Cryptococcosis neetu


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  • Sexual reproduction between partners of the same mating type in Cryptococcus neoformans.
  • 34- to 38-kilodalton Cryptococcus neoformans glycoprotein produced as an exoantigen bearing a glycosylated species-specific epitope
  • Cryptococcosis neetu

    1. 1. Cryptococcosis
    2. 2. Introduction• Causative agent – Cryptococcus spp.• Total number of species – 30• Before AIDS few cases reported• Massive increase in number of cases with advent of – AIDS – Organ transplantation – Other immunosuppresed states
    3. 3. History• 1894 Busse and Buschke - first human case of cryptococcosis• 1894 Sanfelice - peach juice• 1955 Emmons decomposed pigeon droppings• 1990 Ellis and Pfeiffer Cryptococcus neoformans var gatti - Eucalyptus trees• 1975 Kwon-Chung - sexual stage of C. neoformans• 2003 genome of C. neoformans sequenced
    4. 4. Agent• Kingdom: Fungi• Division :Basidiomycota• Class: Basidiomycetes• Order: Filobasidiales• Genus: Filobasidiella (Cryptococcus)
    5. 5. Agent• 30 species - genus Cryptococcus• pathogenic yeasts of cryptococcosis – C neoformans – C gattii• previously classified as three varieties – C neoformans var neoformans – C neoformans var grubii – C neoformans var gattii
    6. 6. AgentSpecies Varieties Serotypes Molecular typesCryptococcus grubii A VNI, VNIIneoformans neoformans D VNIV AD (hybrid VNIII diploid)Cryptococcus B VGI, VGII,gattii VGIII, VGIV C
    7. 7. Life cycle• C neoformans and C gattii are composed of – asexual and sexual stages.• sexual stage of – C neoformans teleomorph – Filobasidiella neoformans – C gattii teleomorph Filobasidiella bacillospora
    8. 8. Life cycle• alpha-mating–type strains (95%) in patients and the environment• recombination also observed between two alpha-mating–type strains - explain the prominent bias of alpha-mating–type strains Nature 2005;434(7036):1017–21
    9. 9. Epidemiology• C neoformans var grubii - 95% of cryptococcal cases - worldwide• C neoformans var neoformans - European countries, such as Denmark, Germany, Italy, France, Switzerland, and the United States• C gattii – tropical and subtropical areas, such as Australia, Southeast Asia, Central Africa, and the tropical and subtropical areas of the Americas.
    10. 10. Epidemiology• C gattii - 44% of patients immunocompetent – limited environment exposure – reduced ability of this species to reactivate in the host• C neoformans infections - 98% were immunocompromised
    11. 11. Epidemiology• Before HIV epidemic - cryptococcal infection uncommon systemic fungal infection• Prior to 1956 - 300 cases documented in medical literature by Littman and Zimmerman• incidence of cryptococcosis in non - AIDS patients 0.2 to J Infect Dis 1999;179(2):449–54. 0.8 per 100,000• HIV infection associated cryptococcosis - more than 80% of cryptococcosis cases worldwide
    12. 12. Epidemiology• pre-HAART era, cryptococcal infection major opportunistic infection and major cause of death• potent antiretroviral treatment became widely available - incidence of cryptococcosis decreased – 66 per 1000 (1992) to 7 per 1000 (2000) in Atlanta, – 24 per 1000 (1993) to 2 per 1000 (2000) in Houston Clin Infect Dis 2003;36(6):789–94. – 1985 through 2001 - 46% decrease in France
    13. 13. Epidemiology in India Epidemiology in India• first report days of the British Raj - Reeves et al, described a case from Calcutta with interlobar empyema and draining chest sinuses• eight cases - late 1940s and 1950s• 48 cases – 1960s• Incidence high in places like Kolkatta, Delhi, Mangalore• Serotype A appears to be the commonest cause of disease (>90% in most reports)
    14. 14. Epidemiology in India• AIIMS, New Delhi 74 cases (5.4 cases per year) 1985-97• NIMHANS, Bangalore, commonest CNS mycotic infection (84.8%, 149 cases 1978-97, 7.5 cases per year)• SGPGI, Lucknow, 54 cases - 5.5 year period between 1996 and 2001 (10 cases / year)
    15. 15. PGIMER, Chandigarh PGIMER, Chandigarh• 1960 through 1972 0.8 cases per year• 1980s (1983-94) suddenly increased to 4.5 cases per year• 1985-97 54 cases, 11.6 cases per year 15 fold increase in incidence since the pre-AIIDS era• 1996-2005, 10 year study 25.2 cases / year – highest incidence recorded in the country
    16. 16. C. laurentii and C. albidus• few reports of infection due to these two species have been recorded (3 from Chandigarh and 1 from Madras)
    17. 17. Ecological Associations India Ecological niches in in India• Cryptococcus neoformans var neoformans – – droppings of pigeons, munia birds and canaries – Vegetables and fruits• Cryptococcus gatti – Eucalyptus trees in the flowering season - Eucalyptus camaldulensis, Eucalyptus tereticornis, Ficus religiosa and Syzigium cumini trees• Cryptococcus neoformans var grubii - Eucalyptus camaldulensis barks• Cryptococcus neoformans var neoformans - Ficus religiosa , Syzigium cumini and Tamarind indica trees.
