Meet the new kid on the block
Cryptococcus gattii
Karen H. Bartlett, PhD
Associate Professor
kbartlet@interchange.ubc.ca
School of Environmental Health
University of British Columbia
The story as it unfolds …

In 2001, veterinary lab first to notice 
cases of cryptococcosis in pets on
Vancouver Island

About the same time, medical
microbiologist also finds  human cases …
What is cryptococcosis?
Cryptococcus is an encapsulated yeast and
opportunistic primary pathogen.
 Cryptococcosis is the infection …may
involve lungs, CNS, or other organ systems.
 Almost exclusively a disease of
immunocompromised hosts.
 It’s the fourth most common cause of life
threatening infections in persons with
AIDS.

Typical cryptococcosis …
Encapsulated yeast,
can identify from
microscope
“Normally”
associated with
pigeons, world
wide distribution
However!
 cases on Vancouver Island were NOT in
immunocompromised humans or animals
 Cryptococcus isolates cultured from human
cases were looked at again and subjected to
serotyping
 Surprise! The Cryptococcus from these
British Columbia cases were serogroup B.

Cryptococcus neoformans




There are 4 serogroups of Cryptococcus
neoformans that are pathogenic to mammals
A and D are found world wide
B and C are only supposed to be found in tropical
or subtropical countries
BUT … none of the new cases reported travel
histories.
Relationship of pathogenic cryptococci
Variety
C. neoformans var.
grubii
C. neoformans var.
neoformans
Neoformans hybrid
Cryptococcus gattii
Serotype
A
D
AD
B
C
Molecular
type
VN I
VN II
VN IV
VN III
VG I
VG II
VG III
VG IV
Outbreak …
Annual incidence of all cryptococcosis
(including AIDS associated) ~ 3-5/106
 As of July 2001:
 Humans (n=38)
 Pets (n=34)
 Porpoises (n=2)
 Incidence on Vancouver Island ~ 20/106

Outbreak …continues
Annual incidence of all cryptococcosis
(including AIDS associated) ~ 3-5/106
 As of December 2008:
 Humans (n=239)
 Pets (estimate = 3 – 4 times diagnosed
human cases)
 Incidence on Vancouver Island ~ 24/106
 Case fatality ratio 8.7 % (19 deaths)

Objective:
Identify Cryptococcus gattii
Objective:
Identify Cryptococcus gattii
Microscopic 1000x
India ink or calcofluor white
Capsule
Budding 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 Agar
Colonies 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 negative
Hydrolysis of urea positive (rapid test available)
Growth on cycloheximide media negative
Carbohydrate assimilation tests
Caffeic Acid disk (rapid test available)
1 kb marker (Gibco-BRL)
R634
R794
F2596
F2932
F3179
F3197
99MR10
1 kb marker (Gibco-BRL)
F2863
F2866
F3016
1 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)
URA5-RFLP unique genotypes
clinical
environmental
Source:
S. Kidd
C. gattii MLST genotyping scheme
URA5 - orotidine monophosphate pyrophosphorylase
CAP59 - capsular associated protein
GPD1- glyceraldehyde-3-phosphate dehydrogenase
LAC1- laccase
PLB1- phospholipase
SOD1- superoxide dismutase
IGS1- rRNA intergenic spacer
Genotyping Cryptococcus at BCCDC
VNIV
1%
VGI
3%
VNII
12%
VNI
27%
VGIIa
47%
VGIIb
10%
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 B
for uncomplicated pulmonary
3 – 12 months
Susceptibility testing C. gattii
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
Source: Dr. Shawn Lockhart
Centers for Disease Control and Prevention
Susceptibility testing C. gattii
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
Source: Dr. Shawn Lockhart
Centers for Disease Control and Prevention
Rates and demographics 1999 -2007




Incidence
236 cases
Mean 24.2 cases per
year
25.1/million
population Vancouver
Island
Source: BC CDC 2008



Demographics
Mean age – 59 years
Age range 2 – 92 y



4 pediatric cases
70 – 79 y highest
incidence
Male 56%
Mortality

19 deaths (case fatality ratio of 8.7%)


Mean age at death: 67.6 (range 26 – 91y)



Cases who died were older than those who did not
Median time from diagnosis to death: 54 days (range 5 –
577 d)
12 (63.2%) had underlying medical conditions





