Cryptococcus neoformans and other Yeast

1. Cryptococcus neoformans and other Yeast Dr Sharon Walmsley University Health Network Toronto

2. Organism • Encapsulated • Heterobasidiomycetous fungi • Asexual stage – simple narrow based budding • Sexual – bipolar system, in-vitro • 19 species

4. Identification • Routine laboratory media • 48-72 hours, 30-35ºC • May be inhibited by cycloheximide • White/cream opaque colonies which become mucoid with prolonged incubation

5. Rapid identification • India ink • Urease test (ureaammoniapH) • Laccase activity (diphenolic compoundsmelanin) – niger seed agar

6. Rapid Urease Test

7. Histopathology • Prominent capsule • Spherical narrow based budding yeast • May have hyphae or pseudohyphae • 5-10 mm diameter • 4 serotypes based on capsule

8. Ecology • Saprobe in nature – fruit, trees, rotting wood, soil • Bird guano – pigeons, turkey, chickens

9. 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

10. Spectrum of Disease Colonization  Asymptomatic  Disease

11. Rates of Disease Pre-AIDS .8/10 6/ year 1992 5/10 6/year HAART 1/10 6/year Africa/HIV 15-45%

12. Rates in Transplant • 18/100,000 • Increased with cell mediated immune inhibitors • Highest in kidney and liver • Rarely carried in through transplanted organ

13. Serotypes - Cryptococcus neoformans • A-D • Commercially available antibody tests • Biochemical tests • PCR

14. 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

15. Pathogenesis • Inhalation • Traumatic inoculation • Human – human – contaminated transplant tissue • Zoonosis?

16. Pathogenicity • Capsule – polysaccharide • Melanin • High temperature growth (37ºC)

17. 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

18. Pathogenesis Host defense Size of Virulence of Inoculation strain

19. 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

20. Pulmonary Cryptococcus

21. Clinical Manifestations CNS • Subacute meningitis or meningo-encephalitis • Headache, fever, cranial nerve palsies, lethargy, coma • Subacute (days)  months HIV • Higher yeast burden •  incidence raised intracranial pressure • Often disseminated • Immune reconstitution disease

22. Cryptococcal meningitis

23. Cryptococcus- Oral Lesions

24. Clinical Manifestations Skin • Papule with ulcerated center • Cellulitis, abscess • Rarely underlying bone lesions Prostrate • Asymptomatic (sanctuary) • Penile, vulvar lesions

25. Cryptococcus, skin lesions

26. Cryptococcus, skin lesions

27. Clinical Manifestations Eye • Ocular palsy, papilledema, optic neuritis • Retinal exudates +/- iritis • endophthalmitis

28. Diagnosis Microscopic • India Ink (50-80% + CSF) • Gram • Calcoflur white • Silver stain Culture • Blood agar • Routine blood culture

29. Cryptococcus, India Ink

30. Diagnosis Serology • Latex agglutination, EIA, 90% sensitive & specific Radiology • CXR – infiltrates, nodules, lymphadenopathy, cavitation, effusion • CT/MRI – 50% normal, hydrocephalus, nodules

31. In vitro susceptibility testing • Low MICs – amphotericin, flucytosine, azole • High MICs – caspofungin • In vitro R demonstrated but most refractory cases are relapses

32. 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

33. Raised ICP • CSF OP > 250mm • Rapidly progressive cerebral edema • Repeated LP, shunt • Corticosteroids not useful

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

35. Poor prognosis • Burden of organism ( + India Ink, crypto Ag > 1:1024, poor CSF inflammatory response < 20 cells/uL) • Sensorium Mortality 10-25%

36. 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

37. Cryptococcus neoformans (var gattii) • Initially described in Australia • Cultured from vegetation around river red gum trees, eucalyptus trees • Recent outbreak Vancouver Island

38. Cryptococcus neoformans var gatti • Outbreak Vancouver Island, January 02 • N = 59, 2 deaths

39. 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

40. Cryptococcus neoformans

41. C.gattiVancouver Island • 1999-2003 • 8.5 – 37/10⁶/year • Australia - endemic • 94 cases/million/year

42. C.gatti • Usually restricted to tropical, subtropical • Now in temperate zone • Able to identify an environmental reservoir • Identified in sea animals

43. 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)