Multilocus Sequence Typing of Clinical Isolates of Cryptococcus from India

Abstract

Cryptococcosis is a life-threatening infection caused by Cryptococcus neoformans and C. gattii species complex. In the present study, to understand the molecular epidemiology of 208 clinical isolates of Cryptococcus from different parts of India, multilocus sequence typing (MLST) using ISHAM MLST consensus scheme for C. neoformans/C. gattii species complex was used. MLST analysis yielded a total of 10 Sequence Types (STs)—7 STs for C. neoformans and 3 for C. gattii species complex. The majority of isolates identified as C. neoformans belonged to molecular type VNI with predominant STs 31 and 93. Only 3 isolates of C. gattii species complex were obtained, belonging to ST58 and ST215 of VGI and ST69 of VGIV. Phylogenetic analysis revealed less diversity among the clinical Indian isolates compared to the global MLST database. No association between prevalent STs and HIV status, geographical origin or minimum inhibitory concentration (MIC) could be established.

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References

  1. 1.

    Heitman J, et al. Cryptococcus: from human pathogen to model yeast. Washington, DC: ASM press; 2011.

    Google Scholar 

  2. 2.

    Matsumoto MT, et al. Genotyping, serotyping and determination of mating-type of Cryptococcus neoformans clinical isolates from Sao Paulo State, Brazil. Rev Inst Med Trop Sao Paulo. 2007;49(1):41–7.

    PubMed  Article  Google Scholar 

  3. 3.

    Franzot SP, Salkin IF, Casadevall A. Cryptococcus neoformans var. grubii: separate varietal status for Cryptococcus neoformans serotype a isolates. J Clin Microbiol. 1999;37(3):838–40.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  4. 4.

    Hagen F, et al. Recognition of seven species in the Cryptococcus gattii/Cryptococcus neoformans species complex. Fungal Genet Biol. 2015;78:16–48.

    CAS  PubMed  Article  Google Scholar 

  5. 5.

    Hagen F, Lumbsch HT, Arsenijevic VA, Badali H, Bertout S, Billmyre RB, Bragulat MR, Cabañes FJ, Carbia M, Chakrabarti A, Chaturvedi S. Importance of resolving fungal nomenclature: the case of multiple pathogenic species in the Cryptococcus genus. Msphere. 2017;2(4).

  6. 6.

    Casadevall A, Perfect JR. Cryptococcus neoformans. Washington, DC: ASM press; 1998.

    Google Scholar 

  7. 7.

    Datta K, Bartlett KH, Marr KA. Cryptococcus gattii: emergence in western North America: exploitation of a novel ecological niche. Interdiscip Perspect Infect Dis. 2009;2009:176532.

    PubMed  PubMed Central  Article  Google Scholar 

  8. 8.

    Byrnes EJ 3rd, et al. A diverse population of Cryptococcus gattii molecular type VGIII in southern Californian HIV/AIDS patients. PLoS Pathog. 2011;7(9):e1002205.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  9. 9.

    Nyazika TK, et al. Cryptococcus tetragattii as a major cause of cryptococcal meningitis among HIV-infected individuals in Harare Zimbabwe. J Infect. 2016;72(6):745–52.

    PubMed  Article  Google Scholar 

  10. 10.

    Litvintseva AP, et al. Multilocus sequence typing reveals three genetic subpopulations of Cryptococcus neoformans var. grubii (serotype A), including a unique population in Botswana. Genetics. 2006;172(4):2223–38.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  11. 11.

    Bovers M, Hagen F, Boekhout T. Diversity of the Cryptococcus neoformans-Cryptococcus gattii species complex. Rev Iberoam Micol. 2008;25(1):S4-12.

    PubMed  Article  Google Scholar 

  12. 12.

    Bovers M, et al. Six monophyletic lineages identified within Cryptococcus neoformans and Cryptococcus gattii by multi-locus sequence typing. Fungal Genet Biol. 2008;45(4):400–21.

