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VGIII Isolates Causing Infections in HIV/AIDS Patients in Southern California: Identification of the Local Environmental Source as Arboreal


The environmentally-acquired human pathogen C. gattii is responsible for ongoing and expanding outbreaks in the Western United States and Canada. C. gattii comprises four distinct molecular types: VGI, VGII, VGIII, and VGIV. Molecular types VGI, VGII, and VGIII have been isolated from patients and animals throughout the Western US. The Pacific Northwest and Canadian outbreak is primarily caused by C. gattii VGII. VGIII is responsible for ongoing infections in HIV/AIDS patients in Southern California. However, only two environmental C. gattii isolates have ever been identified from the Californian environment: CBS7750 (VGII) and WM161 (VGIII). We sought to collect environmental samples from areas that had confirmed reports of clinical or veterinary infections. Here we report the isolation of C. gattii VGI and VGIII from environmental soil and tree samples. C. gattii isolates were obtained from three novel tree species: Canary Island pine, American sweetgum, and a Pohutukawa tree. Genetic analysis provides robust evidence that these environmental isolates are the source of human infections.


Vyšlo v časopise: VGIII Isolates Causing Infections in HIV/AIDS Patients in Southern California: Identification of the Local Environmental Source as Arboreal. PLoS Pathog 10(8): e32767. doi:10.1371/journal.ppat.1004285
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1004285

Souhrn

The environmentally-acquired human pathogen C. gattii is responsible for ongoing and expanding outbreaks in the Western United States and Canada. C. gattii comprises four distinct molecular types: VGI, VGII, VGIII, and VGIV. Molecular types VGI, VGII, and VGIII have been isolated from patients and animals throughout the Western US. The Pacific Northwest and Canadian outbreak is primarily caused by C. gattii VGII. VGIII is responsible for ongoing infections in HIV/AIDS patients in Southern California. However, only two environmental C. gattii isolates have ever been identified from the Californian environment: CBS7750 (VGII) and WM161 (VGIII). We sought to collect environmental samples from areas that had confirmed reports of clinical or veterinary infections. Here we report the isolation of C. gattii VGI and VGIII from environmental soil and tree samples. C. gattii isolates were obtained from three novel tree species: Canary Island pine, American sweetgum, and a Pohutukawa tree. Genetic analysis provides robust evidence that these environmental isolates are the source of human infections.


Zdroje

1. ReedC, BiggerstaffM, FinelliL, KooninLM, BeauvaisD, et al. (2013) Novel framework for assessing epidemiologic effects of influenza epidemics and pandemics. Emerg Infect Dis 19: 85–91.

2. DawoodFS, IulianoAD, ReedC, MeltzerMI, ShayDK, et al. (2012) Estimated global mortality associated with the first 12 months of 2009 pandemic influenza A H1N1 virus circulation: a modelling study. Lancet Infect Dis 12: 687–695.

3. JutlaA, WhitcombeE, HasanN, HaleyB, AkandaA, et al. (2013) Environmental factors influencing epidemic cholera. Am J Trop Med Hyg 89: 597–607.

4. ChristianKA, IjazK, DowellSF, ChowCC, ChitaleRA, et al. (2013) What we are watching–five top global infectious disease threats, 2012: a perspective from CDC's Global Disease Detection Operations Center. Emerg Health Threats J 6: 20632.

5. de Hoog GS, Guarro J, Gene J, Figueras MJ (2000) Atlas of Clinical Fungi. Utrecht: Centraalbureau voor Schimmelcultures/Universitat Rovira i Virgili.

6. FisherFS, BultmanMW, JohnsonSM, PappagianisD, ZaborskyE (2007) Coccidioides niches and habitat parameters in the southwestern United States: a matter of scale. Ann N Y Acad Sci 1111: 47–72.

7. PappagianisD (1988) Epidemiology of coccidioidomycosis. Curr Top Med Mycol 2: 199–238.

8. HowardDH (1984) The epidemiology and ecology of blastomycosis, coccidioidomycosis and histoplasmosis. Zentralbl Bakteriol Mikrobiol Hyg A 257: 219–227.

