Meet the new kid on the block Cryptococcus gattii

Karen H. Bartlett, PhDKaren H. Bartlett, PhD

Associate ProfessorAssociate Professor

kbartlet@interchange.ubc.cakbartlet@interchange.ubc.ca

School of Environmental HealthSchool of Environmental Health

University of British ColumbiaUniversity of British Columbia

The story as it unfolds … In 2001, veterinary lab first to notice In 2001, veterinary lab first to notice cases cases

of cryptococcosis in pets on Vancouver of cryptococcosis in pets on Vancouver Island Island

About the same time, medical About the same time, medical microbiologist also finds microbiologist also finds human cases … human cases …

What is cryptococcosis? CryptococcusCryptococcus is an encapsulated yeast and is an encapsulated yeast and

opportunistic primary pathogen. opportunistic primary pathogen. Cryptococcosis is the infection …may Cryptococcosis is the infection …may

involve lungs, CNS, or other organ systems.involve lungs, CNS, or other organ systems. Almost exclusively a disease of Almost exclusively a disease of

immunocompromised hosts.immunocompromised hosts. It’s the fourth most common cause of life It’s the fourth most common cause of life

threatening infections in persons with threatening infections in persons with AIDS.AIDS.

Typical cryptococcosis …Encapsulated yeast, can identify from microscope

“Normally” associated with pigeons, world wide distribution

However!

cases on Vancouver Island were NOT in cases on Vancouver Island were NOT in immunocompromised humans or animalsimmunocompromised humans or animals

CryptococcusCryptococcus isolates cultured from human isolates cultured from human cases were looked at again and subjected to cases were looked at again and subjected to serotypingserotyping

Surprise! The Surprise! The CryptococcusCryptococcus from these from these British Columbia cases were serogroup B. British Columbia cases were serogroup B.

Cryptococcus neoformans

There are 4 serogroups of There are 4 serogroups of Cryptococcus Cryptococcus neoformans neoformans that are pathogenic to mammalsthat are pathogenic to mammals

A and D are found world wideA and D are found world wide B and C are only supposed to be found in tropical B and C are only supposed to be found in tropical

or subtropical countriesor subtropical countries

BUT … none of the new cases reported travel BUT … none of the new cases reported travel histories.histories.

Variety Serotype Molecular type

C. neoformans var. grubii

A VN I VN II

C. neoformans var. neoformans

D VN IV

Neoformans hybrid AD VN III

Cryptococcus gattii B

C

VG I VG II

VG III VG IV

Relationship of pathogenic cryptococci

Outbreak …

Annual incidence of all cryptococcosis Annual incidence of all cryptococcosis (including AIDS associated) ~ 3-5/10(including AIDS associated) ~ 3-5/1066

As of July 2001:As of July 2001: Humans (n=38) Pets (n=34) Porpoises (n=2)

Incidence on Vancouver Island ~ 20/10Incidence on Vancouver Island ~ 20/1066

Outbreak …continues

Annual incidence of all cryptococcosis Annual incidence of all cryptococcosis (including AIDS associated) ~ 3-5/10(including AIDS associated) ~ 3-5/1066

As of December 2008:As of December 2008: Humans (n=239) Pets (estimate = 3 – 4 times diagnosed

human cases) Incidence on Vancouver Island ~ 24/10Incidence on Vancouver Island ~ 24/1066

Case fatality ratio 8.7 % (19 deaths)Case fatality ratio 8.7 % (19 deaths)

Objective: Identify Cryptococcus gattii

Objective: Identify Cryptococcus gattii

Microscopic 1000x

India ink or calcofluor whiteCapsuleBudding yeast with narrow base

Objective: Identify Cryptococcus gattii

Cryptococcal antigen latex agglutination

- Very sensitive – 10 ng /mL biologic fluid- Positive serum test (1:4) probable infection- Positive test ≥ 1:8 active infection- Can be used to monitor effectiveness of tx- Serum (pretreat with pronase)-CSF (no pretreatment)

