Emilio DeBess, DVM, MPVM

Cryptococcus gattii in Humans and Animals

Oregon Health Authority Portland, OR 97232 emilio.debess@state.or.us

Fun with Fungus

C.gattii in the News

History 1970

First clinical case of Cryptococcus gattii

1984 Epidemiology of C.gattii versus C.neoformans published C.gattii isolated to tropical/subtropical regions

Australia, Brazil, Cambodia, Hawaii, southern California, Mexico, Paraguay, Thailand, Vietnam, Nepal, and countries in central Africa

1999 C.gattii diagnosed in a group of human and animal cases from Vancouver

Island, British Columbia

2004 C.gattii isolated in Oregon

Steady rise in number of cases since 2004 56 C.gattii cases reported in Oregon to date

C. gattii

gattii

B B or C

VGI VGII VGIII VGIV

VGIIa VGIIb VGIIc otherssubtypes

Cryptococcus Taxonomy

Vs.

C. neoformans

grubii neoformansvariety

A DAD ADserotypes

VNI VNII VNBmolecular types (genotypes)

VNIII VNIV

Courtesy of Dr Julie Harris /CDC

Clinical Data from Australia Speed and Dunt: all cases of Cryptococcus from a population-based

registry (1980-1990) analyzed for differences between C.gattii and C. neoformans with regards to clinical and host immune status 71 cases were typed

51 C.neoformans 46 had immunosuppression (23 HIV positive , 23 with another

unspecified immunosuppressing condition) and only 5 described as healthy hosts

20 C.gattii all 20 were HIV seronegative and none with immunosuppressing

conditions Clinical presentations

C.gattii more likely to have pulmonary involvement Both had high rates of meningeal disease (85% and 70%)

intra-cerebral lesions and focal symptomology more common with C.gattii

Outcomes No deaths in C.gattii group versus 30% mortality in C.neoformans group C.gattii patients more likely to suffer neurologic sequelae (39% versus 3%)

and to require either CNS or thoracic surgery

Clinical Data from Australia (2) Mitchell et al: A retrospective review of 118 cases of cryptococcal CNS

disease presenting to Australian teaching hospitals between 1985 and 1992 (all routinely typed as gattii or neoformans)

Immune deficiencies 60 patients with AIDS

only 1 C.gattii case 23 patients with other immune deficiencies (13 heme malignancies, 10 on

immunosuppressing meds) Only 1 C.gattii case

35 patients without AIDS or other immune deficiencies 26 (74%) C.gattii 9 (26%) C.neoformans

Outcomes C. gattii had mortality rate of 15%

C.gattii in Australia Summary

Host risk factors and clinical features of cryptococcosis dependent on the infecting species healthy hosts were primary target of C.gattii infection

C.gattii (vs. C.neoformans) was associated with lower mortality a higher incidence of complications increased long-term sequelae focal lesions

What about C.gattii infection in BC and the Pacific NW US?

Environmental Sampling- BC, Canada

C.gattii found in environmental sampling on Vancouver Island

Increasing diagnoses between 1999 and 2004 in patients on the mainland and in Pacific NW US (without travel exposure)

MacDougal et al: Large scale environmental sampling from mainland BC and Washington State (2001-2005) 3% of environmental samples (soil, air, water, swabs from non-

Eucalyptus trees and other structures) positive for C.gattii

Clinical Epidemiology from BC

Galanis and MacDougal: 218 cases (124 confirmed by culture and 94 probable with positive CrAg, histopathology, or microscopy and HIV seronegative) from lab reporting to BC public health authorities (1999-2006) Rise in incidence of infection in British Columbia over study

period 6 cases in 1999 to 38 cases in 2006

Average age was 58 years, 55% were male 38% immunocompromised (HIV, transplant, cancer, steroids) Presenting clinical features

respiratory syndrome in 76.6% CNS syndrome in 7.8% both in 10%

Clinical Epidemiology from BC #2

Lung nodules (single or multiple) were most common radiographic findings

19 total deaths CFR 8.7% (confirmed and probable) and CFR 12.1%

(confirmed) Mortality associated with older age (p=0.019) and CNS

syndrome (p=0.014) 74% of those who died had underlying medical conditions

cancer, COPD, asthma, liver disease, diabetes, HIV infection, lung transplant, CHF and congenital cardiac disease.

