Medical Microbiology and Immunology

, Volume 203, Issue 5, pp 333–340 | Cite as

Molecular typing of clinical Cryptococcus neoformans isolates collected in Germany from 2004 to 2010

  • Andrea Sanchini
  • Ilka McCormick Smith
  • Ludwig Sedlacek
  • Roman Schwarz
  • Kathrin Tintelnot
  • Volker Rickerts
Original Investigation


Cryptococcosis is a fungal infection mostly caused by Cryptococcus neoformans. We identified agents of cryptococcosis diagnosed in Germany from 2004 to 2010. We used multi-locus sequence typing (MLST) to understand the molecular epidemiology of cryptococcosis. Sero- and mating types of individual patient isolates were determined by PCR. MLST was performed using the seven-locus scheme. Allele and nucleotide diversity was calculated for each locus of C. neoformans var. grubii and C. neoformans var. neoformans. Phylogenetic relations were assessed by dendrograms. Clinical data were compared between infections caused by the two variants. We studied 101 isolates. Eight were identified as hybrids (8 %). All non-hybrids were of the α mating type. Among 78 C. neoformans var. grubii (77 %), 16 sequence types (STs) were identified including three novel STs. They clustered in four groups, previously isolated in Asia, Europe or worldwide. Among 15 C. neoformans var. neoformans (15 %), 10 STs were identified, without clustering. These isolates showed higher allele, and nucleotide diversity compared with C. neoformans var. grubii. C. neoformans var. neoformans was more likely to cause soft-tissue infections (3/9, 33 vs. 1/63, 2 %, p = 0.005) and to affect non-AIDS patients (7/14, 50 vs. 15/76, 20 %, p = 0.036). C. neoformans var. grubii is the predominant agent of cryptococcosis in Germany. MLST suggests that a part of these cases are acquired abroad by immigrants or tourists. C. neoformans var. neoformans isolates represent a greater genetic diversity and are associated with more variable clinical presentations.


Cryptococcus neoformans var. grubii Cryptococcus neoformans var. neoformanMLST Cryptococcosis Molecular epidemiology AD hybrids 



We thank all institutions and physicians providing isolates: Helios Klinikum Heckeshorn Berlin, S.Wagner; Helios Klinikum Buch Berlin; Uniklinik Münster, W. Fegeler; St. Elisabethen KH Frankfurt am Main, J. Bargon; Universität Erlangen, F. Albert; Universität Düsseldorf, Mikrobiologie; Universität Göttingen, U. Groß; Universität Mannheim, H. Hof; Gemeinschaftspraxis Labormedizin Paracelsusstrasse 12, Leverkusen A. Kleiber-Imbeck; Universität Frankfurt am Main, V. Rickerts; Klinikum Chemnitz, ML Kerkmann; Vivantes Klinikum Neukölln; Klinikum Nürnberg, K. Fischer; Universitätsklinik Homburg/Saar; Labor Bertholdt und Kollegen, Frankfurt, Oder; Uniklinik Hannover, Mikrobiologie, L. Sedlacek; Medizinisches Versorgungszentrum Riegel und Partner, Wiesbaden; Laboratorium für Mikrobiologie Mölbis, P. Nenoff; Labor Dr. Gärtner, Ravensburg, T. Schmidt-Wieland; Universität Würzburg Mikrobiologie; Klinikum Traunstein, P. Einring; Universität Mainz, Mikrobiologyie; Med. Versorgungszentrum Dr. Stein und Kollegen, Mönchengladbach, R. Schwarz; Universität Hamburg, Mikrobiologie G. Franke; Krankhenhaus St. Georg, Hamburg, NN; Universität Freiburg, C. Schneider; TU Dresden, Dresden; Städtisches Krankhenhaus Kiel; Uniklinik Heidelberg, Heidelberg; Laborpraxis Osnabrück, Osnabrück; MVZ für Laboratoriumsmedizin Koblenz; Labor Dr. Wittke, Bremen; Klinikum Augsburg; Universität Aachen, G. Haase; Universität Jena, Mikrobiologie. The skilful technical assistance of S. Spodzieja and M. Adam (MVZ Dr. Stein+Kollegen, Mönchengladbach), E. Antweiler, C. Han, H. Losert, W. Pohl, B. Raddatz and M. Seibold is greatly appreciated. The author thanks Aftab Jasir, Androulla Efstratiou and Anton Aebischer for the critical review of the manuscript. We thank Wieland Meyer and Carolina Firacative for the support with the MLST database.

