Knowing where to look: environmental sources of cryptococcal disease in the Pacific Northwest

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1 Knowing where to look: environmental sources of cryptococcal disease in the Pacific Northwest Karen H. Bartlett, PhD Associate Professor School of Environmental Health University of British Columbia

2 Vancouver Island outbreak Annual incidence of all cryptococcosis (including AIDS associated) ~ 3-5/103 6 As of July 2001 B.C. cryptococcosis cases: Humans (n=38) Pets (n=34) Porpoises (n=2) Incidence on Vancouver Island ~ 20/10 6 Source: BC CDC

3 What is cryptococcosis? Cryptococcus is an encapsulated yeast and primary pathogen. Cryptococcosis may involve lungs, CNS, or other organ systems. Usually a disease of immunocompromised hosts. The fourth most common cause of life threatening infections in persons with AIDS.

4 Typical cryptococcosis Encapsulated yeast, can easily identify with microscope Distributed world-wide Associated with aged pigeon faeces & soil contaminated with faecal material

5 Pathogenic Cryptococcus Physiological tests: C. neoformans and C. gattii are indistinguishable: Germ tube test negative Hydrolysis of urea positive (rapid test available) Growth on cycloheximide media negative Carbohydrate assimilation tests Caffeic Acid disk (rapid test available)

6 Cryptococcus gattii! Canavanine-glycine-bromthymol blue (CGB) C. gattii = grows & turns medium blue 24 h 5 days C. neoformans = no growth, medium yellow or green

7 Cryptococcusneoformans primer: Pathogenic cryptococci uc. neoformans var. grubii tserogroup A/ VN I & VN II uc. neoformans var. neoformans tserogroup D/ VN IV uc. neoformans hybrid tserogroup AD/ VN III

8 Cryptococcusgattii primer: C. gattii (formerly known as C. neoformans var gatii) userogroups B or C urflp VG I, VG II, VG III, VG IV uura-5 5 VG IIa, VG IIb, VG IIc umlst multiple sequence types

9 URA5-RFLP unique genotypes 1 kb marker (Gibco-BRL) R634 R794 F2596 F2932 F3179 F MR10 1 kb marker (Gibco-BRL) F2863 F2866 F kb marker (Gibco-BRL) E113 ENV123 ENV124 ENV125 ENV129 ENV130 ENV131 ENV133 ENV152 ENV153 1 kb marker (Gibco-BRL) WM148 VNI Standard WM626 VNII Standard WM628 VNIII Standard WM629 VNIV Standard 1 kb marker (Gibco-BRL) WM179 VGI Standard WM178 VGII Standard WM161 VGIII Standard WM779 VGIV Standard 1 kb marker (Gibco-BRL) clinical environmental Source: S. Kidd

10 Outbreak continues Annual incidence of all cryptococcosis (including AIDS associated) ~ 3-5/103 6 As of 2009 B.C. cases C. gattii: Humans (n=263) Pets (estimate = 3 4 times diagnosed human cases) Incidence on Vancouver Island ~ 24/10 6 Case fatality ratio 8.7 % Source: BC CDC

11 Cryptococcusgattii : Geographic distribution uexposure directly from the environment urestricted distribution (e.g. formerly known as tropical or sub-tropical)

12 Source: BC CDC Sunny Mak

13 Environmental niche: Australia, California Eucalyptus camaldulensis Eucalyptus tereticornis

14 Environmental niche British Columbia Douglas fir Red Alder

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16 Veterinary environmental risk factors Variables Feline Canine n OR 95% CI n OR 95 % CI Soil disturbance within 10 km Logging within 10 km Animal travel to Van. Island Know another crypto. case Owners hiking within 6 months Owner administered supplements Owners visiting a botanical garden Source: Duncan 2006

17 Source: Sunny Mak

18 Source: Sunny Mak

19

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21 Source: Datta et al Emerg Infect Dis 15(8):

22 BC Host tree species Non-native Native, deciduous & nondeciduous

23 positive tree by geographic location Campbell River < 1% Courtenay 6% Parksville 32% Port Alberni 19% Cowichan 8% Victoria 3%

24 C. gattii soil concentration (range CFU/g) Campbell River < LOD Courtenay < to 4x10 3 Parksville < to 3x10 4 Port Alberni < to 5x10 2 Cowichan < to 4x10 4 Gulf Island < to 2x105 Victoria < to 2x10 3

25 Airborne Cryptococcus by month 100 Cryptococcus CFU/m Jan n=18 Feb n=8 Mar n=6 Apr n=8 May n=16 Jun n=24 Jul n=18 Aug n=9 Sep n=7 Oct n=14 Nov n=19 Dec n=7 Month

26 Aerosolization of Cryptococcus Baseline 4x10 2 CFU/m 3 Limbing 2x10 3 CFU/m 3 Chainsawing 2x10 4 CFU/m 3 Chipping 5x10 4 CFU/m 3

27 Mobility of Cryptococcus Air Wood/sawdust Soil Shoes Car tires/wheel wells Water

28 When?

29 Mobility on car wheel wells Direction n Positive C. gattii Genotype From BC Mainland % VG IIa 100% From Vancouver Island % VG IIa VG IIb VG I 86% 5% 10%

