Cryptococcus

Cryptococcus microbiology

1. Cryptococcus neoformans

2. Introduction Cryptococcus neoformans is an encapsulated yeast and an obligate aerobe that can live in both plants and animal C. neoformans usually infects the lungs or the central nervous system (the brain and spinal cord), but it can also affect other parts of the body.

3. Habitat found in soil contaminated with pigeon droppings or eucalyptus trees and decaying wood. Cryptococcus neoformans grows easily in pigeon feces, however the birds are not naturally infected due to high body temp.

4. Speciation: The genus Cryptococcus includes around 37 species. Among these, Cryptococcus neoformans is the only species that is pathogenic to humans. Other species are Cryptococcus albidus , Cryptococcus laurentii , Cryptococcus terreus , and Cryptococcus gastricus .

5. Serotypes-Cryptococcus neoformans It has four serotypes (A to D). Serotyping is based on capsular agglutination reactions. All types can cause human disease Cryptococcus neoformans serotype A is responsible for the majority of human cases.

6. Infection due to serotypes A and D common Serotypes A and D found in excreta of wild and domesticated birds throughout world Serotypes B and C found in flowers Eucalyptus camaldulensis

7. varieties It includes three varieties; var. gattii , var. neoformans and var. grubii . The two varieties, neoformans and gattii are morphologically similar, except that basidiospores of var. neoformans are round and those of var. gattii are more elliptical in shape.

8. Morphology The typical vegetative form of C. neoformans is the yeast form with a cell diameter of 2.5 μm to 10 μm . A characteristic polysaccharide capsule of variable thickness (1-30μm) surrounds these yeasts. In its natural environment the capsule is thinner and the yeast smaller, while thicker capsules tend to be found from infected tissues. The capsules stain pink by the Meyer’s mucicarmine technique.

9. Life cycle Life cycle Asexual: yeast that reproduce by budding Human infections Sexual C. neoformans is heterothallic haploid yeasts that exist in two mating types, MAT α and MATa . MAT α strains are predominant in nature

10. The haploid cells propagate asexually by budding In Sexual life cycle strains of opposite mating type come into contact on substrates conducive for mating In the terminal swollen part of the basidium , the two nuclei from opposite mating types fuse to form a single transient diploidnucleus diploidnucleus undergoes one cycle of meiotic division producing four haploid nuclei

11. Life cycle four haploid nuclei repeatedly undergo mitotic division basipetally produce bud of four long chains of basidiospores (sexual spores) consisting of MAT α and MATa type spores in an equal ratio These basidiospores can cause infection

12. Life Cycle

13. Virulence Factors Virulence The polysaccharide capsule, melanin production, mannitol production, different enzymes :superoxide dismutase, proteases, phospholipase B, and lysophospholipase the phenoloxidase , urease the organism's ability to grow at 37°C these factors increase the degree of pathogenicity of a microbe

14. Melanin Synthesis naturally occurring C. neoformans mutants lacking melanin (Mel-) are less Virulent than melanin-producing strains.

15. mannitol production high concentrations of D- mannitol in the CNS may contribute to brain edema. D- mannitol protect the organism from oxidative damage by hydroxyl radicals

16. Mode of Infection: inhalation of the organism( basidiospores ) from environment. No person to person transmission

17. Disease Cryptococcosis subacute and chronic infection of lung earlier called European blastomycosis considered an opportunistic infections as it affects mainly immunosuppressed individuals.

18. clinical types The clinical types of cryptococcosis are: pulmonary, CNS, visceral, osseous, cutaneous and mucocutaneous .

19. Pulmonary cryptococcosis In healthy individuals, the disease is primarily pulmonary and is asymptomatic. Goes undiagnosed and resolves spontaneously in majority of patients small proportion of cases , haematogeneous spread results in subacute or chronic meningitis or meningoencephalitis All patients with pulmonary disease need a CSF examination to r/o sub clinical meningitis

20. PATHOGENESIS Enters through lungs - inhalation of Basidiospores of C neoformans Enters deep into lungs, Men acquires more infections, and women less infected. Self limiting in most cases, Pulmonary infections can occur. Present as discrete nodules - Cryptococcoma .

21. Disseminated cryptococcosis CNS cryptococcosis Cutaneous and mucocutaneous cryptococcosis In osseous cryptococcosis cranial bones and vertebrae are commonly affected. Visceral cryptococcosis - most commonly heart, testis, prostate and eye.

22. Route of infection

23. Route of cryptococcal meningoencephalitis

24. risk group of cryptococcosis Patients with: AIDS, carcinoma, leukemia, collagen vascular disease, Hodgkin’s disease, sarcoidosis and undergoing immunosuppressive therapy

25. Laboratory diagnosis: The specimens collected according to site of infection Pulmonary : are sputum, blood, lung biopsy CNS: CSF, Serum CRAG Cutaneous and mucocutaneous : skin biopsy, Serum CRAG

26. Wet mount Preparation 10% KOH mount: Globose yeast cells are seen in most clinical materials, such as CSF and pulmonary tissue

27. Negative staining: India ink preparation and Nigrosin

28. Cryptococcus in Lung PAS staining

29. Silver Stain Mucicaramine staining

30. Culture Aspirates and tissues are inoculated onto Sabouraud dextrose agar. Colonies of C.neoformans are fast growing( takes 48 to 72 hours.) Colonies-soft, glistening, smooth, usually mucoid , and cream to yellowish brown in color. It grows well at 25°C as well as 37°C.

31. Bird seed agar

32. Biochemical test phenoloxidase enzyme test: phenoloxidase enzyme that results in production of melanin and thus a brown to black discoloration of the colony when it is grown on caffeic acid agar or bird seed agar. Urease test: Cryptococcus neoformans differs from Candida by hydrolysing urea and not forming pseudohyphae .

33. serological Detection of cryptococcal antigens, particularly polysaccharide capsular antigen by latex-agglutination in CSF, blood or urine. Detection of Serum antibodies by agglutination and immunofluorescence

34. Treatment Amphotericin B combined with 5-fluorocytosine Fluconazole

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