    18. 18. Pathogenesis Pathogenesis• Cryptococcus infects humans from environmental exposures• portal of entry of Cryptococcus - inhalation of the infectious propagules from the environment – either dehydrated yeast cells or basidiospores• Susceptibility of host - latent infection or acute disease
    19. 19. virulence factors Virulence factors• capsule formation• melanin pigment production• ability to grow well at 370c• alpha-mating–type locus• secretory phospholipase B• urease production• myristolyation• enzymes associated with protection against oxidative stresses
    20. 20. Virulence factors• Polysaccharide capsule, comprised of glucuronoxylomannan (GXM)• Antiphagocytic• Ab unresponsiveness• Inhibition of leucocyte migration• Deregulation of cytokine secretion• Interference with antigen presentation• L-selectin & tumor necrosis factor loss
    21. 21. Virulence factors• Melanin• laccase enzyme - encoded by two paralogs, LAC1 and LAC2 genes• catalyzes the conversion of diphenolic compounds to melanin• Laccase regulated by various environmental signals – nutrient starvation, multivalent cations, and temperature stress• mediated through multiple signal transduction pathways
    22. 22. Virulence factors• Melanin• Antioxidant• Cell wall support and integrity• Interference with T-cell response• Reduction of susceptibility to antifungal agents• Abrogation of antibody mediated phagocytosis• Protection from extreme temperature
    23. 23. Virulence factors• Phenotypic switching - during chronic infection• serotype A and D strains of Cryptococcus neoformans• associated with differential gene expression and changes in virulence• polysaccharide capsule and cell wall - yeasts ability to resist phagocytosis• ability of the mucoid colony variant but not the smooth variant to promote increased intracerebral pressure in a rat model of cryptococcal meningitis
    24. 24. Regulation of virulence• (Gpa1)/cyclic adenosine monophosphate pathway – control melanin and capsule production – sense nutrients during mating and disease production• conserved mitogen-activated protein kinase (MAPK) – senses pheromone during mating – regulates haploid fruiting and virulence• RAS (Ras1/Ras2) signaling cascade and the calcineurin- dependent pathway – High-temperature growth in C neoformans
    25. 25. Host immune response• Cell-mediated immunity - most important arm of host defenses • fungus enters alveoli • processed by alveolar macrophages (IL-12, IL-18, (MCP)-1, and MIP 1a) • Th1 response with cytokines (TNF-a, IFN-g, and IL-2) • reduced Th2 cytokines (IL-4 IL-5 and IL-10)
    26. 26. Virulence factors• Cryptococcus spp - attractive for molecular virulence studies – primary fungal pathogens that cause invasive mycoses in healthy and immunocompromised hosts – genome-wide sequencing availability – ease of targeted gene deletions in Cryptococcus – robust animal models
    27. 27. Clinical features• CNS and respiratory tract - most common organs• Other prominent infected organs – skin – prostate – eyes – bone – urinary tract – blood
    28. 28. Predisposing factors of cryptococcosis• HIV infection• Corticosteroids• Solid organ transplantation• Malignancies• CD4+ T-cell lymphopenia• Connective tissue diseases or immunologic diseases• Monoclonal antibodies (etanercept, infliximab, alemtuzumab)• Diabetes mellitus• Chronic pulmonary diseases or lung cancer• Renal failure or peritoneal dialysis• Cirrhosis• Pregnancy
    29. 29. Clinical features• Differences among patients infected with HIV compared with those not infected – more CNS and extrapulmonary involvement – higher rate of positive India ink examinations – Positive blood cultures – fewer CSF inflammatory cells