9. BarkerBM, JewellKA, KrokenS, OrbachMJ (2007) The population biology of coccidioides: epidemiologic implications for disease outbreaks. Ann N Y Acad Sci 1111: 147–163.

10. Neblett FanfairR, BenedictK, BosJ, BennettSD, LoYC, et al. (2012) Necrotizing cutaneous mucormycosis after a tornado in Joplin, Missouri, in 2011. N Engl J Med 367: 2214–2225.

11. ChamanyS, MirzaSA, FlemingJW, HowellJF, LenhartSW, et al. (2004) A large histoplasmosis outbreak among high school students in Indiana, 2001. Pediatr Infect Dis J 23: 909–914.

12. CairnsL, BlytheD, KaoA, PappagianisD, KaufmanL, et al. (2000) Outbreak of coccidioidomycosis in Washington state residents returning from Mexico. Clin Infect Dis 30: 61–64.

13. BrownGD, DenningDW, GowNA, LevitzSM, NeteaMG, et al. (2012) Hidden killers: human fungal infections. Sci Transl Med 4: 165rv113.

14. HagenF, ColomMF, SwinneD, TintelnotK, IattaR, et al. (2012) Autochthonous and dormant Cryptococcus gattii infections in Europe. Emerging Infectious Diseases 18: 1618–1624.

15. MeyerW, AanensenDM, BoekhoutT, CogliatiM, DiazMR, et al. (2009) Consensus multi-locus sequence typing scheme for Cryptococcus neoformans and Cryptococcus gattii. Med Mycol 47: 1–14.

16. CogliatiM (2013) Global molecular epidemiology of Cryptococcus neoformans and Cryptococcus gattii: An atlas of the molecular types. Scientifica 2013: 23.

17. ParkBJ, WannemuehlerKA, MarstonBJ, GovenderN, PappasPG, et al. (2009) Estimation of the current global burden of cryptococcal meningitis among persons living with HIV/AIDS. AIDS 23: 525–530.

18. PyrgosV, SeitzAE, SteinerCA, PrevotsDR, WilliamsonPR (2013) Epidemiology of cryptococcal meningitis in the US: 1997–2009. PLOS ONE 8: e56269.

19. DixonDM, McNeilMM, CohenML, GellinBG, La MontagneJR (1996) Fungal infections: a growing threat. Public Health Rep 111: 226–235.

20. SpringerDJ, ChaturvediV (2010) Projecting global occurrence of Cryptococcus gattii. Emerg Infect Dis 16: 14–20.

21. ByrnesEJ3rd, LiW, LewitY, MaH, VoelzK, et al. (2010) Emergence and pathogenicity of highly virulent Cryptococcus gattii genotypes in the Northwest United States. PLOS Pathogens 6: e1000850.

22. ByrnesEJ3rd, LiW, RenP, LewitY, VoelzK, et al. (2011) A diverse population of Cryptococcus gattii molecular type VGIII in Southern Californian HIV/AIDS patients. PLOS Pathogens 7: e1002205.

23. MeyerW, CastanedaA, JacksonS, HuynhM, CastanedaE (2003) Molecular typing of IberoAmerican Cryptococcus neoformans isolates. Emerging Infectious Diseases 9: 189–195.

24. NgamskulrungrojP, GilgadoF, FaganelloJ, LitvintsevaAP, LealAL, et al. (2009) Genetic diversity of the Cryptococcus species complex suggests that Cryptococcus gattii deserves to have varieties. PLOS ONE 4: e5862.

25. FraserJA, GilesSS, WeninkEC, Geunes-BoyerSG, WrightJR, et al. (2005) Same-sex mating and the origin of the Vancouver Island Cryptococcus gattii outbreak. Nature 437: 1360–1364.

26. ByrnesEJ3rd, BildfellRJ, FrankSA, MitchellTG, MarrKA, et al. (2009) Molecular evidence that the range of the Vancouver Island outbreak of Cryptococcus gattii infection has expanded into the Pacific Northwest in the United States. J Infect Dis 199: 1081–1086.

