, Volume 173, Issue 5–6, pp 337–346 | Cite as

Identification of Novel Hybrids Between Cryptococcusneoformans var. grubii VNI and Cryptococcusgattii VGII

  • Mojgan Aminnejad
  • Mara Diaz
  • Michael Arabatzis
  • Elizabeth Castañeda
  • Marcia Lazera
  • Aristea Velegraki
  • Deborah Marriott
  • Tania C. Sorrell
  • Wieland Meyer


Cryptococcusneoformans and Cryptococcusgattii are pathogenic yeasts causing meningoencephalitis in immunocompromised and immunocompetent hosts. The fungus is typically haploid, and sexual reproduction occurs normally between individuals with opposite mating types, α and a. C. neoformans var. grubii (serotype A) is comprised of molecular types VNI, VNII, and VNB, and C. neoformans var. neoformans (serotype D) contains the molecular type VNIV. Additionally, diploid or aneuploid AD hybrids (VNIII) have been reported. C. gattii contains the molecular types VGI, VGII, VGIII, and VGIV, which encompass both serotypes B and C. To identify possible hybrid strains, URA5-RFLP analysis was performed on 350 globally obtained clinical, environmental, and veterinary isolates. Four clinical isolates from cerebrospinal fluid showed combination patterns of C. neoformans var. grubii and C. gattii: Brazil (n = 2), Colombia (n = 1), and India (n = 1). These strains were monokaryotic and diploid or aneuploid. M13 PCR fingerprinting showed that they contained fragments of both proposed parental groups. Luminex IGS genotyping identified these isolates as hybrids with two different molecular type combinations: three VNI/VGII and one VNI/VGI. Blue color development on CGB agar was delayed in three isolates and absent in one. C. gattii-specific PCR confirmed the presence of C. gattii in the hybrids. CAP59 allele-specific PCR revealed that all the hybrids contained both serotype A and B alleles. Determination of mating-type allelic patterns by PCR revealed that the isolates were αA aB. This is the first study discovering novel natural hybrids between C.neoformans molecular type VNI and C. gattii molecular type VGII.


Cryptococcusneoformans Cryptococcusgattii AB hybrids Serotype Mating type Molecular type 


  1. 1.
    Kwon-Chung KJ, Bennett JE. Epidemiologic differences between the two varieties of Cryptococcus neoformans. Am J Epidemiol. 1984;120:123–30.PubMedGoogle Scholar
  2. 2.
    Speed B, Dunt D. Clinical and host differences between infections with the two varieties of Cryptococcus neoformans. Clin Infect Dis. 1995;21:28–34.PubMedCrossRefGoogle Scholar
  3. 3.
    Emmons CW. Prevalence of Cryptococcus neoformans in pigeon habitats. Public Health Rep. 1960;75:362–4.PubMedCrossRefGoogle Scholar
  4. 4.
    Cafarchia C, Rommito D, Latta R, Camarda A, Montagna MT, Otranto D. Role of birds of prey as carriers and spreaders of Cryptococcus neoformans and other zoonotic yeasts. Med Mycol. 2006;44:458–92.CrossRefGoogle Scholar
  5. 5.
    Randhawa HS, Kowshik T, Chowdhary A, Preeti SK, Khan ZU, Sun S, Xu J. The expanding host tree species spectrum of Cryptococcus gattii and Cryptococcus neoformans and their isolations from surrounding soil in India. Med Mycol. 2008;46:823–33.PubMedCrossRefGoogle Scholar
  6. 6.
    Mitchell TG, Castañeda E, Nielson K, Wanke B, Lazera MS. Environmental niches for Cryptococcus neoformans and Cryptococcus gattii. In: Heitman J, Kozel TR, Kwon-Chung KJ, Perfect JR, Casadevall A, editors. Cryptococcus: from human pathogen to model yeast. Washington: ASM Press; 2011. p. 237–59.Google Scholar
  7. 7.
    Kidd SE, Chow Y, Mak S, Bach PJ, Chen H, Hingston AO, Kronstad JW, Bartlett KH. Characterization of environmental sources of the human and animal pathogen, Cryptococcus gattii, in British Columbia, Canada, and the Pacific Northwest of the United States. Appl Environ Microbiol. 2007;73:1433–43.PubMedCrossRefGoogle Scholar
  8. 8.
    Kwon-Chung KJ, Bennett JE. High prevalence of Cryptococcus neoformans var. gattii in tropical and subtropical regions. Zentralbl Bakteriol Mikrobiol Hyg. 1984;257:213–8.Google Scholar
  9. 9.
