Cryptococcus neoformans for bmit class.pptx

cryptococcus neoformans

1. 1 Cryptococcus neoformans

2. C hronic , subacute to acute pulmonary, systemic or meningitic disease, initiated by the inhalation of the fungus. Primary pulmonary infections have no diagnostic symptoms and are usually subclinical. On dissemination, the fungus usually shows a predilection for the central nervous system, however skin, bones and other visceral organs may also become involved. Distribution: World-wide. Aetiological Agent: Cryptococcus neofor mans . Cryptococcosis 2

3. Risk group Increasing proportions of patients have an underlying immune deficiency HIV/AIDS Accounts for up to 50% cryptococcal infections CD 4 < 200 Prolonged steroid therapy Organ transplantation Malignancy Sarcoidosis Diabetes Organ transplant Peritoneal dialysis Cirrhosis 3

4. Cryptococcus neoformans also called European blastomycosis “Cryptococcus” means hidden sphere . “ neoformans ” was designated in presumption that the fungus caused tumor because it was found in these patients . A Capsulated yeast – A true yeast . A sporadic disease in the past. Most common infection in AIDS patients. 4

5. Morphology A true yeast Round 4 – 10 microns Surrounded by Mucopolysaccharide capsule. Although Gram positive, capsule may interfere with staining of the fungus . Negative staining with India Ink and Nigrosin KoH preparations in Sputum and other tissues, PAS and Mucicaramine staining helps confirmation. 5

6. As Seen in India Ink preparation 6

7. Cryptococcus neoformans Serotypes The species can be divided into: Three varieties C. neoformans var. grubii C. neoformans var. neoformans C. neoformans var. gattii Nine molecular types based on PCR Fingerprinting Five serotypes A, B, C, D and A/D 7

8. Pigeons and Red river gum tress harbors the Cryptococcus in nature 9

9. Life cycle of C.neofromans 10

10. 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. 11

11. Pathogenesis Can infect normal humans Abnormalities of T lymphocyte function aggravates, the clinical manifestations. In AIDS 3- 20% develop Cryptococcosis. Present with Chronic meningitis , Meningo encephalitis Manifest with – headache low grade fever, Visual abnormalities ,Coma – fatal Can manifest with involvement of Skin,mucosa,organs,Bones,and as Disseminated form. Can mimic like Tuberculosis 12

12. 13 Various reports and studies suggest that both internalized and free Cryptococci are able to cross the blood-brain barrier to reach the brain. In the immunocompromised individuals, hematogenous dissemination of neoformans from the lungs to the central nervous system can lead to cryptococcal meningoencephalitis which is a life-threatening complication requiring aggressive chemotherapeutic intervention.

13. Virulence factors Sialic acids : antiphagocytic role Capsular polysaccharides- antiphagocytic role, anti inflammatory, anti complement interaction. Unique among pathogenic fungi, composed of xylose, mannose and glucuronic acid Melanin production : is deposited on the fungal cell wall and thereby protects the fungus from oxidants released by the phagocytic cells. Mannitol production : contributes to increased intracranial pressure in cryptococcal meningitis patients which depress the anticryptococcal immune responses. Phospholipase : invasion from respiratory tract into CNS Superoxide Dismutase secretions Urease production Growth at 37ºC . 14

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15. Clinical Manifestations The incubation period is unknown and could be weeks, months or even longer. The clinical menifestation is as follows Pulmonary cryptococcosis CNS infections (Cryptococcal meningitis) Cutaneous Cryptococcosis Ocular Cryptococcosis Cryptococcosis of Bone (Osteomyelitis ) Other Cryptococcosis 16

16. Clinical Manifestations Lung: pneumonitis CNS: meningitis, meningoencephalitis Bone ; Osteomyelitis Cutaneous: nodules/vesicles (reflects dissemination) Bleeding into the skin, presenting as petechiae or ecchymoses . Ocular : include papilledema (optic disc swelling) and optic atrophy, due to raised intracranial pressure . Disseminated: fungemia Rarer forms: colitis, osteomyelitis, septic arthritis, myocarditis, hepatitis, pancreatitis, adrenal gland, ocular disease and brain abscess. 17

17. Lab diagnosis 18

18. Laboratory diagnosis: 1. Clinical material: Cerebrospinal fluid (CSF), biopsy tissue, sputum, bronchial washings, pus, blood and urine. 2. Direct Microscopy: For exudates and body fluids make a thin wet film under a coverslip using India ink to demonstrate encapsulated yeast cells. Sputum and pus may need to be digested with 10% KOH prior to India ink staining.

19. India ink preparation showing capsules of C. neoformans

20. (c) For tissue sections use PAS stain, GMS and H&E mucicarmine stain is also useful to demonstrate the polysaccharide capsule. Examine for globose to ovoid, budding yeast cells surrounded by wide gelatinous capsules. Note, non encapsulated variants, although rare, may also occur. Tissue section of lung showing showing atypical non-encapsulated yeast cells of C. neoformans

21. Two sets of SDA with antibiotics are inoculated and incubated at 25 ◦ C and 37 ◦ C, separately over a period of 4 weeks. The media should be without cycloheximide For primary isolation following medias and be used Blood agar SDA Brain-heart infusion agar Cysteine-heart hemoglobin agar Birdseed agar Sunflower seed agar 3. Culture

22. On SDA colony is yeast like highly mucoid, cream to buff colored on SDA Organisms are identified by colony characteristics and microscopic appearance in LPCB. Capsule production can be enhanced by growing the organism in chocolate agar at 37ºC in CO2 incubator. Produces brown color effect when grown on medium containing extract of birdseed or sunflower seed . There are brown-colored colonies after a few days due to conversion of the substrate to melanin by phenoloxidase .

23. Birdseed agar Mixed culture of C. neoformans and C. albicans on bird seed agar (Guizotia seeds) showing the distinctive brown colonies of C. neoformans , due to the selective absorption of pigment from the media, compared to the white colonies of Candida albicans . 24

24. Contd.. Creatinine dextrose bromothymol blue thymine (CDBT) agar is the medium of choice for the differentiation of Cryptococcus neoformans var. neoformans . Cryptococcus neoformans var. neoformans grows as bright red colonies , turning the medium a bright orange after 5 days.

25. The suspected yeast isolates are finally identified by standard biochemical tests. All the species of genus Cryptococcus are non-fermentative, assimilate inositol and produce Urease. Confirmation of C. neoformans : browning of the colony on BSA/SSA Growth at 37ºC Hydrolysis of Urea (within 15 min) Inositol and nitrate assimilation 4. Biochemical test

26. Serology: C ryptococcal capsular polysaccharide antigen in spinal fluid is now the method of choice for diagnosing patients with cryptococcal meningitis. In AIDS patients : in nearly 100% of cases. In non-AIDS patients : is less sensitive with only about 60%

27. RFLP Effective tool in taxonomical and epidemiological studies PCR Animal pathogenicity Swiss albino mice Molecular diagnosis

28. Radiological diagnosis 29 Radiographic manifestations of pulmonary cryptococcosis are diverse and depend on immune status. The findings may include single or multiple scattered nodules and masses ,  smaller than 10 mm in diameter, and well-defined with smooth margins. focal areas of segmental or lobar consolidation, and a reticulo nodular pattern of opacities .

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31. MRI 32

32. Treatment Combination therapy of amphotericin B & flucytosine has been considered the standard treatment for cryptococcal meningitis. Amphotericin B (with or without flucytosine ) is curative in AIDS patients. Fluconazole offers excellent penetration of CNS.

33. 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 34

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