Does not differentiate between C. gattii and C. neoformans

Objective: Identify Cryptococcus gattii

Culture techniques:

Primary plating as normal – Cryptococcus grows well on Sabouraud Dextrose AgarColonies visible at 48 – 72 hr Optimal growth 30 – 35 oC (slower ≥ 37)Cream coloured, glistening or matte

Objective: Identify Cryptococcus gattii

Add primary isolation media

Bird Seed Agar or Caffeic Acid Agar

(C. gattii = dark chocolate brown)

(C. neoformans = milk chocolate brown)

Objective: Identify Cryptococcus gattii

Confirmation agar:

Canavanine-glycine-bromthymol blue (CGB)

(C. gattii = grows & turns medium blue 24 h – 5 days)

(C. neoformans = no growth, medium yellow or green)

Objective: Identify Cryptococcus gattii

Physiological tests:C. neoformans and C. gattii are indistinguishable:

Germ tube test negativeHydrolysis of urea positive (rapid test available)Growth on cycloheximide media negativeCarbohydrate assimilation tests Caffeic Acid disk (rapid test available)

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URA5-RFLP unique genotypes

clinical environmental

Source:S. Kidd

URA5 - orotidine monophosphate pyrophosphorylase

CAP59 - capsular associated proteinGPD1- glyceraldehyde-3-phosphate dehydrogenaseLAC1- laccasePLB1- phospholipaseSOD1- superoxide dismutaseIGS1- rRNA intergenic spacer

C. gattii MLST genotyping scheme

Genotyping Cryptococcus at BCCDC

Source: M. Morshed

Susceptibility testing Cryptococcus

- Break points not established for crypto.- E-test used by many laboratories-Micro-broth dilution media not standardized (added glucose ±, shaking ±)

- Fluconazole ± Ampho Bfor uncomplicated pulmonary3 – 12 months

MIC µg/mL fluconzaole

Genotype Range Geo Mean MIC50 MIC90

VG IIa 2 – 8 5.5 8 8

VG IIb 4 – 32 12.5 16 16

VG I & III 0.5 – 16 3.0 4 16

All 0.5 – 32 7.3 8 16

Susceptibility testing C. gattii

Source: Dr. Shawn Lockhart Centers for Disease Control and Prevention

MIC µg/mL Amphotericin B

Genotype Range Geo Mean MIC50 MIC90

VG IIa 0.125 – 0.25 0.19 0.19 0.19

VG IIb 0.06 – 0.25 0.16 0.19 0.25

VG I & III 0.05 – 0.19 0.10 0.125 0.19

All 0.05 – 0.25 0.16 0.19 0.19

Susceptibility testing C. gattii

Source: Dr. Shawn Lockhart Centers for Disease Control and Prevention

Rates and demographics 1999 -2007

IncidenceIncidence 236 cases236 cases Mean 24.2 cases per Mean 24.2 cases per

yearyear 25.1/million 25.1/million

population Vancouver population Vancouver IslandIsland

DemographicsDemographics Mean age – 59 yearsMean age – 59 years Age range 2 – 92 yAge range 2 – 92 y

4 pediatric cases4 pediatric cases 70 – 79 y highest 70 – 79 y highest

incidenceincidence

Male 56%Male 56%

Source: BC CDC 2008

Mortality 19 deaths (case fatality ratio of 8.7%)19 deaths (case fatality ratio of 8.7%)

Underlying cause of death in 9; contributing cause in 10Underlying cause of death in 9; contributing cause in 10

Mean age at death: 67.6 (range 26 – 91y)Mean age at death: 67.6 (range 26 – 91y) Cases who died were older than those who did notCases who died were older than those who did not

Median time from diagnosis to death: 54 days (range 5 – 577 Median time from diagnosis to death: 54 days (range 5 – 577 d)d)