47% of those who died were immunocompromised (p=0.267)

Clinical Epidemiology in Oregon

DeBess et al: 60 cases of C.gattii reported in Pacific NW US (WA, OR, CA, ID) through July 2010 54% male, and 45% of cases were aged 50-69 years 81% (of 47 patients for whom data was available) had a

clinically recognized predisposing condition 29% either HIV or transplant

Mortality of 33% (of 45 patients with known outcomes)

20% of patients died due to C.gattii 13% of patients died with C.gattii (higher than the reported case fatality rates in both BC and

in Australia)

Background Summary

Unique strain in a novel environment

Epidemiology suggests unique clinical features compared to the previously published data

Scarcity of large clinical series of C.gattii infection, therefore lack of detailed clinical and outcome information

The large number of cases reported recently in Oregon provides opportunity to improve our understanding of this infection and the clinical care of these patients

Cryptococcus gattii Human Infection in Oregon

Culture-confirmed C.gattii in Oregon, between November 2004 and November 2010

02468

101214161820

2004 2005 2006 2007 2008 2009 2010

Number of Cases by Year

n=46 cases

Gender and Age

27 female, 19 male

Median age 58 years Range 15-96 No difference by gender

Number (%) of Cases by Site of Infection 21 (46) Pulmonary 12 (26) CNS 9 (20) CNS & Pulmonary 1 (2) Bloodstream alone 3 (6) Other

Clinical Presentations Major Sites of

Infection Symptom onset to diagnosis

Median # days (range) Presenting Complaint(s) &

# of patients

CNS 22 (7-180)

Headache 7

Confusion 4

Seizure 1

Visual field deficit 1

Pulmonary 37 (12-180)

Dyspnea 8

Cough 5

Asymptomatic 4

Fever 2

Weakness 1

Confusion 1

CNS & Pulmonary 33 (6-51)

Headache 3

Cough 3

Hemoptysis 2

Dyspnea 1

Confusion 1

Syncope 1

Predisposing Conditions: Totals**

Predisposing Conditions Number of patients (%)

At least one immune-suppressing condition (Solid organ transplant, autoimmune disease, active malignancy, HIV)

24 (52%)

No immune-suppressing conditions but at least one chronic medical condition (Chronic renal, liver or lung disease, or diabetes)

10 (22%)

No immune-suppressing or chronic medical conditions 12 (26%)

**each patient represented only once

CNS Cases

CSF Feature (n) Median Range

Opening Pressure in cm H2O (n=8)

41 17-55

WBC /mm3 (n=17) 172 1-1090

Percent mononuclear cells (n=17)

73 26-100

Protein mg/dL (n=17) 115 30-571

Glucose mg/dL (n=16) 38 2-100

21 patients had CNS infection 9 of 21 also had pulmonary infection 4 of 21 had focal cryptococcomas

Initial CSF results for 17 patients with meningitis:

(non-focal CNS infection)

Predisposing Conditions Condition # (%)

Patients Details

Autoimmune Disease 12 (26%) SLE (2), Autoimmune hepatitis, Psoriatic Arthritis, Polymyositis, Sarcoidosis, CNS vasculitis, Giant cell arteritis, Microscopic colitis, HSP, Eosinophilic fasciitis

Solid Organ Transplant 9 (20%) 4 Renal, 2 Lung, 1 Cardiac, 1 Liver

Cancer 7 (15%) 3 active (acute leukemia, NHL, CLL) 4 in past (SCC, testicular, uterine, melanoma)

HIV 3 (7%) 2 AIDS (CD4 16, 27) 1 on ART (CD4 290)

Steroids (prior to diagnosis)

24 (52%) 14 on steroids for >3 months prior to diagnosis

Chronic Lung Disease 12 (26%) 4 COPD, 4 asthma, 2 transplant (1 CF), 1 bronchiectasis, 1 sarcoid

Chronic Liver Disease 7 (15%) 5 HCV, 2 HBV, 1 autoimmune hepatitis

Chronic Renal Disease 10 (22%) 4 on HD, 4 transplant, 2 lupus nephritis

Diabetes 17 (37%)

Predisposing Conditions: Totals**

Predisposing Conditions Number of patients (%)

At least one immune-suppressing condition (Solid organ transplant, autoimmune disease, active malignancy, HIV)

24 (52%)