    CAS  PubMed  Article  Google Scholar 

  13. 13.

    Boekhout T, et al. Hybrid genotypes in the pathogenic yeast Cryptococcus neoformans. Microbiology. 2001;147(Pt 4):891–907.

    CAS  PubMed  Article  Google Scholar 

  14. 14.

    Meyer W, et al. Molecular typing of IberoAmerican Cryptococcus neoformans isolates. Emerg Infect Dis. 2003;9(2):189–95.

    PubMed  PubMed Central  Article  Google Scholar 

  15. 15.

    Sukroongreung S, et al. Serotypes of Cryptococcus neoformans isolated from patients prior to and during the AIDS era in Thailand. Mycopathologia. 1996;135(2):75–8.

    CAS  PubMed  Article  Google Scholar 

  16. 16.

    Farrer RA, Chang M, Davis MJ, van Dorp L, Yang DH, Shea T, Sewell TR, Meyer W, Balloux F, Edwards HM, Chanda D. A new lineage of Cryptococcus gattii (VGV) discovered in the central Zambezian Miombo woodlands. Mbio. 2019;10(6).

  17. 17.

    Choi YH, et al. Prevalence of the VNIc genotype of Cryptococcus neoformans in non-HIV-associated cryptococcosis in the Republic of Korea. FEMS Yeast Res. 2010;10(6):769–78.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  18. 18.

    Khayhan K, et al. Geographically structured populations of Cryptococcus neoformans variety grubii in Asia correlate with HIV status and show a clonal population structure. PLoS ONE. 2013;8(9):e72222.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  19. 19.

    Kaocharoen S, et al. Molecular epidemiology reveals genetic diversity amongst isolates of the Cryptococcus neoformans/C. gattii species complex in Thailand. PLoS Negl Trop Dis. 2013;7(7):2297.

    Article  Google Scholar 

  20. 20.

    World Health Organization. Guidelines for the diagnosis, prevention, and management of cryptococcal disease in HIV-infected adults, adolescents and children, March 2018: supplement to the 2016 consolidated guidelines of the use of antiretroviral drugs for treating and preventing HIV infection. 2018; Available from: https://www.who.int/iris/handle/10665/260399.

  21. 21.

    Kumar S, et al. Cryptococcal meningitis in HIV infected: experience from a North Indian tertiary center. Neurol India. 2008;56(4):444–9.

    PubMed  Article  Google Scholar 

  22. 22.

    Jain N, et al. Molecular epidemiology of clinical Cryptococcus neoformans strains from India. J Clin Microbiol. 2005;43(11):5733–42.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  23. 23.

    Baradkar V, et al. Prevalence and clinical presentation of cryptococcal meningitis among HIV seropositive patients. Indian J Sex Transm Dis. 2009;30(1):19–22.

    Article  Google Scholar 

  24. 24.

    Patel AK, et al. Management of cryptococcal meningitis in HIV-infected patients: experience from western India. Indian J Sex Transm Dis. 2010;31(1):22–6.

    Article  Google Scholar 

  25. 25.

    Saha DC, et al. Detection of cryptococcus by conventional, serological and molecular methods. J Med Microbiol. 2009;58(Pt 8):1098–105.

    CAS  PubMed  Article  Google Scholar 

  26. 26.

    Prakash A, et al. Environmental distribution of Cryptococcus species and some other yeast-like fungi in India. Mycoses. 2017;61:305–13.

    Article  CAS  Google Scholar 

  27. 27.

    Chowdhary A, et al. First environmental isolation of Cryptococcus gattii, genotype AFLP5, from India and a global review. Mycoses. 2013;56(3):222–8.

    CAS  PubMed  Article  Google Scholar 

  28. 28.

    Chowdhary A, et al. Environmental prevalence of Cryptococcus neoformans and Cryptococcus gattii in India: an update. Crit Rev Microbiol. 2012;38(1):1–16.

