Advertisement

Mycopathologia

, Volume 175, Issue 5–6, pp 445–454 | Cite as

Molecular Identification of Melanised Non-Sporulating Moulds: A Useful Tool for Studying the Epidemiology of Phaeohyphomycosis

  • Daniel W. C. L. Santos
  • Ana Carolina B. Padovan
  • Analy S. A. Melo
  • Sarah S. Gonçalves
  • Viviane R. Azevedo
  • Marilia M. Ogawa
  • Tainá Veras Sandes Freitas
  • Arnaldo L. Colombo
Article

Abstract

Subcutaneous infections caused by melanised fungi have been increasingly reported among transplant patients, and these infections have the potential for blood and visceral dissemination. Some moulds, such as Mycelia sterilia, cannot grow and sporulate on different media, making their identification impossible by conventional methods. The fast and accurate identification of melanised fungi at the species level is important because species may have tropism to different organs and different susceptibilities to antifungal agents. Molecular tools have been reported to be helpful for the species identification of non-sporulating moulds. Our goal was to identify the species of M. sterilia isolates obtained from clinical samples of transplant patients using sequences of ITS and the D1/D2 regions of rDNA. Clinical samples were obtained from eight kidney transplant recipients who developed subcutaneous fungal infections. The diagnosis was confirmed by histopathology and conventional culture. Histopathology showed septated, melanised hyphae, and the cultures identified non-sporulating fungi. Therefore, the DNA from the M. sterilia isolates was subjected to PCR amplification and sequencing of the ITS and D1/D2 regions. Genus/species identification was obtained by comparison with gene banks. We obtained the following identifications: Alternaria sp. (2), Cochliobolus lunatus/Curvularia lunata (2), Cochliobolus hawaiiensis/Bipolaris hawaiiensis (1), Ochroconis sp. (1), Medicocopsis romeroi/Pyrenochaeta romeroi (1) and Nigrograna mackinnonii/Pyrenochaeta mackinnonii (1).

Keywords

Non-sporulating moulds Melanised fungi Transplant patient Mycelia sterilia 

Notes

Acknowledgments

We are in debt with Professor Dr. José Osmar Medina-Pestana, Head of Kidney and Hypertension Hospital (UNIFESP). We are also grateful to the Infectious Diseases and Dermatology teams that are responsible for the clinical support to transplant recipients. This project was approved by the Ethical Committee of the UNIFESP No. 0524/08. The authors report no conflicts of interest and are responsible for the content and writing of this paper.