    Ellis DH. Cryptococcus neoformans var. gattii in Australia. J Clin Microbiol. 1987;25:430–1.PubMedGoogle Scholar
  10. 10.
    Fraser JA, Subaran RL, Nicholas CB, Heitman J. Recapitulation of the sexual cycle of the primary fungal pathogen Cryptococcus neoformans var. gattii: implications for an outbreak on Vancouver Island, Canada. Eukaryot Cell. 2003;2:1036–45.PubMedCrossRefGoogle Scholar
  11. 11.
    Velegraki A, Kiosses VG, Pitsouni H, Toukas D, Daniilidis VD, Legakis NJ. First report of Cryptococcus neoformans var. gattii serotype B from Greece. Med Mycol. 2001;39:419–22.PubMedGoogle Scholar
  12. 12.
    Romeo O, Scordino F, Criseo G. Environmental isolation of Cryptococcus gattii serotype B, VGI/MATα strains in southern Italy. Mycopathologia. 2011;171:423–30.PubMedCrossRefGoogle Scholar
  13. 13.
    Karstaedt AS, Crewe-Brown HH, Dromer F. Cryptococcal meningitis caused by Cryptococcus neoformans var. gattii, serotype C, in AIDS patients in Soweto, South Africa. Med Mycol. 2002;40:7–11.Google Scholar
  14. 14.
    Colom MF, Frasés S, Ferrer C, Jover A, Andreu M, Reus S, Sánchez M, Torres-Rodríguez JM. First case of human cryptococcosis due to Cryptococcus neoformans var. gattii in Spain. J Clin Microbiol. 2005;43:3548–50.PubMedCrossRefGoogle Scholar
  15. 15.
    Byrnes EJ III, Li W, Lewit Y, Ma H, Voelz K, Ren P, Carter DA, Chaturvedi V, Bildfell RJ, May RC, Heitman J. Emergence and pathogenicity of highly virulent Cryptococcus gattii genotypes in the Northwest United States. PLoS Pathog. 2010;6(4):e1000850. doi:10.1371/journal.ppat.1000850.
  16. 16.
    Lin X, Heitman J. The biology of the Cryptococcus neofomans species complex. Annu Rev Microbiol. 2006;60:69–105.PubMedCrossRefGoogle Scholar
  17. 17.
    Meyer W, Castaneda A, Jackson S, Huynh M, Castaneda E. The Ibro American Cryptococcal Study Group. Molecular typing of IberoAmerican Cryptococcus neoformans isolates. Emerg Infect Dis. 2003;9:189–95.PubMedGoogle Scholar
  18. 18.
    Litvintseva AP, Thakur R, Vilgalys RJ, Mitchell T. Multilocus sequence typing reveals three genetic subpopulations of Cryptococcus neoformans var. grubii (serotype A), including a unique population in Botswana. Genetics. 2006;172:2223–38.PubMedCrossRefGoogle Scholar
  19. 19.
    Ngamskulrungroj P, Gilgado F, Faganello J, Litvintseva AP, Leal AL, Tsui KM, Mitchell TG, Vainstein MH, Meyer W. Genetic diversity of the Cryptococcus species complex suggests that Cryptococcus gattii deserves to have varieties. PLoS One. 2009;4:e5862.PubMedCrossRefGoogle Scholar
  20. 20.
    Kwon-Chung KJ, Boekhout T, Fell JW, Diaz M. Proposal to conserve the name Cryptococcus gattii against C. hondurianus, C bacillisporus (Basidiomycota, Hymenomycetes, Tremellomycetidae). Taxon. 2002;51:804–6.CrossRefGoogle Scholar
  21. 21.
    Currie BP, Casadevall A. Estimation of the prevalence of Cryptococcal infection among patients infected with the human immunodeficiency virus in New York City. Clin Infect Dis. 1994;19:1029–33.PubMedCrossRefGoogle Scholar
  22. 22.
    Franzot SP, Hamdan JS, Currie BP, Casadevall A. Molecular epidemiology of Cryptococcus neoformans in Brazil and the United States: evidence for both local genetic differences and a global clonal population structure. J Clin Microbiol. 1997;35:2243–51.PubMedGoogle Scholar
  23. 23.