    30. 30. Pulmonary cryptococcosis• portal of entry• asymptomatic infection to life-threatening fungal pneumonia• acute pulmonary cryptococcosis – fever, productive cough, chest pain and weight loss• Radiographic presentations are varied - single or multiple pulmonary nodules – most common – Pumonary infiltrates, pleural effusions, hilar lymphadenopathy, diffuse reticulonodular opacities, endobronchial lesion and findings mimicking pulmonary metastasis
    31. 31. Pulmonary cryptococcosis• Immunocompromised - alveolar and interstitial infiltrates tend to be more frequent and potentially mimic pneumocystis pneumonia – present with CNS rather than pulmonary symptoms
    32. 32. Central nervous system• acute, subacute or chronic meningitis, or meningoencephalitis – headache, fever, cranial neuropathy, alteration of consciousness, lethargy, memory loss, meningeal irritation signs, and coma• Meninges covering basal ganglia and thalamus involved• cerebral lesions - most often gelatinous areas of necrosis and cysts
    33. 33. Skin• third most common clinical site• Serotype D strains - propensity to cause cutaneous lesions• primary cutaneous infection from direct inoculation or a secondary lesion as part of disseminated disease
    34. 34. Skin• Primary cutaneous cryptococcosis – Solitary skin lesion - whitlow or phlegmon, – history of skin injury – participation in outdoor activities – exposure to bird droppings, Eucalyptus trees• Secondry lesions – molluscum contagiosum-like lesion – acneiform lesions, purpura, vesicles, nodules, abscesses, ulcers
    35. 35. Prostate• usually asymptomatic• site for yeast sequestration after an occult or treated disseminated infection
    36. 36. Other organs• Bone – Osteolytic lesions with draining sinuses - disseminated infections• Eye – Ocular involvement is common and classified in two categories – Rapid visual loss - 12 hours suggestive of optic neuritis due to invasion of the yeasts in the nerve, usually reversible – Slow visual loss - later in therapy and progresses over weeks to months, due to increased intracerebral pressure and can be halted by shunts and optic nerve fenestration surgery
    37. 37. Immune reconstitution inflammatory syndrome• worsening of clinical or radiographic manifestations – consistent with inflammatory process but negative studies for biomarkers or cultures• 30% to 35% of patients infected with HIV who have cryptococcosis in whom HAART was initiated – 4 to 6 weeks after initiation of HAART – decreasing viral load – increasing CD4 counts
    38. 38. Laboratory Diagnosis Laboratory diagnosis• Samples include – CSF, sputum, BAL, lymph node aspirations, biopsy samples,blood, urine, and expressed prostatic secretion• demonstration of the encapsulated budding round yeasts in a sterile body site by India preparation or histopathology• culture isolation of the fungus from a sterile site• detection of cryptococcal capsular antigen
    39. 39. Laboratory diagnosis• India ink, Modified India Ink (with 2% chromium mercury) and nigrosin stains - negative halo around the budding yeasts – 50% sensitivity immunocompetent hosts – > 80% in immunocompromised hosts.• Mucicarmine and Alcian Blue positive stains for the capsule• Mason Fontana stain which stains melanin and Gomori Methenamine Silver staining - histopathology
    40. 40. Alcian blue stainIndia ink with 2% chromium mercury India ink Gomori’s methenamine silver stain
    41. 41. Culture Culture• CSF should be collected in large amounts (20ml) and centrifuged – Two Sabouraud Dextrose Agar (one each at 370C and 250C), – brain heart infusion agar and – Bird seed agar (or caffeic acid containing media)• Colonies appear in 2-5 days - cream white to shiny in colour• black on niger seed agar in 5 days.• Confirmation - urease test, positive inositol assimilation and negative nitrate assimilation test