    PubMed  Article  Google Scholar 

  29. 29.

    Chowdhary A, et al. In vitro antifungal susceptibility profiles and genotypes of 308 clinical and environmental isolates of Cryptococcus neoformans var. grubii and Cryptococcus gattii serotype B from north-western India. J Med Microbiol. 2011;60(7):961–7.

    PubMed  Article  Google Scholar 

  30. 30.

    Chakrabarti A, et al. Isolation of Cryptococcus neoformans var. gattii from Eucalyptus camaldulensis in India. J Clin Microbiol. 1997;35(12):3340–2.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  31. 31.

    Randhawa HS, Kowshik T, Khan ZU. Decayed wood of Syzygium cumini and Ficus religiosa living trees in Delhi/New Delhi metropolitan area as natural habitat of Cryptococcus neoformans. Med Mycol. 2003;41(3):199–209.

    CAS  PubMed  Article  Google Scholar 

  32. 32.

    Bedi NG, et al. Seasonal prevalence of Cryptococcus neoformans var. grubii and Cryptococcus gattii inhabiting Eucalyptus terreticornis and Eucalyptus camaldulensis trees in Jabalpur city of Madhya Pradesh, Central India. J Mycol Med. 2012;22(4):341–7.

    CAS  PubMed  Article  Google Scholar 

  33. 33.

    Vidotto V, et al. A new caffeic acid minimal synthetic medium for the rapid identification of Cryptococcus neoformans isolates. Rev Iberoam Micol. 2004;21(2):87–9.

    PubMed  Google Scholar 

  34. 34.

    Zimmer B, Roberts GD. Rapid selective urease test for presumptive identification of Cryptococcus neoformans. J Clin Microbiol. 1979;10(3):380–1.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  35. 35.

    Randhawa H, et al. The expanding host tree species spectrum of Cryptococcus gattii and Cryptococcus neoformans and their isolations from surrounding soil in India. Sabouraudia. 2008;46(8):823–33.

    CAS  Article  Google Scholar 

  36. 36.

    Klein K, et al. Identification of Cryptococcus gattii by use of L-canavanine glycine bromothymol blue medium and DNA sequencing. J Clin Microbiol. 2009;47(11):3669–72.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  37. 37.

    Meyer W, et al. Consensus multi-locus sequence typing scheme for Cryptococcus neoformans and Cryptococcus gattii. Med Mycol. 2009;47(6):561–70.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  38. 38.

    Fraser JA, et al. Same-sex mating and the origin of the Vancouver island Cryptococcus gattii outbreak. Nature. 2005;437(7063):1360–4.

    CAS  PubMed  Article  Google Scholar 

  39. 39.

    D’Souza CA, et al. Investigation of the basis of virulence in serotype a strains of Cryptococcus neoformans from apparently immunocompetent individuals. Curr Genet. 2004;46(2):92–102.

    CAS  PubMed  Article  Google Scholar 

  40. 40.

    Clinical and Laboratory Standards Institute. Reference method for broth dilution antifungal susceptibility testing of yeasts; approved standard-third edition M27–A3. CLSI: W., PA, USA; 2008.

    Google Scholar 

  41. 41.

    Simwami SP, et al. Low diversity Cryptococcus neoformans variety grubii multilocus sequence types from Thailand are consistent with an ancestral African origin. PLoS Pathog. 2011;7(4):e1001343.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  42. 42.

    Umeyama T, et al. Determination of epidemiology of clinically isolated Cryptococcus neoformans strains in Japan by multilocus sequence typing. Jpn J Infect Dis. 2013;66(1):51–5.

    PubMed  Article  Google Scholar 

  43. 43.

    Mihara T, et al. Multilocus sequence typing of Cryptococcus neoformans in non-HIV associated cryptococcosis in Nagasaki Japan. Med Mycol. 2013;51(3):252–60.