    Viviani MA, Cogliati M, Esposto MC, Lemmer K, Tintelnot K, Colom Valiente MF, Swinne D, Velegraki A, Velho R. Molecular analysis of 311 Cryptococcus neoformans isolates from a 30-month ECMM survey of cryptococcosis in Europe. FEMS Yeast Res. 2006;6:614–9.PubMedCrossRefGoogle Scholar
  24. 24.
    Bovers M, Hagen F, Kuramae EE, Diaz MR, Spanjaard L, Dromer F, Hoogveld HL, Boekhout T. Unique hybrids between the fungal pathogens Cryptococcus neoformans and Cryptococcus gattii. FEMS Yeast Res. 2006;6:599–607.PubMedCrossRefGoogle Scholar
  25. 25.
    Bovers M, Hagen F, Kuramae EE, Hoogveld HL, Dromer F, St-Germain G, Boekhout T. AIDS patient death caused by novel Cryptococcus neoformans × C. gattii hybrid. Emerg Infect Dis. 2008;14:1105–8.PubMedCrossRefGoogle Scholar
  26. 26.
    Bovers M, Hagen F, Kuramae EE, Boekhout T. Six monophyletic lineages identified within Cryptococcus neoformans, C. gattii by multilocus sequence typing. Fungal Genet Biol. 2008;45:400–21.PubMedCrossRefGoogle Scholar
  27. 27.
    Lin X, Patel S, Litvintseva AP, Floyd A, Mitchell TG, Heitman J. Diploids in the Cryptococcus neofomans serotype A population homozygous for the α mating type originate via unisexual mating. PLoS Pathog. 2009;5:e1000283.PubMedCrossRefGoogle Scholar
  28. 28.
    Franzot SP, Salkin IR, Casadevall A. Cryptococcus neoformans var. grubii: separate varietal status for Cryptococcus neoformans serotype A isolates. J Clin Microbiol. 1999;37:838–40.PubMedGoogle Scholar
  29. 29.
    Viviani MA, Esposto MC, Cogliati M, Montagna MT, Wickes BL. Isolation of a Cryptococcus neoformans serotype A MAT a strain from the Italian environment. Med Mycol. 2001;39:383–6.PubMedGoogle Scholar
  30. 30.
    Kwon-Chung KJ, Edman JC, Wickes BL. Genetic association of mating types and virulence in Cryptococcus neoformans. Infect Immun. 1992;60:602–5.PubMedGoogle Scholar
  31. 31.
    Ngamskulrungroj P, Sorrell TC, Chindamporn A, Chaiprasert A, Poonwan N, Meyer W. Association between fertility and molecular sub-type of global isolates of Cryptococcus gattii molecular type VGII. Med Mycol. 2008;46:665–73.PubMedCrossRefGoogle Scholar
  32. 32.
    Ferrer C, Colom F, Frasés S, Mulet E, Abad JL, Alió JL. Detection and identification of fungal pathogens by PCR and by ITS2 and 5.8S ribosomal DNA typing in ocular infections. J Clin Microbiol. 2001;39:2873–9.PubMedCrossRefGoogle Scholar
  33. 33.
    Dien BS, Peterson MS, Srienc F. Cell cycle analysis in Saccharomyces cerevisiae. In: Darzynkiewicz Z, Robinson JP, Crissman HA, editors. Methods in cell biology, vol. 42. USA: Academic Press; 1994. p. 457–75.Google Scholar
  34. 34.
    Diaz MR, Fell JW. Use of a suspension array for rapid identification of the varieties and genotypes of the Cryptococcus neoformans species complex. J Clin Microbiol. 2005;43:3662–72.PubMedCrossRefGoogle Scholar
  35. 35.
    Diaz MR, Dunbar SA, Jacobson JW. Multiplexed detection of fungal nucleic acid signatures. In: Robinson JP, Darzynkiewicz Z, Hoffman R, Nolan JP, Rabinovitch PS, Watkins S, editors. Current protocols in cytometry. New York: Wiley; 2008. 44:13.9.1–13.9.21. doi:10.1002/0471142956.cy1309s44.
  36. 36.
    Fulton R, McDade R, Smith P, Kienker L, Kettman J. Advanced multiplexed analysis with the Flowmetrixed system. Clin Chem. 1997;43:1749–56.PubMedGoogle Scholar
  37. 37.
    Diaz MR, Fell JW. High-throughput detection of pathogenic yeasts of the genus Trichosporon. J Clin Microbiol. 2004;42:3696–706.PubMedCrossRefGoogle Scholar
  38. 38.