References

  1. 1.
    Revankar SG. Phaeohyphomycosis. Infect Dis Clin N Am. 2006;20:609–6202.CrossRefGoogle Scholar
  2. 2.
    Revankar SG, Sutton DA. Melanized fungi in human disease. Clin Microbiol Rev. 2010;23(4):884–928.PubMedCrossRefGoogle Scholar
  3. 3.
    Badali H, Gueidan C, Najafzadeh MJ, Bonifaz A, Gerrits van den Ende AHG, de Hoog GS. Biodiversity of the genus Cladophialophora. Stud Mycol. 2008;61:175–91.PubMedCrossRefGoogle Scholar
  4. 4.
    Vicente VA, Attili-Angelis D, Pie MR, Queiroz-Telles F, Cruz LM, Najafzadeh MJ, de Hoog GS, Zhao J, Pizzirani-Kleiner A. Environmental isolation of black yeast-like fungi involved in human infection. Stud Mycol. 2008;61:137–44.PubMedCrossRefGoogle Scholar
  5. 5.
    Revankar SG, Patterson JE, Sutton DA, Pullen R, Rinaldi MG. Disseminated phaeohyphomycosis: review of an emerging mycosis. Clin Infect Dis. 2002;34:467–76.PubMedCrossRefGoogle Scholar
  6. 6.
    Robert T, Talarmin JP, Leterrier M, Cassagnau E, Pape PL, Danner-Boucher I, Malard O, Brocard A, Gay-Andrieu F, Miegeville M, Morio F. Phaeohyphomycosis due to Alternaria infectoria: a single-center experience with utility of PCR for diagnosis and species identification. Med Mycol. 2012;50(6):594–600.PubMedCrossRefGoogle Scholar
  7. 7.
    Ogawa MM, Galante NZ, Godoy P, Fischman-Gompertz O, Martelli F, Colombo AL, Tomimori J, Medina-Pestana JO. Treatment of subcutaneous phaeohyphomycosis and prospective follow-up of 17 kidney transplant recipients. J Am Acad Dermatol. 2009;61(6):977–85.PubMedCrossRefGoogle Scholar
  8. 8.
    Pounder JI, Simmon KE, Barton CA, Hohmann SL, Brandt ME, Petti CA. Discovering potential pathogens among fungi identified as non-sporulating molds. J Clin Microbiol. 2007;45:568–71.PubMedCrossRefGoogle Scholar
  9. 9.
    Bagyalakshmi R, Therese KL, Prasanna S, Madhavan HN. Newer emerging pathogens of ocular non-sporulating molds (NSM) identified by polymerase chain reaction (PCR)-based DNA sequencing technique targeting internal transcribed spacer (ITS) region. Curr Eye Res. 2008;33(2):139–47.PubMedCrossRefGoogle Scholar
  10. 10.
    Gerrits van den Ende AHG, de Hoog GS. Variability and molecular diagnostics of the neurotropic species Cladophialophora bantiana. Stud Mycol. 1999;43:152–62.Google Scholar
  11. 11.
    Singh N. Fungal infections in the recipients of solid organ transplantation. Infect Dis Clin North Am. 2003;17(1):113–34.PubMedCrossRefGoogle Scholar
  12. 12.
    Gonçalves SS, Stchigel AM, Cano JF, Godoy-Martinez PC, Colombo AL, Guarro J. Aspergillus novoparasiticus: a new clinical species of the section. Med Mycol. 2012;50(2):152–60.PubMedCrossRefGoogle Scholar
  13. 13.
    Samson RA, Hoekstra ES, Frisvad JC. Introduction to food and airborne fungi. 7th ed. Utrecht: Centraalbureau voor Schimmelcultures; 2004.Google Scholar
  14. 14.
    Kurtzman CP, Robnett CJ. Identification of clinically important ascomycetous yeasts based on nucleotide divergence in the 5′ end of the large-subunit (26S) ribosomal DNA gene. J Clin Microbiol. 1997;35(5):1216–23.PubMedGoogle Scholar
  15. 15.
    White TJ, Bruns T, Lee S, Taylor J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis A, Gelfand DH, Sninsky JJ, White TJ, editors. PCR protocols: a guide to methods and applications. San Diego, CA: Academic Press; 1990. p. 315–22.Google Scholar
  16. 16.
    Ewing B, Hillier L, Wendl MC, Green P. Base-calling of automated sequencer traces using phred I-Accuracy assessment. Genome Res. 1998;8:175–85.PubMedGoogle Scholar
  17. 17.
    Ewing B, Green P. Base-calling of automated sequencer traces using phred II. Error probabilities. Genome Res. 1998;8:186–94.PubMedGoogle Scholar
  18. 18.
