1. Cryptococcosis

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

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. Agent
Species Varieties Serotypes Molecular
types
Cryptococcus grubii A VNI, VNII
neoformans
neoformans D VNIV
AD (hybrid VNIII
diploid)
Cryptococcus B VGI, VGII,
gattii VGIII, VGIV
C

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

9. 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

12. 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.

14. 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

15. 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

16. 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

17. 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)

18. 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)

19. 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

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

21. 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.

22. 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

23. 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

24. 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

25. 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

26. 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

27. 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

29. 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

30. 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)

31. 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

32. Clinical features
• CNS and respiratory tract - most common
organs
• Other prominent infected organs
– skin
– prostate
– eyes
– bone
– urinary tract
– blood

33. 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

34. 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

35. 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

36. Pulmonary cryptococcosis
• Immunocompromised - alveolar and interstitial
infiltrates tend to be more frequent and
potentially mimic pneumocystis pneumonia
– present with CNS rather than pulmonary symptoms

37. 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

38. 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

39. 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

40. Prostate
• usually asymptomatic
• site for yeast sequestration after an occult or
treated disseminated infection

41. 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

42. 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

43. 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

44. 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

45. Alcian blue stain
India ink with 2% chromium mercury
India ink
Gomori’s methenamine silver stain

46. 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

47. 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,

48. 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

49. 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
monoclonal
Sampes used CSF, serum, BAL

50. 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

51. Antigen detection in cryptococcosis
Latex agglutination kits
• False negative results
• Low concentration of antigen
• Prozone phenomena
• Poorly capsulated strain technical error

52. 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

53. 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

54. 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)

55. 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

56. TREATMENT
Treatment

57. 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

58. 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

60. 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

61. 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

62. 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

63. Comparative in vitro activities of posaconazole,
itraconazole, fluconazole, voriconazole, and amphotericinB
against isolates of Cryptococcus spp (271)
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
Antimicrob Agents Chemother. 2006 June; 50(6): 2009–2015

64. 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

65. 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

66. 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

67. 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

68. Thank You