30 C. gattii genotype by sample type Sample n VG IIa VG IIb VG I Clinical 99 89% 7% 4% Air 50 70% 15% 7% Soil 81 67% 18% 6% Swab % 16% 9% Water 8 63% 38% 0 Typing: Sarah Kidd

31 Environmental C. gattii genotype by location Location n VG IIa VG IIb VG I Courtenay % 0 0 Parksville 90 87% 13% 0 Port Alberni 10 90% 10% 0 Nanaimo % 0 0 Duncan % 23% 14% Victoria 9 100% 0 0 Gulf Islands 23 91% 0 9% Typing: Sarah Kidd

32 Veterinary C. gattii genotype by location Location n VG IIa VG IIb VG I Courtenay 25 80% 20% 0 Parksville % 0 0 Port Alberni 2 50% 50% 0 Nanaimo 28 79% 21% 0 Duncan 17 66% 35% 0 Victoria 34 82% 18% 0 Gulf Islands 4 100% 0 0 Mainland BC 18 94% 6% 0 Typing: Sarah Kidd

33 First evidence: C. gattii in the Pacific Northwest Year n Sample Location VG IIa Air Metro Van BC Vet clinical Fraser Valley BC Air Fraser Valley BC Vet clinical Washington USA Swab + soil Washington USA Vet clinical Metro Van BC Vet clinical Metro Van BC Vet clinical Fraser Valley BC Vet clinical Metro Van BC 2 VG IIb

34 Source: Sunny Mak 2010

35 Source: Sunny Mak 2010

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37 Conclusions: Cryptococcus gattii is endemic on the east coast of Vancouver Island

38 Conclusions: Cryptococcus gattii is endemic on the east coast of Vancouver Island Cultured from a wide range of native tree species

39 Conclusions: Cryptococcus gattii is endemic on the east coast of Vancouver Island Cultured from a wide range of native tree species Not homogeneous in the environment (limit of detection hot spots)

40 Conclusions: Human cases preceded by animal cases, veterinarians most likely to see sentinel cases

41 Conclusions: Human cases preceded by animal cases, veterinarians most likely to see sentinel cases C. gattii mis-identified identified as C. neoformans if additional laboratory tests not performed

42 Conclusions: Human cases preceded by animal cases, veterinarians most likely to see sentinel cases C. gattii mis-identified identified as C. neoformans if additional laboratory tests not performed Multiple C. gattii genotypes in Cascadia

43 Conclusions: Best response Education (MDs, DVMs, Public) Cooperation (MDs, DVMs, Epidemiologists, Environmental hygiene) Knowledge Translation/Transfer

44 Team Crypto once and future Vancouver u BC CDC u Epidemiology u L. MacDougall (ex), S. Mak, E. Galanis, Colette Gaulin, Min Li, Marsha Taylor u Laboratory u M. Moreshed, Min Lee, L. Hoang, S. Mithani UBC Sarah Kidd (ex), Yat Chow (ex), Jim Kronstad u UBC

45 Team Crypto once and future Vancouver Island Victoria and Health Authorities Pam Kibsey, Murray Fyfe Centre for Coastal Health Craig Stephen, Colleen Duncan (ex) Animal surveillance Central Laboratory (ex) Canada West Critical Care (Vancouver)

46 Team Crypto once and future Cryptococcus gattii working group of the Pacific Northwest Kieren Marr, Kausik Datta, Rebecca Baer (ex), Edmond Byrnes, Joseph Heitman, Mira Leslie, Shelley Magill, Nicola Marsden-Hoag, Ron Whorle CDC Cryptococcus group Tom Chiller, Shawn Lockhart, Julie Harris

47 Acknowledgements: BC Lung Association Michael Smith Foundation for Health Research Sampling team members: Yat Chow, Tracy Kirkham, Molly Chen, Hua Shen, Caroline Chen, Timothy Ma, Fred Rockwell, Adrian Hingston, Andrea Griffiths BC Parks employees, Vancouver Island EHOs

48 Publications: Hagen, F., Illnait-Zaragozi, M.T., Bartlett, K,, Swinne, D., Geertsen, E., Klaassen, C.H., Boekhout, T., Meis, J.F In vitro antifungal susceptibilities and AFLP genotyping of a worldwide collection of 350 clinical, veterinary and environmental Cryptococcus gattii isolates. Antifungal Agents and Chemotherapy 54(12): Lester, S., Malik, R., Bartlett, K,, Duncan, C. (in press) Cryptococcosis: Literature review and update. Veterinary Clinical Pathology. Mak, S, Klinkenberg, B, Bartlett, K, Fyfe, M Ecological Niche Modeling of Cryptococcus gattii in British Columbia, Canada. Environmental Health Perspectives 118 (5): Chapter: The emergence of Cryptococcus gattii infections on Vancouver Island and expansion in the Pacific Northwest. Karen Bartlett, Edmond Byrnes, Colleen Duncan, Murray Fyfe, Eleni Galanis, Joseph Heitman, Linda Hoang, Sarah Kidd, Laura MacDougall, Sunny Mak, Kieren Marr, Muhammad Morshed, Sarah West, and James Kronstad. In: Cryptococcus: from human pathogen to model yeast.. Eds: J. Kwon-Chun and J. Heitman. ASM Press. (In press).