27. HagenF, CeresiniPC, PolacheckI, MaH, van NieuwerburghF, et al. (2013) Ancient dispersal of the human fungal pathogen Cryptococcus gattii from the Amazon rainforest. PLOS ONE 8: e71148.

28. BoversM, HagenF, KuramaeEE, BoekhoutT (2008) Six monophyletic lineages identified within Cryptococcus neoformans and Cryptococcus gattii by multi-locus sequence typing. Fungal Genet Biol 45: 400–421.

29. SharptonTJ, NeafseyDE, GalaganJE, TaylorJW (2008) Mechanisms of intron gain and loss in Cryptococcus. Genome Biol 9: R24.

30. XuJ, VilgalysR, MitchellTG (2000) Multiple gene genealogies reveal recent dispersion and hybridization in the human pathogenic fungus Cryptococcus neoformans. Mol Ecol 9: 1471–1481.

31. XuJ, YanZ, GuoH (2009) Divergence, hybridization, and recombination in the mitochondrial genome of the human pathogenic yeast Cryptococcus gattii. Mol Ecol 18: 2628–2642.

32. BoversM, HagenF, KuramaeEE, BoekhoutT (2009) Promiscuous mitochondria in Cryptococcus gattii. FEMS Yeast Res 9: 489–503.

33. VoelzK, MaH, PhadkeS, ByrnesEJ, ZhuP, et al. (2013) Transmission of hypervirulence traits via sexual reproduction within and between lineages of the human fungal pathogen Cryptococcus gattii. PLOS Genetics 9: e1003771.

34. MaH, HagenF, StekelDJ, JohnstonSA, SionovE, et al. (2009) The fatal fungal outbreak on Vancouver Island is characterized by enhanced intracellular parasitism driven by mitochondrial regulation. Proc Natl Acad Sci U S A 106: 12980–12985.

35. OlsonA, StenlidJ (2001) Plant pathogens. Mitochondrial control of fungal hybrid virulence. Nature 411: 438.

36. ByrnesEJ3rd, BildfellRJ, DearingPL, ValentineBA, HeitmanJ (2009) Cryptococcus gattii with bimorphic colony types in a dog in western Oregon: additional evidence for expansion of the Vancouver Island outbreak. J Vet Diagn Invest 21: 133–136.

37. WalravenCJ, GersteinW, HardisonSE, WormleyF, LockhartSR, et al. (2011) Fatal disseminated Cryptococcus gattii infection in New Mexico. PLOS ONE 6: e28625.

38. MacDougallL, KiddSE, GalanisE, MakS, LeslieMJ, et al. (2007) Spread of Cryptococcus gattii in British Columbia, Canada, and detection in the Pacific Northwest, USA. Emerg Infect Dis 13: 42–50.

39. UptonA, FraserJA, KiddSE, BretzC, BartlettKH, et al. (2007) First contemporary case of human infection with Cryptococcus gattii in Puget Sound: Evidence for spread of the Vancouver Island outbreak. J Clin Microbiol 45: 3086–3088.

40. ChaturvediS, RenP, NarasipuraSD, ChaturvediV (2005) Selection of optimal host strain for molecular pathogenesis studies on Cryptococcus gattii. Mycopathologia 160: 207–215.

41. OkamotoK, HatakeyamaS, ItoyamaS, NukuiY, YoshinoY, et al. (2010) Cryptococcus gattii genotype VGIIa infection in man, Japan, 2007. Emerging Infectious Diseases 16: 1155–1157.

42. NgamskulrungrojP, SerenaC, GilgadoF, MalikR, MeyerW (2011) Global VGIIa isolates are of comparable virulence to the major fatal Cryptococcus gattii Vancouver Island outbreak genotype. Clinical Microbiology and Infection 17: 251–258.

43. KaocharoenS, NgamskulrungrojP, FiracativeC, TrillesL, PiyabongkarnD, et al. (2013) Molecular epidemiology reveals genetic diversity amongst isolates of the Cryptococcus neoformans/C. gattii species complex in Thailand. PLoS Negl Trop Dis 7: e2297.