12 (63.2%) had underlying medical conditions12 (63.2%) had underlying medical conditions 6 cancer6 cancer 3 COPD3 COPD 2 liver disease2 liver disease 1 HIV, lung transplant, congestive heart failure, congenital heart malformation1 HIV, lung transplant, congestive heart failure, congenital heart malformation

Cases who died were more likely to present with CNS diseaseCases who died were more likely to present with CNS disease

Source: Galanis 2008

Case-Control study 1999 – 2001 (n = 30)Risk factors Matched OR 95% CI

Corticosteroids 8.11 1.74, 37.80

Chronic lung disease 3.21 1.08, 9.52

Recurrent pneumonia 2.71 1.05, 6.98

Cancer 2.03 0.63, 6.81

Current or past smoker 1.18 0.44, 3.20

Diabetes 0.65 0.17, 2.50

Asthma 0.45 0.12, 1.66

Outdoor building repair 4.00 1.00, 16.00

Woods within mile of house 1.70 0.17, 2.02

Camping 1.23 0.23, 2.91

Gardening 1.15 0.47, 2.79

Branch clean-up 0.29 0.10, 0.76

Cutting/chopping wood 0.17 0.04, 0.76

Pruning 0.28 0.09, 0.88

Source: MacDougall & Fyfe 2008

Population-based risk factors 1999 – 2007 (n = 218)

Risk Factor Prevalence in C. gattii cases

Prevalence in BC population

P-value

Age ≥ 50 years 72.4% 31.3% < 0.001

Male 55.8% 49.6% 0.198

Current smoker 41.9% 17.8% < 0.001

Invasive cancer 24.7% 3.6% < 0.001

HIV Infection 3.7% 0.2% < 0.001

COPD 4.1% 8.0% 0.09

Asthma 3.4% 7.8% 0.54

Corticosteroids 27.0% unknown

Source: Galanis 2008

Environmental & host risk factors (canine)

Variables Canine n OR 95% CI Soil disturbance within 10 km 12 17 2.12 – 136.4 Logging within 10 km 12 17 2.12 – 136.4 Activity outdoors 12 9 0.81 – 99.95 Animal travel to Van. Island 20 4.5 1.11 – 18.19 Know other crypto. case 12 9 0.81 – 99.95 Owners visiting botanical garden 12 5 0.33 – 76.81 Owners hiking within 6 months 12 4 0.96 – 16.75 Owner administered supplements 12 2 0.38 – 10.56 Hunting 17 5 0.33 – 76.81 Activity indoors 8 2 0.38 – 10.56 Other pets in household 20 0.33 0.07 – 1.53 Rx in previous year 12 0.05 0.01 – 0.37

Source: Duncan 2006

Environmental & host risk factors (feline)

Variables Feline n OR 95% CI Soil disturbance within 10 km 20 20 1.9 – 52.8 Logging within 10 km 20 6 0.94 – 38.5 Activity outdoors 11 4 0.53 – 30.3 Animal travel to Van. Island 29 7 0.55 – 88.9 Know other crypto. case 20 4.3 1.37 – 13.7 Owners visiting botanical garden 20 4 0.96 – 16.8 Owners hiking within 6 months 20 4 1.24 – 12.9 Owner administered supplements 20 7 1.16 – 42.3 Hunting 27 2.6 0.96 – 7.0 Activity indoors 20 4 0.96 – 16.8 Other pets in household 29 0.4 0.11 – 1.3 Rx in previous year 20 0.4 0.11 – 1.3

Source: Duncan 2006

Airborne Cryptococcus by month

1

10

100

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

Cry

pto

cocc

us

CF

U/m

3

BC Host tree speciesTree n Swab positive Percent positive

Native coniferous/evergreen Arbutus ( A. menziesii) Cedar (Thuja) Douglas fir (Pseudotsuga) Hemlock (Tsuga) Pine (Pinus)

127 329 915 41 81

15 40 95 0 4

22 22 10 0 5

Non-native evergreen Eucalyptus (Eucalyptus) Spruce (Picea)