    Gordon D, Abajian C, Green P. Consed: a graphical tool for sequence finishing. Genome Res. 1998;8(3):195–202.PubMedGoogle Scholar
  19. 19.
    Rossmann SN, Cernoch PL, Davis JR. Dematiaceous fungi are an increasing cause of human disease. Clin Infect Dis. 1996;22(1):73–80.PubMedCrossRefGoogle Scholar
  20. 20.
    Singh N, Chang FY, Gayowski T, Marino IR. Infections due to dematiaceous fungi in organ transplant recipients: case report and review. Clin Infect Dis. 1997;24(3):369–74.PubMedCrossRefGoogle Scholar
  21. 21.
    Garcia-Diaz JB, Baumgarten K. Phaeohyphomycotic infections in solid organ transplant patients. Semin Respir Infect. 2002;17(4):303–9.PubMedCrossRefGoogle Scholar
  22. 22.
    Ben-Ami R, Lewis RE, Raad II, Kontoyiannis DP. Phaeohyphomycosis in a tertiary care cancer center. Clin Infect Dis. 2009;48(8):1033–41.PubMedCrossRefGoogle Scholar
  23. 23.
    Rammaert B, Aguilar C, Bougnoux ME, Noël N, Charlier C, Denis B, Lecuit M, Lortholary O. Success of posaconazole therapy in a heart transplanted patient with Alternaria infectoria cutaneous infection. Med Mycol. 2012;50(5):518–21.PubMedCrossRefGoogle Scholar
  24. 24.
    Chowdhary A, Guarro J, Randhawa HS, Gené J, Cano J, Jain RK, et al. A rare case of chromoblastomycosis in a renal transplant recipient caused by a non-sporulating species of Rhytidhysteron. Med Mycol. 2008;46(2):163–6.PubMedCrossRefGoogle Scholar
  25. 25.
    Dupont C, Duong T, Mallet S, Mamzer-Bruneel M, Thervet E, Bougnoux M, et al. Unusual presentation of chromoblastomycosis due to Cladophialophora carrionii in a renal and pancreas transplant recipient patient successfully treated with posaconazole and surgical excision. Transpl Infect Dis. 2010;12:180–3.PubMedCrossRefGoogle Scholar
  26. 26.
    Cramer RA Jr, Perfect BZ, Pinchai N, Park S, Perlin DS, Asfaw YG, Heitman J, Perfect JR, Steinbach WJ. Calcineurin target CrzA regulates conidial germination, hyphal growth, and pathogenesis of Aspergillus fumigatus. Eukaryot Cell. 2008;7(7):1085–97.PubMedCrossRefGoogle Scholar
  27. 27.
    Chen SC, Lewis RE, Kontoyiannis DP. Direct effects of non-antifungal agents used in cancer chemotherapy and organ transplantation on the development and virulence of Candida and Aspergillus species. Virulence. 2011;2(4):280–95.PubMedCrossRefGoogle Scholar
  28. 28.
    Juvvadi PR, Kuroki Y, Arioka M, Nakajima H, Kitamoto K. Functional analysis of the calcineurin-encoding gene cnaA from Aspergillus oryzae: evidence for its putative role in stress adaptation. Arch Microbiol. 2003;179(6):416–22.PubMedGoogle Scholar
  29. 29.
    Harren K, Schumacher J, Tudzynski B. The Ca(2 +)/Calcineurin-Dependent Signaling Pathway in the Gray Mold Botrytis cinerea: the Role of Calcipressin in Modulating Calcineurin Activity. PLoS ONE. 2012;7(7):e41761.PubMedCrossRefGoogle Scholar
  30. 30.
    De Gruyter J, Woudenberg JHC, Aveskamp MM, Verkley GJM, Groenewald JZ, Crous PW. Rediposition of Phoma-like anamorphs in Pleosporales. Stud Mycol. 2012;75:1–36.Google Scholar
  31. 31.
    De Albornoz MB, Rodríguez-Garcilazo YG, Urdaneta-González D. Foot mycetoma of double etiology. Sabouraudia. 1977;15(2):187–93.PubMedCrossRefGoogle Scholar
  32. 32.
    Cerar D, Malallah YM, Howard SJ, Bowyer P, Denning DW. Isolation, identification and susceptibility of Pyrenochaeta romeroi in a case of eumycetoma of the foot in the UK. Int J Antimicrob Agents. 2009;34(6):617–8.PubMedCrossRefGoogle Scholar
  33. 33.
    Badali H, Chander J, Gulati N, Attri A, Chopra R, Najafzadeh MJ, Chhabra S, Meis JF, de Hoog GS. Subcutaneous phaeohyphomycotic cyst caused by Pyrenochaeta romeroi. Med Mycol. 2010;48(5):763–8.PubMedCrossRefGoogle Scholar
  34. 34.
