Cryptococcus neoformans and other Yeast

Dr Sharon Walmsley

University Health Network

Toronto

Organism

• Encapsulated

• Heterobasidiomycetous fungi

• Asexual stage – simple narrow based budding

• Sexual – bipolar system, in-vitro

• 19 species

Identification

• Routine laboratory media

• 48-72 hours, 30-35ºC

• May be inhibited by cycloheximide

• White/cream opaque colonies which become mucoid with prolonged incubation

Rapid identification

• India ink

• Urease test (ureaammoniapH)

• Laccase activity (diphenolic compoundsmelanin) – niger seed agar

Rapid Urease Test

Histopathology

• Prominent capsule

• Spherical narrow based budding yeast

• May have hyphae or pseudohyphae

• 5-10 mm diameter

• 4 serotypes based on capsule

Ecology

• Saprobe in nature – fruit, trees, rotting wood, soil

• Bird guano – pigeons, turkey, chickens

Epidemiology

• HIV• Lymphoproliferative disroders• Sarcoidosis• Corticosteroids• Hyper IgM or IgE syndrome• Monoclonal antibodies (infliximab)• SLE• CD4 T-cell lymphoma (idiopathic)• Diabetes• Organ transplant• Peritoneal dialysis• Cirrhosis

• 20% without HIV have no underlying comorbidity

Spectrum of Disease

Colonization Asymptomatic Disease

Rates of Disease

Pre-AIDS .8/10 6/ year

1992 5/10 6/year

HAART 1/10 6/year

Africa/HIV 15-45%

Rates in Transplant

• 18/100,000

• Increased with cell mediated immune inhibitors

• Highest in kidney and liver

• Rarely carried in through transplanted organ

Serotypes - Cryptococcus neoformans

• A-D

• Commercially available antibody tests

• Biochemical tests

• PCR

Serotypes - Cryptococcus neoformans

Serotype

A – 80% clinical cases

B – tropical, subtropical – S. California, Hawaii, Brazil, Australia, SE Asia

C – rare

D – Europe – Denmark, Germany, Italy, France, Switzerland

Pathogenesis

• Inhalation

• Traumatic inoculation

• Human – human – contaminated transplant tissue

• Zoonosis?

Pathogenicity

• Capsule – polysaccharide

• Melanin

• High temperature growth (37ºC)

Host Response

• Cellular immune response, granulomatous inflammation

• Th – 1 polarized

• Cytokines – TNF, 1F-8, 1L-2

• Proinflammatory 1L-12, 1L-18, MCP-1, MIP

• NK cells

Pathogenesis

Host defense

Size of Virulence of

Inoculation strain

Clinical Manifestations

Lung- Portal of entry- asymptomatic (1/3) life threatening

pneumonia (ARDS)- Endobronchial colonization underlying

chronic lung disease- Single pulmonary nodule- Symptomatic – acute, subacute

Pulmonary Cryptococcus

Clinical Manifestations

CNS- Subacute meningitis or meningo-encephalitis- Headache, fever, cranial nerve palsies, lethargy, coma- Subacute (days) monthsHIV- Higher yeast burden incidence raised intracranial pressure- Often disseminated- Immune reconstitution disease

Cryptococcal meningitis

Cryptococcus- Oral Lesions

Clinical Manifestations

Skin

- Papule with ulcerated center

- Cellulitis, abscess

- Rarely underlying bone lesions

Prostrate

- Asymptomatic (sanctuary)

- Penile, vulvar lesions

Cryptococcus, skin lesions

Cryptococcus, skin lesions

Clinical Manifestations

Eye

- Ocular palsy, papilledema, optic neuritis

- Retinal exudates +/- iritis

- endophthalmitis

Diagnosis

Microscopic– India Ink (50-80% + CSF)– Gram– Calcoflur white– Silver stain

Culture– Blood agar– Routine blood culture

Cryptococcus, India Ink

Diagnosis

Serology– Latex agglutination, EIA, 90% sensitive &

specific

Radiology– CXR – infiltrates, nodules, lymphadenopathy,

cavitation, effusion– CT/MRI – 50% normal, hydrocephalus,

nodules

In vitro susceptibility testing

• Low MICs – amphotericin, flucytosine, azole

• High MICs – caspofungin

• In vitro R demonstrated but most refractory cases are relapses

Therapy – Cryptococcal meningitis

• Amphotericin B +/- flucytosine

• Fluconazole

• Amphotercin x 2 wk then fluconazole 400-800 mg/d x 8-10 wk

• Chronic suppression fluconazole 200 mg/d

Raised ICP

• CSF OP > 250mm

• Rapidly progressive cerebral edema

• Repeated LP, shunt

• Corticosteroids not useful

Prognosis

• Need to be able to control underlying disease immunosuppression prednisone– HAART– ? Adjunctive cytokines – interferon, GCSF

Poor prognosis

Burden of organism( + India Ink, crypto Ag > 1:1024, poor CSF

inflammatory response < 20 cells/uL)

Sensorium

Mortality 10-25%

Prevention

• Fluconazole prophylaxis

• Active immunization- cryptococcal GXM-tetanus toxoid conjugate vaccine- in animal models, no human trials

• Monoclonal antibodies- would require repeated injections

• Avoid high risk environments

Cryptococcus neoformans (var gattii)

• Initially described in Australia

• Cultured from vegetation around river red gum trees, eucalyptus trees

• Recent outbreak Vancouver Island

Cryptococcus neoformans var gatti

• Outbreak Vancouver Island, January 02

• N = 59, 2 deaths

Cryptococcus neoformans var gatti

• 75% primarily pulmonary disease

• 25% CNS

• 58% male, 5.3% Asian

• Mean age 60

• Certain geographic locations

• Never cultured from bird guano

• May be associated with certain trees

Cryptococcus neoformans

C ryp to co cu s n eo fo rm a ns

va r g ru b ii

(n o w C .g a tt i)

va r ga tti va r e n o fo rm a ns

C.gattiVancouver Island

• 1999-2003– 8.5 – 37/10⁶/year

• Australia - endemic– 94 cases/million/year

C.gatti

• Usually restricted to tropical, subtropical

• Now in temperate zone

• Able to identify an environmental reservoir

• Identified in sea animals

Cryptococcus

• Global epidemiology• Study – Canada 1984• N = 78• 7.7% C.gatti• 79.5% C.neoformans v grubii• 6.4% C.neoformans v neoformans (serotype D)• 6.4% C.neoformans v neoformans (hybid AD)