25 41

0 2

0 2

Native deciduous Alder (Alnus) Garry Oak (Quercus) Maple (Acer)

252 113 228

27 16 12

11 14 5

Other: Cut log Stump Other trees, non-native

23 83 159

2 3 7

9 4 4

Tree swab by geographic locationTree Location Distance from

Victoria (km)Swabs

examinedSwabs

positive% positive

Campbell River N 245 20 0 0%

Courtenay N 200 220 13 6%

Parksville N 125 391 126 32%

Nanaimo N 100 307 3 1%

Cowichan N 50 168 14 8%

Gulf Islands E 20 117 7 1%

Victoria 353 12 3%

Distance from Parksville (km)

Port Alberni W 40 57 11 19%

West Coast W 140 45 0 0

Mainland E 90 - 100 231 0 0

Cryptococcus in soil by geographic location

Location Distance from Victoria (km)

n CFU/gram soilGM GSD

Range

Campbell River N 245 6 0 0 0

Courtenay N 200 139 1.5 5.2 0 – 4365

Parksville N 125 127 4.7 12.1 0 – 29512

Nanaimo N 100 136 1.3 3.1 0 – 708

Cowichan N 50 96 2.6 13.2 0 – 36308

Gulf Islands E 20 104 1.6 5.4 0 – 181970

Victoria 167 1.2 3.0 0 – 2042

Distance from Parksville (km)

Port Alberni W 40 48 2.3 5.0 0 – 537

Mainland E 90-100 124 0 0 0

Mobility of Cryptococcus

AirAir Wood/sawdustWood/sawdust SoilSoil ShoesShoes Car tires/wheel wellsCar tires/wheel wells WaterWater

Mobility on car wheel wells

Source n Positive Genotype

BC Mainland 50 2/50 4% VG IIa 100%

Vancouver Island

70 21/70 30% VG IIa VG IIb VG I

86% 5% 10%

Environmental sampling: Mainland and beyond

Date Sample type Location Related case Genotype October 2002 Air Langley Tapir Tapir = VGIII

Air = VG IIa November 2003 Clinical Langley German Shepard VG IIa December 2003 Clinical Columbia Valley Llama VG IIa July 2004 Air Langley Mountain View VG IIa July 2004 Air Columbia Valley Llama VG IIa Clinical Lynden, WA

Custer, WA Cat Cat = VG IIa

Cat = VG II b October 2005 Swab Lynden, WA City park VG IIa October 2005 Soil Lynden, WA City park VG IIa December 2005 Clinical Richmond Cat January 2006 Clinical Abbotsford Cat February 2006 Clinical Surrey Dog June 2006 Clinical New Westminster Dog September 2006 Clinical Chilliwack Cat December 2006 Clinical Chilliwack Dog January 2007 Clinical New Westminster Dog June 2007 Clinical Coquitlam Dog

Source: S. Mak 2010

Conclusions:

Cryptococcus gattiiCryptococcus gattii is endemic on the east is endemic on the east coast of Vancouver Islandcoast of Vancouver Island

Cultured from a wide range of native tree Cultured from a wide range of native tree speciesspecies

Not homogeneous in the environment (hot Not homogeneous in the environment (hot spots)spots)

Conclusions: Human cases preceded by animal cases, Human cases preceded by animal cases,

veterinarians most likely to see sentinel veterinarians most likely to see sentinel casescases

C. gattii C. gattii mis-identified as mis-identified as C. neoformans C. neoformans if if additional laboratory tests not performedadditional laboratory tests not performed

Multiple Multiple C. gattii C. gattii genotypes in Cascadia genotypes in Cascadia

Team Crypto once and future … VancouverVancouver

BC CDCBC CDC EpidemiologyEpidemiology

L. MacDougall (ex), S. Mak, E. Galanis , L. MacDougall (ex), S. Mak, E. Galanis , Colette Gaulin, Min Li

LaboratoryLaboratory M. Moreshed, Min Lee, L. Hoang, S. MithaniM. Moreshed, Min Lee, L. Hoang, S. Mithani