Conflict of interest

The authors report no conflict of interest.

Supplementary material

430_2014_341_MOESM1_ESM.docx (98 kb)
Supplementary material 1 (DOCX 98 kb)


  1. 1.
    Park BJ, Wannemuehler KA, Marston BJ, Govender N, Pappas PG, Chiller TM (2009) Estimation of the current global burden of cryptococcal meningitis among persons living with HIV/AIDS. Aids 23:525–530. doi: 10.1097/QAD.0b013e328322ffac PubMedCrossRefGoogle Scholar
  2. 2.
    Idnurm A, Bahn YS, Nielsen K, Lin X, Fraser JA, Heitman J (2005) Deciphering the model pathogenic fungus Cryptococcus neoformans. Nat Rev Microbiol 3:753–764. doi: 10.1038/nrmicro1245 PubMedCrossRefGoogle Scholar
  3. 3.
    Ma H, May RC (2009) Virulence in Cryptococcus species. Adv Appl Microbiol 67:131–190. doi: 10.1016/S0065-2164(08)01005-8 PubMedCrossRefGoogle Scholar
  4. 4.
    Lester SJ, Malik R, Bartlett KH, Duncan CG (2011) Cryptococcosis: update and emergence of Cryptococcus gattii. Vet Clin Pathol 40:4–17. doi: 10.1111/j.1939-165X.2010.00281.x PubMedCrossRefGoogle Scholar
  5. 5.
    Fraser JA, Giles SS, Wenink EC et al (2005) Same-sex mating and the origin of the Vancouver island Cryptococcus gattii outbreak. Nature 437:1360–1364. doi: 10.1038/nature04220 PubMedCrossRefGoogle Scholar
  6. 6.
    Lin X, Heitman J (2006) The biology of the Cryptococcus neoformans species complex. Annu Rev Microbiol 60:69–105. doi: 10.1146/annurev.micro.60.080805.142102 PubMedCrossRefGoogle Scholar
  7. 7.
    Viviani MA, Cogliati M, Esposto MC et al (2006) Molecular analysis of 311 Cryptococcus neoformans isolates from a 30-month ECMM survey of cryptococcosis in Europe. FEMS Yeast Res 6:614–619. doi: 10.1111/j.1567-1364.2006.00081.x PubMedCrossRefGoogle Scholar
  8. 8.
    Meyer W, Aanensen DM, Boekhout T et al (2009) Consensus multi-locus sequence typing scheme for Cryptococcus neoformans and Cryptococcus gattii. Med Mycol 47:561–570. doi: 10.1080/13693780902953886 PubMedCrossRefPubMedCentralGoogle Scholar
  9. 9.
    Li W, Averette AF, Desnos-Ollivier M, Ni M, Dromer F, Heitman J (2012) Genetic diversity and genomic plasticity of Cryptococcus neoformans ad hybrid strains. G32:83–97. doi: 10.1534/g3.111.001255
  10. 10.
    Librado P, Rozas J (2009) Dnasp v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25:1451–1452. doi: 10.1093/bioinformatics/btp187 PubMedCrossRefGoogle Scholar
  11. 11.
    Simonsen KL, Churchill GA, Aquadro CF (1995) Properties of statistical tests of neutrality for DNA polymorphism data. Genetics 141:413–429PubMedPubMedCentralGoogle Scholar
  12. 12.
    Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) Mega5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739. doi: 10.1093/molbev/msr121 PubMedCrossRefPubMedCentralGoogle Scholar
  13. 13.
    Litvintseva AP, Thakur R, Vilgalys R, Mitchell TG (2006) Multilocus sequence typing reveals three genetic subpopulations of Cryptococcus neoformans var. grubii (serotype a), including a unique population in Botswana. Genetics 172:2223–2238. doi: 10.1534/genetics.105.046672 PubMedCrossRefPubMedCentralGoogle Scholar
  14. 14.
    Desnos-Ollivier M, Patel S, Spaulding AR et al (2010) Mixed infections and in vivo evolution in the human fungal pathogen Cryptococcus neoformans. MBio 1. doi: 10.1128/mBio.00091-10