No immune-suppressing conditions but at least one chronic medical condition (Chronic renal, liver or lung disease, or diabetes)

10 (22%)

No immune-suppressing or chronic medical conditions 12 (26%)

**each patient represented only once

Predisposing Conditions and Disease Manifestations

Predisposing Condition

Lung (n=21)

CNS (n=12)

Lung and CNS (n=9)

Solid Organ Transplant 3 2 4

Autoimmune Disease 9 0 2

Hematologic Malignancy 2 1 0

HIV 2 1 0

ESRD on HD 3 0 1

Chronic Lung Disease 6 1 1

Chronic Liver Disease 4 0 0

Diabetes 9 1 5

None 1 7 3

Type Number of Cases (n= 46)

Died Survived

VGIIa 25 10 (40%) 15 (60%) VGIIb 3 2 (66%) 1 (33%) VGIIc 15 5 (33%) 10 (66%) VGI 3 1(33%) 2 (66%)

Type Immune Suppressed

Chronic Medical Conditions

No Predisposing Conditions

VGIIa 11 6 8 VGIIb 1 2 0 VGIIc 11 2 2 VGI 1 0 2

C gattii VG Subtype

Typing by Clinical Syndrome

Primary Site of Infection VGIIa (n=25)

VGIIb (n=3)

VGIIc (n=15)

VGI (n=3)

Lungs* 12 2 7

CNS 7 1 3 1

CNS and Lungs 3 4 2

Blood 1

Other 2 1

*4 pts with lung infection had asymptomatic nodules: 2 VGIIa and 2 VGIIc

Mortality by Infection Manifestation

Manifestation # Patients (%)

Death (n= 18)

Survival (n= 28)

Pulmonary (n=21) 12 (57) 9 (43)

- Asymptomatic pulmonary nodules 0 4 (100)

CNS (n=12) 1 (8) 11 (92)

- Solitary CNS lesion 0 4 (100)

Pulmonary and CNS (n=9) 4 (44) 5 (56)

Bloodstream only (n=1) 1 (100) 0

Other (urine, throat, toenail) (n=3) 0 3 (100)

Mortality by Predisposing Condition

# Patients (%)

Predisposing Condition Death (n= 18)

Survival (n= 28)

Solid Organ Transplant (n= 9) 5 (56) 4 (44) Autoimmune Disease (n=12) 7 (58) 5 (42) Hematologic Malignancy (n=3) 2 (67) 1 (33) HIV (n=3) 0 3 (100) Chronic Lung Disease (n=8) 5 (63) 3 (37) Chronic Liver Disease (n=9) 6 (67) 3 (33) Diabetes (n=16) 10 (69) 6 (31) No underlying medical conditions (n=12)

0 12 (100)

Summary of Mortality Feature # patients (%) P-value

Death (n= 18)

Survival (n= 28)

Mean age in years 62 56

Male 8/18 11/28 0.77

Predisposing Conditions

Immune-suppressing Conditions 12 12 0.14

Chronic Medical Conditions Only 6 4 0.16

No Predisposing Conditions 0 12 0.0012

Site of Infection

Pulmonary 12 9 0.034

CNS 1 11 0.015

CNS and Pulmonary 4 5 0.72

Bloodstream (only site) 1 0 0.39

Other 0 3 0.27

Positive C.gattii Blood Cultures (n=23) 5 0 0.046

Conclusions and Discussion Increasing number of diagnosed cases of C.gattii in Oregon over past 6 years

Increased proportion of patients with C.gattii had underlying comorbidities than previously reported (74%)

Serum cryptococcal antigen only 68% sensitive for pulmonary infection and 94% sensitive for CNS infection in this series (culture as gold standard)

Both site of infection and mortality correlated strongly with host immune/medical status Mortality occurred exclusively in patients with underlying conditions CNS disease more likely in patients without underlying conditions Relative good baseline health of those with CNS disease may explain why CNS disease

associated with better outcomes than pulmonary disease

Fungemia was a particularly poor prognostic indicator

Cryptococcosis in Animals

Port of entry = respiratory tract via inhalation of spores

Yeast generally precipitate into the upper respiratory tract, as they are too large to be immediately delivered into the lungs

Based on presentation- oral exposure may be a factor in animals

Predisposing factors to infection: Commercial environmental disturbances Immunosuppression