    Vassart G, Georges M, Monsieur R, Brocas H, Lequarre AS, Christophe D. A sequence in M13 phage detects hypervariable minisatellites in human and animal DNA. Science. 1987;235:683–4.PubMedCrossRefGoogle Scholar
  39. 39.
    Kwon-Chung KJ, Polacheck I, Bennett JE. Improved diagnostic medium for separation of Cryptococcus neoformans var. neoformans (serotypes A and D) and Cryptococcus neoformans var. gattii (serotypes B and C). J Clin Microbiol. 1982;15:535–7.PubMedGoogle Scholar
  40. 40.
    Halliday CL, Bui T, Krockenberger M, Malik R, Ellis DH, Carter DA. Presence of α and a mating type in environmental and clinical collections of Cryptococcus neoformans var. gattii strains from Australia. J Clin Microbiol. 1999;37:2920–6.PubMedGoogle Scholar
  41. 41.
    Enache-Angoulvant A, Chandenier J, Symoens F, Lacube P, Bolognini J, Douchet C, Poirot JL, Hennequin C. Molecular identification of Cryptococcus neoformans serotypes. J Clin Microbiol. 2007;45:1261–5.PubMedCrossRefGoogle Scholar
  42. 42.
    Lengeler KB, Cox GM, Heitman J. Serotype AD strains of Cryptococcus neoformans are diploid or aneuploid and are heterozygous at the mating-type locus. Infect Immun. 2001;69(1):115–22.PubMedCrossRefGoogle Scholar
  43. 43.
    Lin X, Litvintseva AP, Nielsen K, Patel S, Floyd A, Mitchell TG, Heitman J. αADα hybrids of Cryptococcus neofomans: evidence of same-sex mating in nature and hybrid fitness. PLoS Genet. 2007;3:e186.CrossRefGoogle Scholar
  44. 44.
    Chaturvedi S, Rodeghier B, Fan J, McClelland CM, Wickes BL, Chaturvedi V. Direct PCR of Cryptococcus neoformans MATα and MAT a pheromones to determine mating type, ploidy, and variety: a tool for epidemiological and molecular pathogenesis studies. J Clin Microbiol. 2000;38:2007–9.PubMedGoogle Scholar
  45. 45.
    Kwon-Chung KJ, Varma A. Do major species concepts support one, two or more species within Cryptococcus neoformans? FEMS Yeast Res. 2006;6:574–87. doi:10.1111/j.1567-1364.2006.00088.x.PubMedCrossRefGoogle Scholar
  46. 46.
    Kellis M, Birren BW, Lander ES. Proof and evolutionary analysis of ancient genome duplication in the yeast Saccharomyces cerevisiae. Nature. 2004;428:617–24.PubMedCrossRefGoogle Scholar
  47. 47.
    Hunter PR, Fraser CA. Application of the theory of adaptive polymorphism to the ecology and epidemiology of pathogenic yeasts. Appl Environ Microbiol. 1990;56:2219–22.PubMedGoogle Scholar
  48. 48.
    Seehausen O. Hybridization and adaptive radiation. Trends Ecol Evol. 2004;19:198–207.Google Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Mojgan Aminnejad
    • 1
    • 2
  • Mara Diaz
    • 3
  • Michael Arabatzis
    • 4
  • Elizabeth Castañeda
    • 5
  • Marcia Lazera
    • 6
  • Aristea Velegraki
    • 4
  • Deborah Marriott
    • 7
  • Tania C. Sorrell
    • 2
  • Wieland Meyer
    • 2
    • 8
  1. 1.Molecular Mycology Research LaboratoryWestmeadAustralia
  2. 2.Centre for Infectious Diseases and Microbiology, Sydney Medical School-Westmead, Westmead Millennium Institute, Sydney Emerging Infections and Biosecurity Institute, University of SydneySydneyAustralia
  3. 3.Rosenstiel School of Marine and Atmospheric Sciences, University of MiamiMiamiUSA
  4. 4.Mycology Laboratory, Department of MicrobiologyMedical School, National and Kapodistrian University of AthensAthensGreece
  5. 5.Grupo de Microbiología, Instituto Nacional de SaludBogotáColombia
  6. 6.Mycology Laboratory, Instituto de Pesquisa Clínica Evandro Chagas, Fundação Oswaldo CruzRio de JaneiroBrazil
  7. 7.Department of Microbiology and Clinical Infectious DiseasesSt. Vincent’s HospitalSydneyAustralia
  8. 8.Molecular Mycology Research Laboratory, CIDM, ICPMR, Level 3, Room 3114AWestmead HospitalWestmeadAustralia

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