  15. 15.
    Khayhan K, Hagen F, Pan W et al (2013) Geographically structured populations of Cryptococcus neoformans variety grubii in Asia correlate with HIV status and show a clonal population structure. PLoS One 8:e72222. doi: 10.1371/journal.pone.0072222 PubMedCrossRefPubMedCentralGoogle Scholar
  16. 16.
    Tintelnot K, Lemmer K, Losert H, Schar G, Polak A (2004) Follow-up of epidemiological data of cryptococcosis in Austria, Germany and Switzerland with special focus on the characterization of clinical isolates. Mycoses 47:455–464. doi: 10.1111/j.1439-0507.2004.01072.x PubMedCrossRefGoogle Scholar
  17. 17.
    Byrnes EJ 3rd, Bartlett KH, Perfect JR, Heitman J (2011) Cryptococcus gattii: an emerging fungal pathogen infecting humans and animals. Microbes Infect 13:895–907. doi: 10.1016/j.micinf.2011.05.009 PubMedCrossRefPubMedCentralGoogle Scholar
  18. 18.
    Hagen F, Colom MF, Swinne D et al (2012) Autochthonous and dormant Cryptococcus gattii infections in Europe. Emerg Infect Dis 18:1618–1624. doi: 10.3201/eid1810.120068 PubMedCrossRefPubMedCentralGoogle Scholar
  19. 19.
    Dromer F, Mathoulin-Pelissier S, Launay O, Lortholary O, French Cryptococcosis Study G (2007) Determinants of disease presentation and outcome during cryptococcosis: the CryptoA/D study. PLoS Med 4:e21. doi: 10.1371/journal.pmed.0040021 PubMedCrossRefPubMedCentralGoogle Scholar
  20. 20.
    Simwami SP, Khayhan K, Henk DA et al (2011) Low diversity Cryptococcus neoformans variety grubii multilocus sequence types from Thailand are consistent with an ancestral African origin. PLoS Pathog 7:e1001343. doi: 10.1371/journal.ppat.1001343 PubMedCrossRefPubMedCentralGoogle Scholar
  21. 21.
    Mihara T, Izumikawa K, Kakeya H et al (2012) Multilocus sequence typing of Cryptococcus neoformans in non-HIV associated cryptococcosis in Nagasaki, japan. Med Mycol 51:252–260. doi: 10.3109/13693786.2012.708883 PubMedCrossRefGoogle Scholar
  22. 22.
    Umeyama T, Ohno H, Minamoto F et al (2013) Determination of epidemiology of clinically isolated Cryptococcus neoformans strains in Japan by multilocus sequence typing. Jpn J Infect Dis 66:51–55PubMedCrossRefGoogle Scholar
  23. 23.
    Garcia-Hermoso D, Janbon G, Dromer F (1999) Epidemiological evidence for dormant Cryptococcus neoformans infection. J Clin Microbiol 37:3204–3209PubMedPubMedCentralGoogle Scholar
  24. 24.
    Perfect JR, Dismukes WE, Dromer F et al (2010) Clinical practice guidelines for the management of cryptococcal disease: 2010 update by the infectious diseases society of America. Clin Infect Dis 50:291–322. doi: 10.1086/649858 PubMedCrossRefGoogle Scholar
  25. 25.
    Pan W, Khayhan K, Hagen F et al (2012) Resistance of Asian Cryptococcus neoformans serotype a is confined to few microsatellite genotypes. PLoS One 7:e32868. doi: 10.1371/journal.pone.0032868 PubMedCrossRefPubMedCentralGoogle Scholar
  26. 26.
    Litvintseva AP, Carbone I, Rossouw J, Thakur R, Govender NP, Mitchell TG (2011) Evidence that the human pathogenic fungus Cryptococcus neoformans var. grubii may have evolved in Africa. PloS One 6:e19688. doi: 10.1371/journal.pone.0019688
  27. 27.
