, Volume 183, Issue 2, pp 371–380 | Cite as

Molecular Characterization and Antifungal Susceptibility Testing of Sequentially Obtained Clinical Cryptococcus deneoformans and Cryptococcus neoformans Isolates from Ljubljana, Slovenia

  • Rok Tomazin
  • Tadeja MatosEmail author
  • Jacques F. Meis
  • Ferry Hagen



To retrospectively investigate the epidemiology of cryptococcosis in Ljubljana, Slovenia.


Forty-six sequentially obtained isolates from 19 patients were subjected to amplified fragment length polymorphism (AFLP) genotyping, microsatellite typing, mating- and serotype PCRs and antifungal susceptibility testing.


Majority of the isolates were Cryptococcus deneoformans (n = 29/46; 63%) followed by Cryptococcus neoformans (n = 16/46; 34.8%) and their interspecies hybrid (n = 1/46; 2.2%). Mating-type α was predominant, two mating-type a C. deneoformans isolates and one mating-type a/α isolate were observed. Several mixed infections were found by microsatellite typing; one patient had a persisting C. deneoformans infection for > 2.5 years. For C. deneoformans, the in vitro antifungal MIC90 and susceptibility ranges were for amphotericin B 0.25 µg/ml (0.031–0.25 µg/ml), 5-fluorocytosine 0.25 µg/ml (0.063–4 µg/ml), fluconazole 8 µg/ml (0.5–16 µg/ml), voriconazole 0.063 µg/ml (0.008–0.125 µg/ml), posaconazole 0.063 µg/ml (0.008–0.063 µg/ml) and itraconazole 0.063 µg/ml (0.031–0.125 µg/ml). For C. neoformans, these values were for amphotericin B 0.25 µg/ml (0.063–0.5 µg/ml), 5-fluorocytosine 1 µg/ml (0.063–1 µg/ml), fluconazole 16 µg/ml (0.5–64 µg/ml), voriconazole 0.125 µg/ml (0.008–0.25 µg/ml), posaconazole 0.063 µg/ml (0.008–0.063 µg/ml) and itraconazole 0.063 µg/ml (0.031–0.125 µg/ml).


Majority of the cases were caused by C. deneoformans; mating-type α was predominant. Several mixed infections were identified by AFLP genotyping and microsatellite typing. Despite antifungal therapy, a cryptococcal isolate could persist for years. Voriconazole, itraconazole and posaconazole were the most potent antifungal drugs.


Cryptococcosis Cryptococcus deneoformans Cryptococcus neoformans AFLP genotyping Microsatellite typing Epidemiology 



The research visit of Rok Tomazin to the Canisius-Wilhelmina Hospital was supported with an ESCMID Observership (Grant Number 516).

Compliance with Ethical Standards

Conflict of interest

JF.M received grants from Astellas, Basilea, F2G and Merck. He has been a consultant to Astellas, Basilea and Merck and received speaker’s fees from Merck, Gilead and United Medical.