FeLV/FIV in cats Chronic immunosuppressive therapy

Cryptococcosis

Clinical signs in Cats

Nasal/Pharyngeal/Sinus

involvement

Sneezing

Mucoid nasal discharge

Proliferative soft tissue

mass on nasal planum

Cutaneous ulceration

Cryptococcosis CNS involvement

For brain signs

Depression, seizures, altered mentation, circling,

ataxia, head-pressing

Ocular signs

Blindness

Chorioretinitis

Retinal detachment

Anterior uveitis

Cryptococcosis Lower Respiratory Disease

Smaller desiccated yeast inhaled directly into the lungs

Dyspnea, tachypnea, cough

*Skin/subcutaneous involvement*

Present in 40-50% of cases

Papules

Nodules

Ulceration and drainage

Regional lymphadenopathy

Anorexia, lethargy, fever (uncommon)

Cryptococcosis

Canine Cryptococcosis: Dogs < 4 years

CNS involvement (50-80%)

Upper respiratory tract (50%)

Ocular disease (20-40%)

Cutaneous disease (10-20%)

Head, feet, nail beds, oral mucous membranes

Cryptococcus gattii Animal Data, Oregon 2007-2011 Subtypes Isolated (N=40)

Animal Type VGIIa VGIIb VGIIc VGIII

Canine 7 1 2 -

Feline 4 1 5 1

Elk 1 1 - -

Goat 5 - - -

Sheep - - 1 -

Dolphin 1 - - -

Ferret 2

Alpaca 7 - - -

Horse - 1** - -

Total 27 (68%) 4 (10%) 8 (20%) 1(2%)

Site of initial infection at presentation in Animals

Tissue/sample n

Tissue/sample n

lung 11 head swelling 1

nasal discharge 8 periorbital swelling 1

brain 7

skin abscess 6

oral cavity swelling 4

fecal 2

liver 2

renal mass 2

Laboratory Identification

Culture on CGB agar distinguishes species • C. gattii stains blue

Molecular analysis identifies genotype and subtypes (@CDC by Dr Lockhart)

India ink preparation showing capsules of

Cryptococcus Color reaction on CGB agar RFLP or MLST distinguishes

subtypes

Treatment of Cryptococcosis

Treatment

Fluconazole = treatment of choice

Good CNS penetration

50mg/cat PO q 12 hrs

5mg/kg PO q 12-24hrs (dogs)

Itraconazole = good second choice

Effective in cats and dogs

Despite lack of CNS penetration, has been used to successfully treat CNS

cryptococcus (BBB not intact due to inflammation)

10mg/kg PO q 24 hrs

Ketoconazole = variably effective, ineffective in CNS dz

Lipid complex amphotericin B for severe or refractory cases

Susceptibilities…….

Location Location Location

Cases by geographical location

Geographic location of Cryptococcus gattii, Oregon 2008-2010

GILLIAM

MAN

WASCO

CLATSOP

BAKER

CROOK

DESCHUTES

DOUGLAS

GRANT

LAKE

MALHEUR

MORROW

UMATILLA

UNION

WALLOWA

WHEELER

SHER-

COLUMBIA

CURRY

POLK

TILLAMOOK

LINCOLN

WASHINGTON

CLACKAMAS

JACKSON

LANE

BENTON

JOSEPHINE

HARNEY

JEFFERSON

MULTNOMAH

MARION

KLAMATH

LINN

HOOD RIVER

COOS

YAMHILL

Model based on 32 domestic veterinary cases in the US, 2004-2010. The data were split into 50% training and 50% testing subsets and applied against seven environmental layers: January minimum and maximum temperatures, temperature seasonality, minimum temperature of coldest month, annual temperature range, and mean temperature of coldest quarter. The mean training and testing accuracy of the model were 82% and 78%, respectively. Map created November 23, 2010 by Julie Harris, CDC, with assistance from Sunny Mak, BC CDC.

Ecological Niche Modeling of Cryptococcus gattii in the Pacific Northwest

91-100%81-90%71-80%61-70%51-60%41-50%31-40%21-30%11-20%1-10%0%

Acknowledgments

Centers for Disease Control and prevention

Dr Chiller, Dr Harris and Dr Lockhart

Oregon Health Authority – and the OSPHL

Dr Paul Cieslak , Dr Lynn Fitzgibbons, Rob Vega

Oregon State University –

Veterinary Diagnostics laboratory