    CAS  PubMed  Article  Google Scholar 

  44. 44.

    Liaw SJ, Wu HC, Hsueh PR. Microbiological characteristics of clinical isolates of Cryptococcus neoformans in Taiwan: serotypes, mating types, molecular types, virulence factors, and antifungal susceptibility. Clin Microbiol Infect. 2009;16(6):696–703.

    PubMed  Article  CAS  Google Scholar 

  45. 45.

    Tay ST, et al. Determination of molecular types and genetic heterogeneity of Cryptococcus neoformans and C gattii in Malaysia. Med Mycol. 2006;44(7):617–22.

    CAS  PubMed  Article  Google Scholar 

  46. 46.

    Chen M, et al. Molecular epidemiology of Cryptococcus neoformans species complex isolates from HIV-positive and HIV-negative patients in southeast China. Front Med China. 2010;4(1):117–26.

    Article  Google Scholar 

  47. 47.

    Cogliati M. Global molecular epidemiology of Cryptococcus neoformans and Cryptococcus gattii: an atlas of the molecular types. Scientifica (Cairo). 2013;2013:675213.

    Google Scholar 

  48. 48.

    Dou HT, et al. Molecular epidemiology of Cryptococcus neoformans and Cryptococcus gattii in China between 2007 and 2013 using multilocus sequence typing and the diversiLab system. Eur J Clin Microbiol Infect Dis. 2015;34(4):753–62.

    PubMed  Article  Google Scholar 

  49. 49.

    Park SH, et al. Molecular epidemiology of clinical Cryptococcus neoformans isolates in Seoul Korea. Mycobiology. 2014;42(1):73–8.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  50. 50.

    Wu SY, et al. Molecular characterisation of clinical Cryptococcus neoformans and Cryptococcus gattii isolates from Sichuan province China. Mycoses. 2015;58(5):280–7.

    PubMed  Article  Google Scholar 

  51. 51.

    Hatthakaroon C, et al. Molecular epidemiology of cryptococcal genotype VNIc/ST5 in siriraj hospital, Thailand. PLoS ONE. 2017;12(3):e0173744.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  52. 52.

    Smith KD, et al. Increased antifungal drug resistance in clinical isolates of Cryptococcus neoformans in Uganda. Antimicrob Agents Chemother. 2015;59(12):7197–204.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  53. 53.

    Ferreira-Paim K, et al. MLST-based population genetic analysis in a global context reveals clonality amongst Cryptococcus neoformans var. grubii VNI isolates from HIV patients in southeastern Brazil. PLoS Negl Trop Dis. 2017;11(1):e0005223.

    PubMed  PubMed Central  Article  Google Scholar 

  54. 54.

    Prakash A, et al. Genotypic diversity in clinical and environmental isolates of Cryptococcus neoformans from India using multilocus microsatellite and multilocus sequence typing. Mycoses. 2020;63(3):284–93.

    CAS  PubMed  Article  Google Scholar 

  55. 55.

    Wiesner DL, Moskalenko O, Corcoran JM, McDonald T, Rolfes MA, Meya DB, Kajumbula H, Kambugu A, Bohjanen PR, Knight JF, Boulware DR. Cryptococcal genotype influences immunologic response and human clinical outcome after meningitis. MBio. 2012;3(5).

  56. 56.

    Chen J, et al. Cryptococcus neoformans strains and infection in apparently immunocompetent patients. China Emerg Infect Dis. 2008;14(5):755–62.

    CAS  PubMed  Article  Google Scholar 

  57. 57.

    Trilles L, et al. Regional pattern of the molecular types of Cryptococcus neoformans and Cryptococcus gattii in Brazil. Mem Inst Oswaldo Cruz. 2008;103(5):455–62.

    CAS  PubMed  Article  Google Scholar 

  58. 58.