    42. 42. Distinguish between varieties distinguish between varieties• Canavanine Glycine Bromothymol Blue test – C gatti turns the medium blue while var neoformans and var. grubii do not• D-proline assimilation test are used• all isolate - tested for the mouse pathogenicity test by the IV / intracerebral / intraperitoneal route,
    43. 43. Antigen detection in cryptococcosis Antigen detection in cryptococcosis• Antigens – Capsular polysaccharide – Protein antigen• Methods – Pollysacharide antigens • Latex agglutination • Elisa • Co-agglutination – Protein antigens • Elisa • Western blot
    44. 44. Antigen detection in cryptococcosis Latex agglutination kits Latex agglutination kits• Crypto-La (International Biologicals NJ) polyclonal Ab• Myco-immune (American Microscan NJ) polyclonal Ab• IMMY (Immuno-Mycologics, Okla) polyclonal Ab• CALAS (Meridian diagnostics, Ohio) polyclonal Ab• Eiken tet (Eiken Co, Tokyo)• Pastorex Cryptococcus (Sanofi Diagnostic Pasteur, France) monoclonal Ab• Murex Cryptococcus (Murex Diagnostics, Ga) IgM based monoclonalSampes used CSF, serum, BAL
    45. 45. Antigen detection in cryptococcosis Latex agglutination kits• False-positive results• Rheumatoid factor – heating serum specimens at 560C for 30 minutes and CSF specimens at 1000C for 10 minutes or – pretreating with dithiothreitol – 2-b-mercaptoethanol – protease enzyme• T richosporon beigelii, Stomatococcus mucilaginosus, C apnocytophaga canimorsus and K lebsiella pneumoniae• contamination by syneresis fluid
    46. 46. Antigen detection in cryptococcosis Latex agglutination kits• False negative results• Low concentration of antigen• Prozone phenomena• Poorly capsulated strain technical error
    47. 47. Antigen detection in cryptococcosis ELISA kits ELISA kits• Meridian Premier ELISA kits ( polyclonal capture, monoclonal detector)• Monoclonal Ab of different isotypes for both capturing and detection• Biotin amplified sandwich ELISA
    48. 48. Correlation of Ag detection with outcome• Latex agglutination test for detection of antigen in CSF – diagnosis – predicting the outcome of the patients• geometric mean of antigen titre in CSF was significantly higher (p0.001) in patients who died compared to those who recovered IJMM 1995; 13: 65-69
    49. 49. Protein antigen in cryptococcosis• False positivity of detection of capsular polysaccharide• Species specific culture supernatant, exo-Ag identified• Mab 3C2 – Reactive to cytoplasm and cell membrane – 34-38 KD antigen• MAb to 7C9 – 110-112 KD antigen, 65-70, 45-50, 36-38 KD• Helps in AIDS (low titer, acapsular strains)
    50. 50. Antibody detection in cryptococcosis• cryptococcal antibodies not helpful in diagnosing and deciding treatment for cryptococcosis• poor sensitivity and specificity performance• positive in the absence of overt disease• immunologically paralyzed status of patients infected with HIV and those who are severely immunosuppressed Infect Dis Clin N Am 20 (2006) 507–544
    51. 51. TREATMENT Treatment
    52. 52. Cryptococcal disease in HIV-negative patients• Central nervous system• Induction/consolidation or clearance therapy: – Amphotericin B, 0.7 to 1 mg/kg/d (preferably 0.7mg/kg/d), plus flucytosine, 100 mg/kg/d (assuming normal renal function), for 2weeks, then fluconazole, 400-800mg/d, forminimum10weeks