    De Hoog GS, Horré R. Molecular taxonomy of the Alternaria and Ulocladium species from humans and their identification in the routine laboratory. Mycoses. 2002;45(8):259–76.PubMedCrossRefGoogle Scholar
  35. 35.
    Andrew M, Peever TL, Pryor BM. An expanded multilocus phylogeny does not resolve morphological species within the small-spored Alternaria species complex. Mycologia. 2009;101(1):95–109.PubMedCrossRefGoogle Scholar
  36. 36.
    Schoch CL, Seifert KA, Huhndorf S, Robert V, Spouge JL, Levesque CA, Chen W. Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for fungi. Proc Natl Acad Sci USA. 2012;109(16):6241–6.PubMedCrossRefGoogle Scholar
  37. 37.
    Martin-Sanchez PM, Nováková A, Bastian F, Alabouvette C, Saiz-Jimenez C. Two new species of the genus Ochroconis, O. lascauxensis and O. anomala isolated from black stains in Lascaux Cave, France. fungal. Biol. 2012;116(5):574–89.Google Scholar
  38. 38.
    Najafzadeh MJ, Gueidan C, Badali H, Van Den Ende AH, Xi L, De Hoog GS. Genetic diversity and species delimitation in the opportunistic genus Fonsecaea. Med Mycol. 2009;47(1):17–25.PubMedCrossRefGoogle Scholar
  39. 39.
    Balajee SA, Houbraken J, Verweij PE, Hong SB, Yaghuchi T, Varga J, Samson RA. Aspergillus species identification in the clinical setting. Stud Mycol. 2007;59:39–46.PubMedCrossRefGoogle Scholar
  40. 40.
    Begerow D, Nilsson H, Unterseher M, Maier W. Current state and perspectives of fungal DNA bar coding and rapid identification procedures. Appl Microbiol Biotechnol. 2010;87(1):99–108.PubMedCrossRefGoogle Scholar
  41. 41.
    Aveskamp MM, Gruyter JD, Woudenberg JHC, Verkley GJM, Crous PW. Highlights of the Didymellaceae: a polyphasic approach to characterize Phoma and related pleosporalean genera. Stud Mycol. 2010;65:1–60.PubMedCrossRefGoogle Scholar
  42. 42.
    Sutton DA. Coelomycetous fungi in human disease. A review: clinical entities, pathogenesis, identification and therapy. Rev Iberoam Micol. 1999;16(4):171–9.PubMedGoogle Scholar
  43. 43.
    Girard C, Dereure O, Rispail P, Durand L, Guilhou JJ. Subcutaneous phaeohyphomycosis due to Pyrenochaeta romeroi in a patient with leprosy. Acta Derm Venereol. 2004;84(2):154–5.PubMedCrossRefGoogle Scholar
  44. 44.
    Khan Z, Ahmad S, Kapila K, Ramaswamy NV, Alath P, Joseph L, Chandy R. Pyrenochaeta romeroi: a causative agent of phaeohyphomycotic cyst. J Med Microbiol. 2011;60:842–6.PubMedCrossRefGoogle Scholar
  45. 45.
    Young NA, Kwon-Chung KJ, Greeman J. Subcutaneous abscess caused by Phoma sp. resembling Pyrenochaeta romeroi. Unique fungal infection occurring in immunosuppressed recipient of renal allograft. Amer J Clin Pathol. 1973;59:810–6.Google Scholar
  46. 46.
    Mathuram Thiyagarajan U, Bagul A, Nicholson ML. A nodulo-cystic eumycetoma caused by Pyrenochaeta romeroi in a renal transplant recipient: a case report. J Med Case Rep. 2011;5:460.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • Daniel W. C. L. Santos
    • 1
    • 2
  • Ana Carolina B. Padovan
    • 1
  • Analy S. A. Melo
    • 1
  • Sarah S. Gonçalves
    • 1
  • Viviane R. Azevedo
    • 1
  • Marilia M. Ogawa
    • 3
  • Tainá Veras Sandes Freitas
    • 4
  • Arnaldo L. Colombo
    • 1
    • 5
  1. 1.Special Mycology Laboratory—LEMI, Department of MedicineFederal University of São PauloSão PauloBrazil
  2. 2.Institute of Infectious Diseases Emílio RibasSão PauloBrazil
  3. 3.Department of DermatologyFederal University of São PauloSão PauloBrazil
  4. 4.Department of Medicine, Division of NephrologyHospital do Rim e HipertensãoSão PauloBrazil
  5. 5.Laboratório Especial de Micologia, Disciplina de InfectologiaUniversidade Federal de São PauloSão PauloBrazil

Personalised recommendations