UBCUBC• Sarah Kidd (ex), Yat Chow, Jim KronstadSarah Kidd (ex), Yat Chow, Jim Kronstad

Team Crypto once and future … Vancouver IslandVancouver Island

Victoria and Health AuthoritiesVictoria and Health Authorities Pam Kibsey, Murray FyfePam Kibsey, Murray Fyfe

Centre for Coastal HealthCentre for Coastal Health Craig Stephen, Colleen Duncan (ex)Craig Stephen, Colleen Duncan (ex)

Animal surveillanceAnimal surveillance Central Laboratory (ex)Central Laboratory (ex) Canada West Critical Care (Vancouver)Canada West Critical Care (Vancouver)

Team Crypto once and future … Cryptococcus gattii Cryptococcus gattii working group of the Pacific working group of the Pacific

NorthwestNorthwest Kieren Marr, Kausik Datta, Rebecca Baer, Kieren Marr, Kausik Datta, Rebecca Baer,

Edmond Byrnes, Joseph Heitman, Mira Leslie, Edmond Byrnes, Joseph Heitman, Mira Leslie, Shelley MagillShelley Magill

CDC CDC CryptococcusCryptococcus group group Tom Chiller, Shawn Lockhart, Julie Harris, Tom Chiller, Shawn Lockhart, Julie Harris,

Ron WohrleRon Wohrle

Acknowledgements: BC Lung AssociationBC Lung Association Michael Smith Foundation for Health Michael Smith Foundation for Health

ResearchResearch Sampling team members:Sampling team members:

Yat Chow, Tracy Kirkham, Molly Chen, Yat Chow, Tracy Kirkham, Molly Chen, Hua Shen, Caroline Chen, Timothy Ma, Hua Shen, Caroline Chen, Timothy Ma, Fred RockwellFred Rockwell

BC Parks employees, Vancouver Island BC Parks employees, Vancouver Island EHOsEHOs

Publications: in press or in preparation …

Mak, S, Klinkenberg, B, Bartlett, K, Fyfe, M. (in press) Ecological Niche Mak, S, Klinkenberg, B, Bartlett, K, Fyfe, M. (in press) Ecological Niche Modeling of Modeling of Cryptococcus gattii Cryptococcus gattii in British Columbia, Canada. Environmental in British Columbia, Canada. Environmental Health Perspectives Health Perspectives 118 (5):653- 658 118 (5):653- 658

Chapter: Chapter: “The emergence of “The emergence of Cryptococcus gattiiCryptococcus gattii infections on Vancouver infections on Vancouver Island and expansion in the Pacific Northwest.” Karen Bartlett, Edmond Island and expansion in the Pacific Northwest.” Karen Bartlett, Edmond Byrnes, Colleen Duncan, Murray Fyfe, Eleni Galanis, Joseph Heitman, Linda Byrnes, Colleen Duncan, Murray Fyfe, Eleni Galanis, Joseph Heitman, Linda Hoang, Sarah Kidd, Laura MacDougall, Sunny Mak, Kieren Marr, Hoang, Sarah Kidd, Laura MacDougall, Sunny Mak, Kieren Marr, Muhammad Morshed, Sarah West, and James Kronstad. Muhammad Morshed, Sarah West, and James Kronstad. InIn: : Cryptococcus: Cryptococcus: from human pathogen to model yeastfrom human pathogen to model yeast. Eds: J. Kwon-Chun and J. Heitman. . Eds: J. Kwon-Chun and J. Heitman. ASM Press. Expected publication date 2010.ASM Press. Expected publication date 2010.