44. ChaturvediS, DyavaiahM, LarsenRA, ChaturvediV (2005) Cryptococcus gattii in AIDS patients, southern California. Emerging Infectious Diseases 11: 1686–1692.

45. LockhartSR, IqbalN, HarrisJR, GrossmanNT, DebessE, et al. (2013) Cryptococcus gattii in the United States: genotypic diversity of human and veterinary isolates. PLOS ONE 8: e74737.

46. TrillesL, Lazera MdosS, WankeB, OliveiraRV, BarbosaGG, et al. (2008) Regional pattern of the molecular types of Cryptococcus neoformans and Cryptococcus gattii in Brazil. Mem Inst Oswaldo Cruz 103: 455–462.

47. FiracativeC, TorresG, RodriguezMC, EscandonP (2011) First environmental isolation of Cryptococcus gattii serotype B, from Cucuta, Colombia. Biomedica 31: 118–123.

48. ChoiYH, NgamskulrungrojP, VarmaA, SionovE, HwangSM, et al. (2010) Prevalence of the VNIc genotype of Cryptococcus neoformans in non-HIV-associated cryptococcosis in the Republic of Korea. FEMS Yeast Res 10: 769–778.

49. ChowdharyA, PrakashA, RandhawaHS, KathuriaS, HagenF, et al. (2013) First environmental isolation of Cryptococcus gattii, genotype AFLP5, from India and a global review. Mycoses 56: 222–228.

50. OlivaresLR, MartinezKM, CruzRM, RiveraMA, MeyerW, et al. (2009) Genotyping of Mexican Cryptococcus neoformans and C. gattii isolates by PCR-fingerprinting. Med Mycol 47: 713–721.

51. GrosseP, TintelnotK, SollnerO, SchmitzB (2001) Encephalomyelitis due to Cryptococcus neoformans var gattii presenting as spinal tumour: case report and review of the literature. J Neurol Neurosurg Psychiatry 70: 113–116.

52. ByrnesEJ3rd, MarrKA (2011) The outbreak of Cryptococcus gattii in western North America: epidemiology and clinical issues. Current Infectious Disease Reports 13: 256–261.

53. EscandonP, SanchezA, FiracativeC, CastanedaE (2010) Isolation of Cryptococcus gattii molecular type VGIII, from Corymbia ficifolia detritus in Colombia. Med Mycol 48: 675–678.

54. MazzaM, RefojoN, Bosco-BorgeatME, TavernaCG, TroveroAC, et al. (2013) Cryptococcus gattii in urban trees from cities in North-eastern Argentina. Mycoses

55. CallejasA, OrdonezN, RodriguezMC, CastanedaE (1998) First isolation of Cryptococcus neoformans var. gattii, serotype C, from the environment in Colombia. Med Mycol 36: 341–344.

56. HallidayCL, BuiT, KrockenbergerM, MalikR, EllisDH, et al. (1999) Presence of alpha and a mating types in environmental and clinical collections of Cryptococcus neoformans var. gattii strains from Australia. J Clin Microbiol 37: 2920–2926.

57. SaracliMA, YildiranST, SenerK, GonlumA, DoganciL, et al. (2006) Genotyping of Turkish environmental Cryptococcus neoformans var. neoformans isolates by pulsed field gel electrophoresis and mating type. Mycoses 49: 124–129.

58. MontagnaMT (2002) A note on the isolation of Cryptococcus neoformans serotype A MATa strain from the Italian environment. Med Mycol 40: 593–595.

59. KiddSE, HagenF, TscharkeRL, HuynhM, BartlettKH, et al. (2004) A rare genotype of Cryptococcus gattii caused the cryptococcosis outbreak on Vancouver Island (British Columbia, Canada). Proc Natl Acad Sci USA 101: 17258–17263.

