassezia yeasts using amplified fragment length polymorphism (AFLP), random amplified polymorphic DNA (RAPD) and denaturing gradient gel electrophoresis (DGGE). FEMS Yeast Res. 2001. 1:79–86.

17. Gandra RF, Simão RC, Matsumoto FE, da Silva BC, Ruiz LS, da Silva EG, et al. Genotyping by RAPD-PCR analyses of Malassezia furfur strains from pityriasis versicolor and seborrhoeic dermatitis patients. Mycopathologia. 2006. 162:273–280.

18. Gaitanis G, Velegraki A, Alexopoulos EC, Chasapi V, Tsigonia A, Katsambas A. Distribution of Malassezia species in pityriasis versicolor and seborrhoeic dermatitis in Greece. Typing of the major pityriasis versicolor isolate M. globosa. Br J Dermatol. 2006. 154:854–859.

19. Gemmer CM, DeAngelis YM, Theelen B, Boekhout T, Dawson TL Jr. Fast, noninvasive method for molecular detection and differentiation of Malassezia yeast species on human skin and application of the method to dandruff microbiology. J Clin Microbiol. 2002. 40:3350–3357.

20. Guillot J, Deville M, Berthelemy M, Provost F, Guého E. A single PCR-restriction endonuclease analysis for rapid identification of Malassezia species. Lett Appl Microbiol. 2000. 31:400–403.

21. Makimura K, Tamura Y, Kudo M, Uchida K, Saito H, Yamaguchi H. Species identification and strain typing of Malassezia species stock strains and clinical isolates based on the DNA sequences of nuclear ribosomal internal transcribed spacer 1 regions. J Med Microbiol. 2000. 49:29–35.

22. Gaitanis G, Velegraki A, Frangoulis E, Mitroussia A, Tsigonia A, Tzimogianni A, et al. Identification of Malassezia species from patient skin scales by PCR-RFLP. Clin Microbiol Infect. 2002. 8:162–173.

23. Mirhendi H, Makimura K, Zomorodian K, Yamada T, Sugita T, Yamaguchi H. A simple PCR-RFLP method for identification and differentiation of 11 Malassezia species. J Microbiol Methods. 2005. 61:281–284.

24. Sugita T, Kodama M, Saito M, Ito T, Kato Y, Tsuboi R, et al. Sequence diversity of the intergenic spacer region of the rRNA gene of Malassezia globosa colonizing the skin of patients with atopic dermatitis and healthy individuals. J Clin Microbiol. 2003. 41:3022–3027.

25. Lim SW, Shin MG, Lim JY, Yun SJ, Kim SJ, Lee SC, et al. Nested PCR for detection of Malassezia species from patient skin scales and clinical strains. Korean J Dermatol. 2008. 46:446–452.

26. Jang SJ, Lim SH, Ko JH, Oh BH, Kim SM, Song YC, et al. The investigation on the distribution of malassezia yeasts on the normal Korean skin by 26S rDNA PCR-RFLP. Ann Dermatol. 2009. 21:18–26.

27. Oh BH, Song YC, Lee YW, Choe YB, Ahn KJ. Comparison of nested PCR and RFLP for identification and classification of malassezia yeasts from healthy human skin. Ann Dermatol. 2009. 21:352–357.