49 Datta, K. Bartlett, K, Baer, R, Byrnes, E, Galanis, E, Heitman, J, Hoang, L, Leslie, M, MacDougall, L, Magill, S, Morshed, M, Marr, K Cryptococcus gattii: : an Emerging Pathogenic Fungus in the Pacific Northwest. Emerging Infectious Diseases 15(8): Datta, K., Bartlett, KH, Marr, KA Cryptococcus gattii emergence in Western North America: Exploitation of a novel ecological niche. Interdisciplinary Perspectives on Infectious Diseases v. 2009, Article ID , 8 pages. doi: /2009/ Galanis, E, Hoang, L, Kibsey, P, Morshed, M, Phillips, P Clinical presentation, diagnosis and managment of Cryptococcus gattii cases: Lessons learned from British Columbia. Can J Infect Dis Med Microbiol 20(1): Fyfe, M, MacDougall, L, Romney, M, Starr, M, Pearce M, Mak, S, Mithani, M S, Kibsey, P Cryptococcus gattii infections on Vancouver Island, British Columbia, Canada: Emergence ence of a tropical fungus in a temperate environment. CCDR 34(6): Chambers, C, MacDougall, L, Li, M, Galanis, E Using tourism data to define specific risk areas for Cryptococcus gattii on Vancouver Isalnd, Canada. Emerg Infecg Dis 14(11): Bartlett, KH, Kidd, SE, Kronstad, J The emergence of Cryptococcus gattii in British Columbia and the Pacific Northwest. Current Infectious Disease Reports, 10(1): Nicol, A-M, A Hurrell, C, McDowall, W, Bartlett, K, Elmieh, N Communicating the risks of a new, emerging pathogen: the case of Cryptococcus gattii. Journal of Risk Analysis 28(2): doi: /j x. Bartlett, KH, Kidd, SE,, Kronstad, J The emergence of Cryptococcus gattii in British Columbia and the Pacific Northwest. Current Fungal Infection Reports, 1: Upton, A, Fraser, JA, Kidd, SE, Bretz, C, Heitman, J, Bartlett, KH, Raverty, S. Marr, KA First Contemporary Case of Human Infection with Cryptococcus gattii in Puget Sound: Evidence for Spread of the Vancouver Island Outbreak. Journal of Clinical Microbiology,, 45(9): Kidd, SE, Chow, Y, Mak, S, Bach, PJ, Chen, H, Hingston, AO,, Kronstad, JW, and Bartlett, KH Characterization of Environmental Sources of Cryptococcus gattii in British Columbia, Canada, and the Pacific Northwest. Applied and Environmental Microbiology, 73 (5):

50 MacDougall, L, Kidd, SE, Galanis, E., Mak, S., Leslie, MJ, Cieslak, PR, Kronstad, JW, Morshed, MG, and Bartlett, KH Spread of Cryptococcus gattii in British Columbia, Canada, and detection in the Pacific Northwest, USA. Emerging Infectious Diseases 13(1): Kidd, SE, Bach, PJ, Hingston, AO, Mak, S, Chow, Y, MacDougall, L, Kronstad, JW, Bartlett, KH Cryptococcus gattii Dispersal Mechanisms, British Columbia, Canada. Emerging Infectious Diseases 13(1): Duncan, C, Schwantje, H, Stephen, C, Campbell, J, Bartlett K Cryptococcus gattii in wildlife of Vancouver Island, BC, Canada. Journal of Wildlife Diseases 42: Duncan, C, Stephen, C, Lester, S, Bartlett, KH Sub-clinical infection and asymptomatic carriage of Cryptococcus gattii in dogs and cats during an outbreak of cryptococcosis. Medical Mycology 43: Kidd, SE, Guo, H, Bartlett, KH, Kronstad, JW, Xu, J Comparative gene genealogies indicate that two clonal lineages of Cryptococcus gattii in British Columbia resemble strains from other geographical areas. Eucaryotic Cell 4: Duncan C, Stephen C, Lester S and Bartlett, KH Follow-up study of dogs and cats with asymptomatic Cryptococcus gattii infection or nasal colonization. Medical Mycology 43: Kidd S, Hagen F, Tscharke M, Huynh M, Bartlett KH, Fyfe, M, MacDougall L, Boekhout T, Kwon- Chung KJ, Meyer W A rare genotype of Cryptococcus gattii caused the cryptococcosis outbreak on Vancouver Island (British Columbia, Canada). Proceedings of the National Academy of Sciences 101(49): Lester S, Kowalewich N, Bartlett KH, Krockenberger MB, Fairfax, TM, Malik R Clinicopathologic features of cryptococcosis in dogs, cats, ferrets, and a bird: 38 Cases (January to July 2003). Journal of the American Veterinary Medical Association, 225 (11):