  1. 1.
    Fonseca Á, Boekhout T, Fell JW. Cryptococcus. In: Kurtzman CP, Fell JW, Boekhout T, editors. The yeasts—a taxonomic study, vol. 3. Amsterdam: Elsevier Science; 2011. p. 1661–17347 (Chapter 138).Google Scholar
  2. 2.
    La Hoz RM, Pappas PG. Cryptococcal infections: changing epidemiology and implications for therapy. Drugs. 2013;73:495–504. doi: 10.1007/s40265-013-0037-z.CrossRefPubMedGoogle Scholar
  3. 3.
    Rajasingham R, Smith RM, Park BJ, Jarvis JN, Govender NP, et al. Global burden of disease of HIV-associated cryptococcal meningitis: an updated analysis. Lancet Infect Dis. 2017;17:873–81. doi: 10.1016/S1473-3099(17)30243-8.CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Cogliati M. Global molecular epidemiology of Cryptococcus neoformans and Cryptococcus gattii: an atlas of the molecular types. Scientifica (Cairo). 2013;2013:675213. doi: 10.1155/2013/675213.Google Scholar
  5. 5.
    Hagen F, Khayhan K, Theelen B, Kolecka A, Polacheck I, et al. Recognition of seven species in the Cryptococcus gattii/Cryptococcus neoformans species complex. Fungal Genet Biol. 2015;78:16–48. doi: 10.1016/j.fgb.2015.02.009.CrossRefPubMedGoogle Scholar
  6. 6.
    Meyer W, Aanensen DM, Boekhout T, Cogliati M, Diaz MR, et al. Consensus multi-locus sequence typing scheme for Cryptococcus neoformans and Cryptococcus gattii. Med Mycol. 2009;47:561–70. doi: 10.1080/13693780902953886.CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Hagen F, Lumbsch HT, Arsic Arsenijevic V, Badali H, Bertout S et al. The importance of resolving fungal nomenclature: the case of the multiple pathogenic species in the genus Cryptococcus. mSphere. 2017;2 (in press).Google Scholar
  8. 8.
    Kwon-Chung KJ, Bennett JE, Wickes BL, Meyer W, Cuomo CA, et al. The case for adopting the “species complex” nomenclature for the etiologic agents of cryptococcosis. mSphere. 2017;2:e00357-16. doi: 10.1128/mSphere.00357-16.CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Aminnejad M, Diaz M, Arabatzis M, Castañeda E, Lazera M, et al. Identification of novel hybrids between Cryptococcus neoformans var. grubii VNI and Cryptococcus gattii VGII. Mycopathologia. 2012;173:337–46. doi: 10.1007/s11046-011-9491-x.CrossRefPubMedGoogle Scholar
  10. 10.
    Bovers M, Hagen F, Kuramae EE, Diaz MR, Spanjaard L, et al. Unique hybrids between the fungal pathogens Cryptococcus neoformans and Cryptococcus gattii. FEMS Yeast Res. 2006;6:599–607. doi: 10.1111/j.1567-1364.2006.00082.x.CrossRefPubMedGoogle Scholar
  11. 11.
    Bovers M, Hagen F, Kuramae EE, Hoogveld HL, Dromer F, et al. AIDS patient death caused by novel Cryptococcus neoformans × C. gattii hybrid. Emerg Infect Dis. 2008;14:1105–8. doi: 10.3201/eid1407.080122.CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Hagen F, Hare Jensen R, Meis JF, Arendrup MC. Molecular epidemiology and in vitro antifungal susceptibility testing of 108 clinical Cryptococcus neoformans sensu lato and Cryptococcus gattii sensu lato isolates from Denmark. Mycoses. 2016;59:576–84. doi: 10.1111/myc.12507.CrossRefPubMedGoogle Scholar
  13. 13.
    Cogliati M, D’Amicis R, Zani A, Montagna MT, Caggiano G, et al. Environmental distribution of Cryptococcus neoformans and C. gattii around the Mediterranean basin. FEMS Yeast Res. 2016;16:fow045. doi: 10.1093/femsyr/fow045.CrossRefPubMedGoogle Scholar
  14. 14.
    Viviani MA, Cogliati M, Esposto MC, Lemmer K, Tintelnot K, et al. Molecular analysis of 311 Cryptococcus neoformans isolates from a 30-month ECMM survey of cryptococcosis in Europe. FEMS Yeast Res. 2006;6:614–9. doi: 10.1111/j.1567-1364.2006.00081.x.CrossRefPubMedGoogle Scholar
  15. 15.
    Dromer F, Mathoulin S, Dupont B, Letenneur L, Ronin O. Individual and environmental factors associated with infection due to Cryptococcus neoformans serotype D. Clin Infect Dis. 1996;23:91–6.CrossRefPubMedGoogle Scholar
  16. 16.
    Boekhout T, Theelen B, Diaz M, Fell JW, Hop WC, et al. Hybrid genotypes in the pathogenic yeast Cryptococcus neoformans. Microbiology. 2001;147:891–907. doi: 10.1099/00221287-147-4-891.CrossRefPubMedGoogle Scholar
  17. 17.
    Nyazika TK, Hagen F, Meis JF, Robertson VJ. Cryptococcus tetragattii as a major cause of cryptococcal meningitis among HIV-infected individuals in Harare, Zimbabwe. J Infect. 2016;72:745–52. doi: 10.1016/j.jinf.2016.02.018.CrossRefPubMedGoogle Scholar
  18. 18.
    Arsic Arsenijevic V, Pekmezovic MG, Meis JF, Hagen F. Molecular epidemiology and antifungal susceptibility of Serbian Cryptococcus neoformans isolates. Mycoses. 2014;57:380–7. doi: 10.1111/myc.12171.PubMedGoogle Scholar
  19. 19.
    Illnait-Zaragozí MT, Martínez-Machín GF, Fernández-Andreu CM, Boekhout T, Meis JF, et al. Microsatellite typing of clinical and environmental Cryptococcus neoformans var. grubii isolates from Cuba shows multiple genetic lineages. PLoS ONE. 2010;5:e9124. doi: 10.1371/journal.pone.0009124.CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Hagen F, Illnait-Zaragozí MT, Meis JF, Chew WH, Curfs-Breuker I, et al. Extensive genetic diversity within the Dutch clinical Cryptococcus neoformans population. J Clin Microbiol. 2012;50:1918–26. doi: 10.1128/JCM.06750-11.CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Mlinaric-Missoni E, Hagen F, Chew WH, Vazic-Babic V, Boekhout T, et al. In vitro antifungal susceptibilities and molecular typing of sequentially isolated clinical Cryptococcus neoformans strains from Croatia. J Med Microbiol. 2011;60:1487–95. doi: 10.1099/jmm.0.031344-0.CrossRefPubMedGoogle Scholar