    Litvintseva AP, Mitchell TG (2012) Population genetic analyses reveal the African origin and strain variation of Cryptococcus neoformans var. grubii. PLoS Pathog 8:e1002495. doi: 10.1371/journal.ppat.1002495 PubMedCrossRefPubMedCentralGoogle Scholar
  28. 28.
    Cogliati M, Zamfirova RR, Tortorano AM, Viviani MA (2013) Molecular epidemiology of Italian clinical Cryptococcus neoformans var. grubii isolates. Med Mycol 51:499–506. doi: 10.3109/13693786.2012.751642 PubMedCrossRefGoogle Scholar
  29. 29.
    Hagen F, Illnait-Zaragozi MT, Meis JF et al (2012) Extensive genetic diversity within the Dutch clinical Cryptococcus neoformans population. J Clin Microbiol 50:1918–1926. doi: 10.1128/JCM.06750-11 PubMedCrossRefPubMedCentralGoogle Scholar
  30. 30.
    Ngamskulrungroj P, Gilgado F, Faganello J et al (2009) Genetic diversity of the cryptococcus species complex suggests that Cryptococcus gattii deserves to have varieties. PLoS One 4:e5862. doi: 10.1371/journal.pone.0005862 PubMedCrossRefPubMedCentralGoogle Scholar
  31. 31.
    Neuville S, Dromer F, Morin O et al (2003) Primary cutaneous cryptococcosis: a distinct clinical entity. Clin Infect Dis 36:337–347. doi: 10.1086/345956 PubMedCrossRefGoogle Scholar
  32. 32.
    Dromer F, Mathoulin S, Dupont B, Letenneur L, Ronin O (1996) Individual and environmental factors associated with infection due to Cryptococcus neoformans serotype D. Clin Infect Dis 23:91–96PubMedCrossRefGoogle Scholar
  33. 33.
    Cogliati M (2012) Global molecular epidemiology of Cryptococcus neoformans and Cryptococcus gattii: an atlas of the molecular types. Scientifica (Cairo) 2013:675213. doi: 10.1155/2013/675213 Google Scholar
  34. 34.
    Miglia KJ, Govender NP, Rossouw J et al (2011) Analysis of pediatric isolates of Cryptococcus neoformans from South Africa. J Clin Microbiol 49:307–314. doi: 10.1128/JCM.01277-10 PubMedCrossRefPubMedCentralGoogle Scholar
  35. 35.
    Lin X, Hull CM, Heitman J (2005) Sexual reproduction between partners of the same mating type in Cryptococcus neoformans. Nature 434:1017–1021. doi: 10.1038/nature03448 PubMedCrossRefGoogle Scholar
  36. 36.
    Lin X, Patel S, Litvintseva AP, Floyd A, Mitchell TG, Heitman J (2009) Diploids in the Cryptococcus neoformans serotype a population homozygous for the alpha mating type originate via unisexual mating. PLoS Pathog 5:e1000283. doi: 10.1371/journal.ppat.1000283 PubMedCrossRefPubMedCentralGoogle Scholar
  37. 37.
    Wiesner DL, Moskalenko O, Corcoran JM et al (2012) Cryptococcal genotype influences immunologic response and human clinical outcome after meningitis. MBio 3. doi: 10.1128/mBio.00196-12

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Andrea Sanchini
    • 1
    • 2
  • Ilka McCormick Smith
    • 1
  • Ludwig Sedlacek
    • 3
  • Roman Schwarz
    • 4
  • Kathrin Tintelnot
    • 1
  • Volker Rickerts
    • 1
  1. 1.Robert Koch-InstitutBerlinGermany
  2. 2.European Public Health Microbiology (EUPHEM) Training ProgrammeEuropean Centre for Disease Prevention and Control (ECDC)StockholmSweden
  3. 3.Institute for Medical Microbiology and Hospital EpidemiologyHannover Medical SchoolHannoverGermany
  4. 4.MVZ Labor Dr. Stein+KollegenMönchengladbachGermany

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