60. KiddSE, ChowY, MakS, BachPJ, ChenH, et al. (2007) Characterization of environmental sources of the human and animal pathogen Cryptococcus gattii in British Columbia, Canada, and the Pacific Northwest of the United States. Applied Environmental Microbiology 73: 1433–1443.

61. Byrnes EJ, III (2010) Examination of the molecular epidemiology, expansion, population structure, and virulence of the emerging fungal pathogen Cryptococcus gattii in the United States [Ph.D.]. Ann Arbor: Duke University. 311 p.

62. MagditchDA, LiuTB, XueC, IdnurmA (2012) DNA mutations mediate microevolution between host-adapted forms of the pathogenic fungus Cryptococcus neoformans. PLoS Pathog 8: e1002936.

63. ToffalettiDL, NielsenK, DietrichF, HeitmanJ, PerfectJR (2004) Cryptococcus neoformans mitochondrial genomes from serotype A and D strains do not influence virulence. Curr Genet 46: 193–204.

64. ZhuP, ZhaiB, LinX, IdnurmA (2013) Congenic strains for genetic analysis of virulence traits in Cryptococcus gattii. Infect Immun 81: 2616–2625.

65. YanZ, XuJ (2003) Mitochondria are inherited from the MATa parent in crosses of the basidiomycete fungus Cryptococcus neoformans. Genetics 163: 1315–1325.

66. YanZ, SunS, ShahidM, XuJ (2007) Environment factors can influence mitochondrial inheritance in the fungus Cryptococcus neoformans. Fungal Genet Biol 44: 315–322.

67. D'SouzaCA, KronstadJW, TaylorG, WarrenR, YuenM, et al. (2011) Genome variation in Cryptococcus gattii, an emerging pathogen of immunocompetent hosts. mBio 2: e00342–00310.

68. SpringerDJ, RenP, RainaR, DongY, BehrMJ, et al. (2010) Extracellular fibrils of pathogenic yeast Cryptococcus gattii are important for ecological niche, murine virulence and human neutrophil interactions. PLOS ONE 5: e10978.

69. Kwon-ChungKJ, BennettJE (1984) Epidemiologic differences between the two varieties of Cryptococcus neoformans. Am J Epidemiol 120: 123–130.

70. Kwon-ChungKJ, BennettJE (1984) High prevalence of Cryptococcus neoformans var. gattii in tropical and subtropical regions. Zentralbl Bakteriol Mikrobiol Hyg [A] 257: 213–218.

71. PfeifferT, EllisD (1991) Environmental isolation of Cryptococcus neoformans var. gattii from California. J Infect Dis 163: 929–930.

72. ReedKD, MeeceJK, ArcherJR, PetersonAT (2008) Ecologic niche modeling of Blastomyces dermatitidis in Wisconsin. PLoS ONE 3: e2034.

73. McCulloughMJ, DiSalvoAF, ClemonsKV, ParkP, StevensDA (2000) Molecular epidemiology of Blastomyces dermatitidis. Clin Infect Dis 30: 328–335.

74. MandelMA, BarkerBM, KrokenS, RounsleySD, OrbachMJ (2007) Genomic and population analyses of the mating type loci in Coccidioides species reveal evidence for sexual reproduction and gene acquisition. Eukaryot Cell 6: 1189–1199.

75. ByrnesEJ3rd, LiW, LewitY, PerfectJR, CarterDA, et al. (2009) First reported case of Cryptococcus gattii in the Southeastern USA: implications for travel-associated acquisition of an emerging pathogen. PLOS ONE 4: e5851.

76. SellersB, HallP, Cine-GowdieS, HaysAL, PatelK, et al. (2012) Cryptococcus gattii: an emerging fungal pathogen in the Southeastern United States. Am J Med Sci 343: 510–511.

77. LitvintsevaAP, KestenbaumL, VilgalysR, MitchellTG (2005) Comparative analysis of environmental and clinical populations of Cryptococcus neoformans. J Clin Microbiol 43: 556–564.

78. Loperena-AlvarezY, RenP, LiX, Schoonmaker-BoppDJ, RuizA, et al. (2010) Genotypic characterization of environmental isolates of Cryptococcus gattii from Puerto Rico. Mycopathologia 170: 279–285.