  22. 22.
    Kwon-Chung KJ, Edman JC, Wickes BL. Genetic association of mating types and virulence in Cryptococcus neoformans. Infect Immun. 1992;60:602–5.PubMedPubMedCentralGoogle Scholar
  23. 23.
    Nielsen K, Marra RE, Hagen F, Boekhout T, Mitchell TG, et al. Interaction between genetic background and the mating-type locus in Cryptococcus neoformans virulence potential. Genetics. 2005;171:975–83.CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Nielsen K, Cox GM, Litvintseva AP, Mylonakis E, Malliaris SD, et al. Cryptococcus neoformans alpha strains preferentially disseminate to the central nervous system during coinfection. Infect Immun. 2005;73:4922–33. doi: 10.1128/IAI.73.8.4922-4933.2005.CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Desnos-Ollivier M, Patel S, Spaulding AR, Charlier C, Garcia-Hermoso D, Nielsen K, Dromer F. Mixed infections and in vivo evolution in the human fungal pathogen Cryptococcus neoformans. mBio. 2010;1. pii: e00091-10. doi: 10.1128/mBio.00091-10.
  26. 26.
    Gago S, Serrano C, Alastruey-Izquierdo A, Cuesta I, Martín-Mazuelos E, et al. Molecular identification, antifungal resistance and virulence of Cryptococcus neoformans and Cryptococcus deneoformans isolated in Seville, Spain. Mycoses. 2017;60:40–50. doi: 10.1111/myc.12543.CrossRefPubMedGoogle Scholar
  27. 27.
    Kassi FK, Bellet V, Doumbia A, Krasteva D, Drakulovski P, et al. First case of mixed infection with Cryptococcus deuterogattii and Cryptococcus neoformans VNI in an Ivorian HIV-positive patient. JMM Case Rep. 2016;3:e005037. doi: 10.1099/jmmcr.0.005037.CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Bertout S, Drakulovski P, Kouanfack C, Krasteva D, Ngouana T, et al. Genotyping and antifungal susceptibility testing of Cryptococcus neoformans isolates from Cameroonian HIV-positive adult patients. Clin Microbiol Infect. 2013;19:763–9. doi: 10.1111/1469-0691.12019.CrossRefPubMedGoogle Scholar
  29. 29.
    Fries BC, Casadevall A. Serial isolates of Cryptococcus neoformans from patients with AIDS differ in virulence for mice. J Infect Dis. 1998;178(6):1761–6.CrossRefPubMedGoogle Scholar
  30. 30.
    Illnait-Zaragozí MT, Martínez-Machín GF, Fernández-Andreu CM, Hagen F, Boekhout T, et al. Microsatellite typing and susceptibilities of serial Cryptococcus neoformans isolates from Cuban patients with recurrent cryptococcal meningitis. BMC Infect Dis. 2010;10:289. doi: 10.1186/1471-2334-10-289.CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Kammalac Ngouana T, Drakulovski P, Krasteva D, Kouanfack C, Reynes J, et al. Cryptococcus neoformans isolates from Yaoundé human immunodeficiency virus-infected patients exhibited intra-individual genetic diversity and variation in antifungal susceptibility profiles between isolates from the same patient. J Med Microbiol. 2016;65:579–89. doi: 10.1099/jmm.0.000265.CrossRefPubMedGoogle Scholar
  32. 32.
    Mandal P, Banerjee U, Casadevall A, Nosanchuk JD. Dual infections with pigmented and albino strains of Cryptococcus neoformans in patients with or without human immunodeficiency virus infection in India. J Clin Microbiol. 2005;43:4766–72.CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Rhodes J, Beale MA, Vanhove M, Jarvis JN, Kannambath S, Simpson JA, Ryan A, Meintjes G, Harrison TS, Fisher MC, Bicanic T. A population genomics approach to assessing the genetic basis of within-host microevolution underlying recurrent cryptococcal meningitis infection. G3 (Bethesda). 2017;7:1165–76. doi: 10.1534/g3.116.037499.CrossRefGoogle Scholar
  34. 34.
    Casadevall A, Spitzer ED, Webb D, Rinaldi MG. Susceptibilities of serial Cryptococcus neoformans isolates from patients with recurrent cryptococcal meningitis to amphotericin B and fluconazole. Antimicrob Agents Chemother. 1993;37:1383–6.CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Espinel-Ingroff A, Aller AI, Canton E, Castañón-Olivares LR, Chowdhary A, et al. Cryptococcus neoformans-Cryptococcus gattii species complex: an international study of wild-type susceptibility endpoint distributions and epidemiological cutoff values for fluconazole, itraconazole, posaconazole and voriconazole. Antimicrob Agents Chemother. 2012;56:5898–906. doi: 10.1128/AAC.01115-12.CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Pfaller MA, Castanheira M, Diekema DJ, Messer SA, Jones RN. Wild-type MIC distributions and epidemiologic cutoff values for fluconazole, posaconazole, and voriconazole when testing Cryptococcus neoformans as determined by the CLSI broth microdilution method. Diagn Microbiol Infect Dis. 2011;71:252–9. doi: 10.1016/j.diagmicrobio.2011.07.007.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2017

Authors and Affiliations

  1. 1.Institute of Microbiology and Immunology, Faculty of MedicineUniversity of LjubljanaLjubljanaSlovenia
  2. 2.Department of Medical Microbiology and Infectious DiseasesCanisius-Wilhelmina HospitalNijmegenThe Netherlands
  3. 3.Centre of Expertise in Mycology Radboudumc/CWZNijmegenThe Netherlands

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