Cryptococcosis neetu

Cryptococcosis

• 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

Introduction

• 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

History

• Kingdom: Fungi• Division :Basidiomycota• Class: Basidiomycetes• Order: Filobasidiales • Genus: Filobasidiella (Cryptococcus)

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

Agent

Species Varieties Serotypes Molecular types

Cryptococcus neoformans

grubii A VNI, VNII

neoformans D VNIV

AD (hybrid diploid)

VNIII

Cryptococcus gattii

B VGI, VGII, VGIII, VGIV

C

Agent

• 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

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

Life cycle

• 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.

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

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

0.8 per 100,000

• HIV infection associated cryptococcosis - more than 80%

of cryptococcosis cases worldwide

J Infect Dis 1999;179(2):449–54.

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

– 1985 through 2001 - 46% decrease in France

Clin Infect Dis 2003;36(6):789–94.

Epidemiology

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)



• 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)


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

PGIMER, Chandigarh

C. laurentii and C. albidus

• few reports of infection due to these two species have been recorded (3 from Chandigarh and 1 from Madras)

Ecological Associations 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.

Ecological niches in India

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

Pathogenesis

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

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

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

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

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 - yeast's 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

Virulence factors

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

Regulation of virulence

• 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)

Host immune response

• 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

Virulence factors

• CNS and respiratory tract - most common organs

• Other prominent infected organs – skin – prostate– eyes – bone – urinary tract – blood

Clinical features

• 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

Predisposing factors of cryptococcosis

• 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

Clinical features

• 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

Pulmonary cryptococcosis

• Immunocompromised - alveolar and interstitial infiltrates tend to be more frequent and potentially mimic pneumocystis pneumonia

– present with CNS rather than pulmonary symptoms

Pulmonary cryptococcosis

• 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

Central nervous system

• 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

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

Skin

• usually asymptomatic

• site for yeast sequestration after an occult or treated disseminated infection

Prostate

• 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

Other organs

• 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

Immune reconstitution inflammatory syndrome

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

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

Laboratory diagnosis

India ink with 2% chromium mercury

Alcian blue stain

India ink

Gomori’s methenamine silver stain

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

Culture

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,

Distinguish between varieties

Antigen detection in cryptococcosis

• Antigens– Capsular polysaccharide– Protein antigen

• Methods– Pollysacharide antigens

• Latex agglutination• Elisa• Co-agglutination

– Protein antigens• Elisa• Western blot

Antigen detection in cryptococcosis

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

monoclonal

Sampes used CSF, serum, BAL

Antigen detection in cryptococcosisLatex 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

• Trichosporon beigelii, Stomatococcus mucilaginosus, Capnocytophaga canimorsus and Klebsiella pneumoniae

• contamination by syneresis fluid


• False negative results • Low concentration of antigen• Prozone phenomena• Poorly capsulated strain technical error


ELISA kits

• Meridian Premier ELISA kits ( polyclonal

capture, monoclonal detector)

• Monoclonal Ab of different isotypes for both

capturing and detection

• Biotin amplified sandwich ELISA

Antigen detection in cryptococcosisELISA kits

• 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

Correlation of Ag detection with outcome

• 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)

Protein antigen 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

Antibody detection in cryptococcosis

Infect Dis Clin N Am 20 (2006) 507–544

TREATMENTTreatment

• 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

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

Cryptococcal disease in HIV-negative patients

Cryptococcal disease in patients infected 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

Cryptococcal disease in HIV-positive patients


• 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



• 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


MICs

POS ITC FLC VRC AMB

50% 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

Comparative in vitro activities of posaconazole, itraconazole, fluconazole, voriconazole, and amphotericinB

against isolates of Cryptococcus spp (271)

Antimicrob Agents Chemother. 2006 June; 50(6): 2009–2015

Management of elevated intracranial 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

Management of elevated ICP


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



Follow-up for elevated pressure• Repeated drainage daily until opening pressure

and symptoms/signs are stable

If elevated pressure persists• Lumbar drain• Ventriculoperitoneal shunt


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

Prevention

Thank You

Health & Medicine

Cryptococcosis neetu