79. LitvintsevaAP, ThakurR, VilgalysR, MitchellTG (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.

80. VelagapudiR, HsuehYP, Geunes-BoyerS, WrightJR, HeitmanJ (2009) Spores as infectious propagules of Cryptococcus neoformans. Infection and Immunity 77: 4345–4355.

81. SuC, EvansD, ColeRH, KissingerJC, AjiokaJW, et al. (2003) Recent expansion of Toxoplasma through enhanced oral transmission. Science 299: 414–416.

82. WendteJM, MillerMA, LambournDM, MagargalSL, JessupDA, et al. (2010) Self-mating in the definitive host potentiates clonal outbreaks of the apicomplexan parasites Sarcocystis neurona and Toxoplasma gondii. PLoS Genet 6: e1001261.

83. JanbonG, OrmerodKL, PauletD, ByrnesEJ3rd, YadavV, et al. (2014) Analysis of the genome and transcriptome of Cryptococcus neoformans var. grubii reveals complex RNA expression and microevolution leading to virulence attenuation. PLoS Genet 10: e1004261.

84. PriceJR, GolubchikT, ColeK, WilsonDJ, CrookDW, et al. (2014) Whole-genome sequencing shows that patient-to-patient transmission rarely accounts for acquisition of Staphylococcus aureus in an intensive care unit. Clin Infect Dis 58: 609–618.

85. SteenbergenJN, ShumanHA, CasadevallA (2001) Cryptococcus neoformans interactions with amoebae suggest an explanation for its virulence and intracellular pathogenic strategy in macrophages. Proc Natl Acad Sci U S A 98: 15245–15250.

86. SteenbergenJN, NosanchukJD, MalliarisSD, CasadevallA (2003) Cryptococcus neoformans virulence is enhanced after growth in the genetically malleable host Dictyostelium discoideum. Infect Immun 71: 4862–4872.

87. MylonakisE, AusubelFM, PerfectJR, HeitmanJ, CalderwoodSB (2002) Killing of Caenorhabditis elegans by Cryptococcus neoformans as a model of yeast pathogenesis. Proc Natl Acad Sci U S A 99: 15675–15680.

88. McClellandEE, PerrineWT, PottsWK, CasadevallA (2005) Relationship of virulence factor expression to evolved virulence in mouse-passaged Cryptococcus neoformans lines. Infect Immun 73: 7047–7050.

89. CurrieB, SanatiH, IbrahimAS, EdwardsJEJr, CasadevallA, et al. (1995) Sterol compositions and susceptibilities to amphotericin B of environmental Cryptococcus neoformans isolates are changed by murine passage. Antimicrob Agents Chemother 39: 1934–1937.

90. FranzotSP, MukherjeeJ, CherniakR, ChenL, HamdanJS, et al. (1998) Microevolution of a standard strain of Cryptococcus neoformans resulting in differences in virulence and other phenotypes. Infect Immun 66: 89–97.

91. TamuraK, PetersonD, PetersonN, StecherG, NeiM, et al. (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28: 2731–2739.

92. ClementM, PosadaD, CrandallKA (2000) TCS: a computer program to estimate gene genealogies. Mol Ecol 9: 1657–1659.

93. TanakaR, TaguchiH, TakeoK, MiyajiM, NishimuraK (1996) Determination of ploidy in Cryptococcus neoformans by flow cytometry. J Med Vet Mycol 34: 299–301.

94. DePristoMA, BanksE, PoplinR, GarimellaKV, MaguireJR, et al. (2011) A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat Genet 43: 491–498.

95. LiH, DurbinR (2009) Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25: 1754–1760.

96. CingolaniP, PlattsA, Wang leL, CoonM, NguyenT, et al. (2012) A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. Fly (Austin) 6: 80–92.

97. LassmannT, SonnhammerEL (2005) Kalign–an accurate and fast multiple sequence alignment algorithm. BMC Bioinformatics 6: 298.

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