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 19925/10 6 /year HAART1/10 6 /year Africa/HIV15-45%

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 ofVirulence of Inoculationstrain

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

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.gatti Vancouver Island 1999-2003 –8.5 – 37/10 ⁶ /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)