Lester, S, Bartlett, K, and others 2010 A review of Lester, S, Bartlett, K, and others 2010 A review of C. gattii C. gattii cryptococcosis in cryptococcosis in the North American Pacific Northwest. the North American Pacific Northwest. Vet Clin Path Vet Clin Path

Datta, K. Bartlett, K, Baer, R, Byrnes, E, Galanis, E, Heitman, J, Hoang, L, Leslie, M, MacDougall, L, Datta, K. Bartlett, K, Baer, R, Byrnes, E, Galanis, E, Heitman, J, Hoang, L, Leslie, M, MacDougall, L, Magill, S, Morshed, M, Marr, K. 2009. Magill, S, Morshed, M, Marr, K. 2009. Cryptococcus gattiiCryptococcus gattii: an Emerging Pathogenic Fungus in the Pacific : an Emerging Pathogenic Fungus in the Pacific Northwest. Northwest. Emerging Infectious DiseasesEmerging Infectious Diseases 15(8): 1185-1191. 15(8): 1185-1191.

Datta, K., Bartlett, KH, Marr, KA. 2009. Datta, K., Bartlett, KH, Marr, KA. 2009. Cryptococcus gattiiCryptococcus gattii emergence in Western North America: emergence in Western North America: Exploitation of a novel ecological niche. Exploitation of a novel ecological niche. Interdisciplinary Perspectives on Infectious DiseasesInterdisciplinary Perspectives on Infectious Diseases v. 2009, v. 2009, Article ID 176532, 8 pages. Article ID 176532, 8 pages. doi:10.1155/2009/176532 doi:10.1155/2009/176532..

Galanis, E, Hoang, L, Kibsey, P, Morshed, M, Phillips, P. 2009. Clinical presentation, diagnosis and Galanis, E, Hoang, L, Kibsey, P, Morshed, M, Phillips, P. 2009. Clinical presentation, diagnosis and managment of managment of Cryptococcus gattii Cryptococcus gattii cases: Lessons learned from British Columbia. Can J Infect Dis Med cases: Lessons learned from British Columbia. Can J Infect Dis Med Microbiol 20(1): 23-28.Microbiol 20(1): 23-28.

Fyfe, M, MacDougall, L, Romney, M, Starr, M, Pearce M, Mak, S, Mithani, S, Kibsey, P. 2008. Fyfe, M, MacDougall, L, Romney, M, Starr, M, Pearce M, Mak, S, Mithani, S, Kibsey, P. 2008. Cryptococcus gattii Cryptococcus gattii infections on Vancouver Island, British Columbia, Canada: Emergence of a tropical infections on Vancouver Island, British Columbia, Canada: Emergence of a tropical fungus in a temperate environment. CCDR 34(6): 1 – 12.fungus in a temperate environment. CCDR 34(6): 1 – 12.

Chambers, C, MacDougall, L, Li, M, Galanis, E. 2008. Using tourism data to define specific risk areas for Chambers, C, MacDougall, L, Li, M, Galanis, E. 2008. Using tourism data to define specific risk areas for Cryptococcus gattii Cryptococcus gattii on Vancouver Isalnd, Canada. Emerg Infecg Dis 14(11): 1781 – 3.on Vancouver Isalnd, Canada. Emerg Infecg Dis 14(11): 1781 – 3.

Bartlett, KH, Kidd, SE, Kronstad, J. 2008. The emergence of Bartlett, KH, Kidd, SE, Kronstad, J. 2008. The emergence of Cryptococcus gattiiCryptococcus gattii in British Columbia and in British Columbia and the Pacific Northwest. the Pacific Northwest. Current Infectious Disease Reports, Current Infectious Disease Reports, 10(1): 58-65.10(1): 58-65.

Nicol, A-M, Hurrell, C, McDowall, W, Bartlett, K, Elmieh, N. 2008. Communicating the risks of a new, Nicol, A-M, Hurrell, C, McDowall, W, Bartlett, K, Elmieh, N. 2008. Communicating the risks of a new, emerging pathogen: the case of emerging pathogen: the case of Cryptococcus gattiiCryptococcus gattii. . Journal of Risk AnalysisJournal of Risk Analysis 28(2): 373-386 28(2): 373-386 doi:10.1111/j.1539-6924.2008.01024.x.