Underlying cause of death in 9; contributing cause in 10
6 cancer
3 COPD
2 liver disease
1 HIV, lung transplant, congestive heart failure, congenital heart malformation
Cases 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
Soil disturbance within 10 km
Logging within 10 km
Activity outdoors
Animal travel to Van. Island
Know other crypto. case
Owners visiting botanical garden
Owners hiking within 6 months
Owner administered supplements
Hunting
Activity indoors
Other pets in household
Rx in previous year
Canine
n
12
12
12
20
12
12
12
12
17
8
20
12
OR
17
17
9
4.5
9
5
4
2
5
2
0.33
0.05
95% CI
2.12 – 136.4
2.12 – 136.4
0.81 – 99.95
1.11 – 18.19
0.81 – 99.95
0.33 – 76.81
0.96 – 16.75
0.38 – 10.56
0.33 – 76.81
0.38 – 10.56
0.07 – 1.53
0.01 – 0.37
Source: Duncan 2006
Environmental & host risk factors
(feline)
Variables
Soil disturbance within 10 km
Logging within 10 km
Activity outdoors
Animal travel to Van. Island
Know other crypto. case
Owners visiting botanical garden
Owners hiking within 6 months
Owner administered supplements
Hunting
Activity indoors
Other pets in household
Rx in previous year
Feline
n
20
20
11
29
20
20
20
20
27
20
29
20
OR
20
6
4
7
4.3
4
4
7
2.6
4
0.4
0.4
95% CI
1.9 – 52.8
0.94 – 38.5
0.53 – 30.3
0.55 – 88.9
1.37 – 13.7
0.96 – 16.8
1.24 – 12.9
1.16 – 42.3
0.96 – 7.0
0.96 – 16.8
0.11 – 1.3
0.11 – 1.3
Source: Duncan 2006
Airborne Cryptococcus by month
Cryptococcus CFU/m3
100
10
1
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
BC Host tree species
Tree
n
Swab positive
Percent positive
127
329
915
41
81
15
40
95
0
4
22
22
10
0
5
25
41
0
2
0
2
252
113
228
27
16
12
11
14
5
23
83
159
2
3
7
9
4
4
Native coniferous/evergreen
Arbutus ( A. menziesii)
Cedar (Thuja)
Douglas fir (Pseudotsuga)
Hemlock (Tsuga)
Pine (Pinus)
Non-native evergreen
Eucalyptus (Eucalyptus)
Spruce (Picea)
Native deciduous
Alder (Alnus)
Garry Oak (Quercus)
Maple (Acer)
Other:
Cut log
Stump
Other trees, non-native
Tree swab by geographic location
Tree Location
Distance from
Victoria (km)
Swabs
examined
Swabs
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%
353
12
3%
Victoria
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
Campbell River
N 245
6
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
167
1.2
3.0
0 – 2042
40
48
2.3
5.0
0 – 537
90-100
124
0
0
Victoria
CFU/gram soil
GM
GSD
Range
0
Distance from Parksville
(km)
Port Alberni
W
Mainland
E
0
Mobility of Cryptococcus






Air
Wood/sawdust
Soil
Shoes
Car tires/wheel wells
Water
Mobility on car wheel wells
Source
n
Positive
Genotype
BC Mainland 50
2/50
4%
VG IIa
100%
Vancouver
Island
21/70
30%
VG IIa
VG IIb
VG I
86%
5%
10%
70
Environmental sampling: Mainland
and beyond
Date
October 2002
Sample type
Location
Air
Langley
Related case
Tapir
November 2003
December 2003
July 2004
July 2004
Clinical
Clinical
Air
Air
Clinical
German Shepard
Llama
Mountain View
Llama
Cat
October 2005
October 2005
December 2005
January 2006
February 2006
June 2006
September 2006
December 2006
January 2007
June 2007
Swab
Soil
Clinical
Clinical
Clinical
Clinical
Clinical
Clinical
Clinical
Clinical
Langley
Columbia Valley
Langley
Columbia Valley
Lynden, WA
Custer, WA
Lynden, WA
Lynden, WA
Richmond
Abbotsford
Surrey
New Westminster
Chilliwack
Chilliwack
New Westminster
Coquitlam
City park
City park
Cat
Cat
Dog
Dog
Cat
Dog
Dog
Dog
Genotype
Tapir = VGIII
Air = VG IIa
VG IIa
VG IIa
VG IIa
VG IIa
Cat = VG IIa
Cat = VG II b
VG IIa
VG IIa
Source: S. Mak 2010
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 (hot
spots)
Conclusions:

Human cases preceded by animal cases,
veterinarians most likely to see sentinel
cases

C. gattii mis-identified as C. neoformans if
additional laboratory tests not performed

Multiple C. gattii genotypes in Cascadia
Team Crypto once and future …

Vancouver
 BC CDC



Epidemiology
 L. MacDougall (ex), S. Mak, E. Galanis ,
Colette Gaulin, Min Li
Laboratory
 M. Moreshed, Min Lee, L. Hoang, S. Mithani
UBC
• Sarah Kidd (ex), Yat Chow, Jim Kronstad
Team Crypto once and future …

Vancouver Island
 Victoria and Health Authorities


Centre for Coastal Health


Pam Kibsey, Murray Fyfe
Craig Stephen, Colleen Duncan (ex)
Animal surveillance
 Central Laboratory (ex)
 Canada West Critical Care (Vancouver)
Team Crypto once and future …


Cryptococcus gattii working group of the Pacific
Northwest
 Kieren Marr, Kausik Datta, Rebecca Baer,
Edmond Byrnes, Joseph Heitman, Mira Leslie,
Shelley Magill
CDC Cryptococcus group
 Tom Chiller, Shawn Lockhart, Julie Harris,
Ron Wohrle
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
 BC Parks employees, Vancouver Island
EHOs

Publications: in press or in preparation …

Mak, S, Klinkenberg, B, Bartlett, K, Fyfe, M. (in press) Ecological Niche
Modeling of Cryptococcus gattii in British Columbia, Canada. Environmental
Health Perspectives 118 (5):653- 658

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. Expected publication date 2010.

Lester, S, Bartlett, K, and others 2010 A review of C. gattii cryptococcosis in
the North American Pacific Northwest. Vet Clin Path
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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. Cryptococcus gattii: an Emerging Pathogenic Fungus in
the Pacific Northwest. Emerging Infectious Diseases 15(8): 1185-1191.
Datta, K., Bartlett, KH, Marr, KA. 2009. Cryptococcus gattii emergence in Western North America:
Exploitation of a novel ecological niche. Interdisciplinary Perspectives on Infectious Diseases v.
2009, Article ID 176532, 8 pages. doi:10.1155/2009/176532.
Galanis, E, Hoang, L, Kibsey, P, Morshed, M, Phillips, P. 2009. Clinical presentation, diagnosis and
managment of Cryptococcus gattii cases: Lessons learned from British Columbia. Can J Infect Dis
Med Microbiol 20(1): 23-28.
Fyfe, M, MacDougall, L, Romney, M, Starr, M, Pearce M, Mak, S, Mithani, S, Kibsey, P. 2008.
Cryptococcus gattii infections on Vancouver Island, British Columbia, Canada: Emergence of a
tropical 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 Cryptococcus gattii on Vancouver Isalnd, Canada. Emerg Infecg Dis 14(11): 1781 – 3.
Bartlett, KH, Kidd, SE, Kronstad, J. 2008. The emergence of Cryptococcus gattii in British
Columbia and the Pacific Northwest. Current Infectious Disease Reports, 10(1): 58-65.
Nicol, A-M, Hurrell, C, McDowall, W, Bartlett, K, Elmieh, N. 2008. Communicating the risks of a
new, emerging pathogen: the case of Cryptococcus gattii. Journal of Risk Analysis 28(2): 373-386
doi:10.1111/j.1539-6924.2008.01024.x.
Bartlett, KH, Kidd, SE, Kronstad, J. 2007. The emergence of Cryptococcus gattii in British
Columbia and the Pacific Northwest. Current Fungal Infection Reports, 1:108 - 115
Upton, A, Fraser, JA, Kidd, SE, Bretz, C, Heitman, J, Bartlett, KH, Raverty, S. Marr, KA. 2007.
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):3086-8.
Kidd, SE, Chow, Y, Mak, S, Bach, PJ, Chen, H, Hingston, AO, Kronstad, JW, and Bartlett, KH.
2007 Characterization of Environmental Sources of Cryptococcus gattii in British Columbia, Canada,
and the Pacific Northwest. Applied and Environmental Microbiology, 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: 511516.
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.