    Lockhart SR, et al. Cryptococcus gattii in the United States: genotypic diversity of human and veterinary isolates. PLoS ONE. 2013;8(9):e74737.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  59. 59.

    Ngamskulrungroj P, et al. Genetic diversity of the cryptococcus species complex suggests that Cryptococcus gattii deserves to have varieties. PLoS ONE. 2009;4(6):e5862.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  60. 60.

    Castanon-Olivares LR, et al. Crytococcus neoformans var. gattii in an AIDS patient: first observation in Mexico. J Med Vet Mycol. 1997;35(1):57–9.

    CAS  PubMed  Article  Google Scholar 

  61. 61.

    Escandon P, et al. Isolation of Cryptococcus gattii molecular type VGIII, from Corymbia ficifolia detritus in Colombia. Med Mycol. 2010;48(4):675–8.

    CAS  PubMed  Article  Google Scholar 

  62. 62.

    Lizarazo J, et al. Retrospective study of the epidemiology and clinical manifestations of Cryptococcus gattii infections in Colombia from 1997–2011. PLoS Negl Trop Dis. 2014;8(11):e3272.

    PubMed  PubMed Central  Article  Google Scholar 

  63. 63.

    Litvintseva AP, et al. Prevalence of clinical isolates of Cryptococcus gattii serotype C among patients with AIDS in sub-Saharan Africa. J Infect Dis. 2005;192(5):888–92.

    CAS  PubMed  Article  Google Scholar 

  64. 64.

    Brandt ME, et al. Trends in antifungal drug susceptibility of Cryptococcus neoformans isolates in the United States: 1992 to 1994 and 1996 to 1998. Antimicrob Agents Chemother. 2001;45(11):3065–9.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  65. 65.

    Govender NP, et al. Trends in antifungal drug susceptibility of Cryptococcus neoformans isolates obtained through population-based surveillance in South Africa in 2002–2003 and 2007–2008. Antimicrob Agents Chemother. 2011;55(6):2606–11.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  66. 66.

    Tewari A, et al. Comparative analysis of the Vitek 2 antifungal susceptibility system and E-test with the CLSI M27–A3 broth microdilution method for susceptibility testing of Indian clinical isolates of Cryptococcus neoformans. Mycopathologia. 2012;173(5–6):427–33.

    CAS  PubMed  Article  Google Scholar 

  67. 67.

    Datta K, et al. Fluconazole and itraconazole susceptibility of clinical isolates of Cryptococcus neoformans at a tertiary care centre in India: a need for care. J Antimicrob Chemother. 2003;52(4):683–6.

    CAS  PubMed  Article  Google Scholar 

  68. 68.

    Capoor MR, et al. Current scenario of cryptococcosis and antifungal susceptibility pattern in India: a cause for reappraisal. Mycoses. 2008;51(3):258–65.

    CAS  PubMed  Article  Google Scholar 

  69. 69.

    Pfaller MA, et al. 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(3):252–9.

    CAS  PubMed  Article  Google Scholar 

  70. 70.

    Espinel-Ingroff A, et al. Cryptococcus neoformans-Cryptococcus gattii species complex: an international study of wild-type susceptibility endpoint distributions and epidemiological cutoff values for amphotericin B and flucytosine. Antimicrob Agents Chemother. 2012;56(6):3107–13.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

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Acknowledgements

The authors are grateful to laboratory technologist Mr. Bhaskar Rana for maintaining the cultures

Funding

This work was supported by Department of Biotechnology, India (BT/PR5193/MED/29/463/2012), and All India Institute of Medical Sciences, New Delhi, India (A-08).

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Xess, I., Pandey, M., Dabas, Y. et al. Multilocus Sequence Typing of Clinical Isolates of Cryptococcus from India. Mycopathologia (2021). https://doi.org/10.1007/s11046-020-00500-6

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Keywords

  • Cryptococcus
  • Multilocus sequence typing (MLST)
  • Antifungal susceptibility
  • India