    53. 53. Cryptococcal disease in HIV-negative patients• Alternative regimens: – Amphotericin B, 0.3 mg/kg/d, plus flucytosine, 100 mg/kg/d, for 6 to 10 weeks – Amphotericin B, 0.4 to 1 mg/kg/d, for 6 to 10 weeks – Lipid formulation of amphotericin B, 4 to 6 mg/kg/d, for 6 to 10 weeks, with or without 2 weeks of flucytosine (100 mg/kg/d)• Suppressive therapy: – Fluconazole 200 to 400 mg/d, for completion of 1 year of therapy
    54. 54. Cryptococcaldisease in in patients infected Cryptococcal disease HIV-positive patients with HIV• Pulmonary• Mild-to-moderate symptoms or asymptomatic with culture positive from the lungs: – Fluconazole, 200 to 400 mg/d, for 1 to 2 years (depending on response to HAART)• Alternative regimen: – Itraconazole, 200 to 400 mg/d, for 1 to 2 years (depending onresponse to HAART) – Fluconazole, 200 to 400 mg/d, and flucytosine, 100 to 150 mg/kg/d, for 10 weeks• Severe symptoms: – Treat like CNS disease
    55. 55. Cryptococcaldisease in in patients infected Cryptococcal disease HIV-positive patients with HIV• Central nervous system• Induction/consolidation or clearance therapy: – Amphotericin B, 0.7 to 1 mg/kg/d (preferably 0.7 mg/kg/d), plus flucytosine,100 mg/kg/d, for 2 weeks, then fluconazole, 400 to 800 mg/d, for minimum 10 weeks
    56. 56. Cryptococcaldisease in in patients infected Cryptococcal disease HIV-positive patients with HIV• Alternatives regimens: – Fluconazole, 400 to 800 mg/d, for 10 to 12 weeks – Fluconazole, 400 to 800 mg/d, plus flucytosine, 100 to 150 mg/kg/d, for 6 to 10 weeks – Lipid formulation of amphotericin B, 4 to 6 mg/kg/d, for 6 to 10 weeks, with or without flucytosine• Maintenance or suppressive therapy:1 to 2 years and may consider stopping if response to HAART – Fluconazole, 200 to 400 mg/d• Alternatives regimens: – Itraconazole, 200 mg/d – Amphotericin B, 1 mg/kg intravenously, one to three times per week
    57. 57. Comparative in vitro activities of posaconazole,itraconazole, fluconazole, voriconazole, and amphotericinB against isolates of Cryptococcus spp (271) MICs POS ITC FLC VRC AMB50% 90% 50% 90% 50% 90% 50% 90% 50% 90%0.125 0.25 0.125 0.5 4.0 8.0 0.063 0.125 1.0 1.0 Antimicrob Agents Chemother. 2006 June; 50(6): 2009–2015
    58. 58. Management of elevated intracranial Management of elevated ICP pressure• Managing increased intracranial pressure is equally important as using direct antifungal therapy• opening pressure of 250 mm H2O - elevated intracranial pressure• high intracranial pressure after 2 weeks of treatment predicted a poorer clinical response in patients infected with HIV who had cryptococcal meningitis
    59. 59. Management of elevated intracranial Management of elevated ICP pressure Before treatment• Focal neurologic signs, obtunded – Radiographic imaging before lumbar puncture to exclude contraindications• Normal opening pressure – Initiate medical therapy, with follow-up lumbar puncture at 2 weeks• Opening pressure 250 mm H2O or more with signs or symptoms – Lumbar drainage sufficient to achieve closing pressure less than 200 mm H2O or 50% of initial opening pressure
    60. 60. Management of elevated intracranial Management of elevated ICP pressure Follow-up for elevated pressure• Repeated drainage daily until opening pressure and symptoms/signs are stable If elevated pressure persists• Lumbar drain• Ventriculoperitoneal shunt
    61. 61. Prevention• pre-HAART era - fluconazole prophylaxis in patients with AIDS and CD4 counts under 100 cells/μL – use of HAART and concern about drug resistance - reduced enthusiasm for this approach• immunization with a vaccine in high-risk patients – cryptococcal GXM–tetanus toxoid conjugate vaccine – new potential protective antigens have been identified• protective serotherapy with specific monoclonal antibodies• avoid high-risk environments
    62. 62. Thank You
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