Bartlett, KH, Bartlett, KH, Kidd, SE, Kronstad, J. 2007. The emergence of , Kronstad, J. 2007. The emergence of Cryptococcus gattiiCryptococcus gattii in British Columbia and in British Columbia and the Pacific Northwest. the Pacific Northwest. Current Fungal Infection Reports, Current Fungal Infection Reports, 1:108 - 1151:108 - 115

Upton, A, Fraser, JA, Kidd, SE, Bretz, C, Heitman, J, Bartlett, KH, Raverty, S. Marr, KA.Upton, A, Fraser, JA, Kidd, SE, Bretz, C, Heitman, J, Bartlett, KH, Raverty, S. Marr, KA. 2007. 2007. First First Contemporary Case of Human Infection with Contemporary Case of Human Infection with Cryptococcus gattiiCryptococcus gattii in Puget Sound: Evidence for Spread of in Puget Sound: Evidence for Spread of the Vancouver Island Outbreak. the Vancouver Island Outbreak. Journal of Clinical Microbiology Journal of Clinical Microbiology, 45(9):3086-8., 45(9):3086-8.

Kidd, SE, Chow, Y, Mak, S, Bach, PJ, Kidd, SE, Chow, Y, Mak, S, Bach, PJ, Chen,Chen, HH, , Hingston, AOHingston, AO, Kronstad, JW, and , Kronstad, JW, and Bartlett, KH. 2007 . 2007

Characterization of Environmental Sources of Characterization of Environmental Sources of Cryptococcus gattiiCryptococcus gattii in British Columbia, Canada, and the in British Columbia, Canada, and the Pacific Northwest. Pacific Northwest. Applied and Environmental Microbiology,Applied and Environmental Microbiology, 73 (5):1433 – 1443. 73 (5):1433 – 1443.

MacDougall, L, Kidd, SE, Galanis, E., Mak, S., Leslie, MJ, Cieslak, PR, Kronstad, JW, Morshed, MG, and Bartlett, KH. 2007. Spread of Cryptococcus gattii in British Columbia, Canada, and detection in the Pacific Northwest, USA. Emerging Infectious Diseases 13(1):42-50. Kidd, SE, Bach, PJ, Hingston, AO, Mak, S, Chow, Y, MacDougall, L, Kronstad, JW, Bartlett, KH. 2007. Cryptococcus gattii Dispersal Mechanisms, British Columbia, Canada. Emerging Infectious Diseases 13(1):51-57.Duncan, C, Schwantje, H, Stephen, C, Campbell, J, Bartlett K. 2006. Cryptococcus gattii in wildlife of Vancouver Island, BC, Canada. Journal of Wildlife Diseases 42: 175-178.Duncan, C, Stephen, C, Lester, S, Bartlett, KH. 2005. Sub-clinical infection and asymptomatic carriage of Cryptococcus gattii in dogs and cats during an outbreak of cryptococcosis. Medical Mycology 43: 511-516. Kidd, SE, Guo, H, Bartlett, KH, Kronstad, JW, Xu, J. 2005. Comparative gene genealogies indicate that two clonal lineages of Cryptococcus gattii in British Columbia resemble strains from other geographical areas. Eucaryotic Cell 4: 1629-1638. Duncan C, Stephen C, Lester S and Bartlett, KH. 2005 Follow-up study of dogs and cats with asymptomatic Cryptococcus gattii infection or nasal colonization. Medical Mycology 43: 663-666. Kidd S, Hagen F, Tscharke M, Huynh M, Bartlett KH, Fyfe, M, MacDougall L, Boekhout T, Kwon-Chung KJ, Meyer W. 2004. 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): 17258-17263. Lester S, Kowalewich N, Bartlett KH, Krockenberger MB, Fairfax, TM, Malik R. 2004. 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): 1716-1722.