Cryptococcosis
Reference:
Monograph on Cryptococcus and Cryptococcosis In man, animals and birds
A guide for postgraduate students in developing countries By
Mohamed Refai, Mahmoud El-Hariri and Randa Alarousy
History and nomenclature of
the genus Cryptococcus
• The name Cryptococcus was created from
the Greek kryptus meaning ‘hidden’ for a
group of yeasts that lacked the ability to
produce endospores by Kützing (1833) .
Morphology of Cryptococcus
Macro- and microscopic morphology
• On solid media most species have
a slimy appearance, carotenoid
pigments may be produced.
• The cells of Cryptococcus species
are spheroidal, ovoidal, elongate,
amoeboid or polymorphic.
• Cells of most strains are covered
by a thin layer of glycoprotein
capsular material that has a
gelatin-like consistency and that,
among other functions, serves to
help extract nutrients from the
soil.
Ultra structure
• The
electron
microscopy
of
Cryptococcus cell reveals the presense
of a capsule, cell wall, plasma
membrane and nucleus, as well as
other intracytoplasmic organelles.
• The nucleus shows a clear membrane
and generally has disintegrated
chromatin material, instead of a
definite nucleolus.
• Mitochondria are consistent in size and
shape.
• Lipid granules with glycogen in
transition, large numbers of vacuoles,
well
preserved
ribosomes
and
endoplasmic reticulum are observed.
• Bud separation demonstrates a
simulated break-off appearance from
mother cells.
Biochemical characteristics
of Cryptococcus species
Biochemically, the members of the genus
Cryptococcus are unable to ferment sugars, but do
assimilate different sugars, inositol and produce
urease.
Carotenoid pigment production is extremely
variable. Cryptococcus differs from Rhodotorula*
by assimilating inositol, and from Candida
glabrata by assimilating inositol and being urease
positive.
Pathogenicity
•
•
•
•
Cryptococcus neoformans is an opportunistic fungi.
Virulent isolates of Cryptococcus must be able to:
produce small particles that can get into the alveolar spaces,
Grow at 37oC at a pH of 7.3 to 7.4 in an atmosphere of
approximately 5% CO2
• Have the ability to produce a large capsule and shed great
amounts of capsular material into the body fluids makes the
organism highly virulent.
• Other factors, such as melanin, mannitol, superoxide
dismutase, protease, and phospholipase production, may
enhance the pathogenicity of C. neoformans.
• The effectiveness of many of these cryptococcal virulence
factors depends on the status of the host's defensive
mechanisms.
Epidemiology of
Cryptococcosis
• Emmons (1952) was the first one who
isolated C. neoformans from the pigeon droppings after an
outbreak occurred in new York squares.
• Inhalation is the most common mode of transmission.
• Incubation period is unknown.
• Person-to-person transmission has not been documented
other than through transplanted organs.
• Host range: humans and various domestic and wild animals.
• C. neoformans serotypes A and D are distributed worldwide
and cause the vast majority of cryptococcal infections,
predominately in immunocompromised individuals. Serotype
A is responsible for over 95% of cryptococcosis cases
worldwide.
Cryptococcosis in man
• Pulmonary Cryptococcosis :
• Radiographic features. In general
several CT patterns that can be seen:
• clustered nodular
pattern - most prevalent
• solitary pulmonary nodular
• scattered nodular
bronchopneumonic
• The most common CT findings in immunocompetent patients with
pulmonary cryptococcosis are pulmonary nodules. The nodules are most
often multiple, smaller than 10 mm in diameter, and well defined with
smooth margins.
• Associated cavitation may be seen in up to 40% of cases
Central Nervous System: Dissemination to the brain and
meninges is the most common clinical manifestation of
cryptococcosis and includes meningitis, meningoencephalitis
or expanding cryptococcoma.
i. Meningitis is the most common clinical form, accounting for
up to 85% of the total number of cases, however the clinical
signs are rarely dramatic.
Symptoms usually develop slowly over several months, and
initially include headache, followed by drowsiness, dizziness,
irritability, confusion, nausea, vomiting, neck stiffness and focal
neurological defects, such as ataxia. Diminishing visual acuity
and coma may also occur in later stages of the infection. Acute
onset cases may also occur, especially in patients with
widespread disease, and these patients may deteriorate rapidly
and die in a matter of weeks.
(a) Gross photograph of the brain showing opaque meninges in cryptococcal
meningitis; (b) dense lymphomononuclear inflammation over meninges
with scattered yeast like fungal organisms. (H and E; ×400); (c) yeast like
forms of Cryptococcus highlighted by mucicarmine stain (×400).
Cryptococcosis in man
ii. Meningoencephalitis
due to invasion of the
cerebral cortex, brain
stem and cerebellum is an
uncommon, rapid
infection, often leading to
coma and death within a
short time. Symptoms
include slow response to
treatment and signs of
cerebral edema or
hydrocephalitis.
iii. Cryptococcoma
Cryptococcoma is a rare entity, characterized by
localized, solid, tumor-like masses, usually
found in the cerebral hemispheres or
cerebellum, or more rarely in the spinal cord.
Symptoms are consistent with an expanding
intracranial mass and include headache,
drowsiness, nausea, vomiting, mental changes,
slurred speech, double vision, unsteadiness of
gait, coma, paralysis and hemiparalysis. These
symptoms may mimic cerebral neoplasm which
may delay a true diagnosis
 Cutaneous Cryptococcosis:
In patients with AIDS, skin manifestations
represent the second most common site of
disseminated cryptococcosis.
Lesions often occur on the head and neck and
may present as papules, nodules, plaques,
ulcers, abscesses, cutaneous ulcerated plaques.
Anal ulceration may also occur.
Cutaneous Cryptococcosis
 Cryptococcal peritonitis:
Cryptococcal peritonitis has been well- described. It may present in the
ascites of chronic liver disease or in the peritoneal dialysate of patients
with chronic renal disease.
 Cryptococcal pyelonephritis:
Cryptococcuria occurs and even pyelonephritis is described but the
urinary tract site is less common than one would expect with severely
disseminated disease and may simply reflect the length of routine
culture methods.
 Prostatic cryptococcosis:
Prostatic cryptococcosis is generally asymptomatic and has been
considered a site for occult or sequestered infection that is protected
from antifungal treatments.
 Ocular cryptococcosis:
It is reported in up to 45% of patients with
cryptococcosis and thus becomes a significant site for
disease .
• The most common manifestations are ocular
palsies and blindness but in severely
immunosuppressed patients retinal lesions may
appear.
• Loss of vision without evidence of endophthalmitis
also occurs.
 Cryptococcal endocarditis
Despite frequent cryptococcemia, the reported cases
of cryptococcal endocarditis are less than a dozen.
Diagnosis of cryptococcosis
• The workup in patients with suspected cryptococcosis includes
the following:
• Cutaneous lesions: biopsy with fungal stains and cultures
• Blood: fungal culture, cryptococcal serology, cryptococcal
antigen testing and molecular biological assays.
• Cerebrospinal fluid: India ink smear, fungal culture,
cryptococcal antigen testing and molecular biological assays .
• Urine and sputum: cultures, even if renal or pulmonary
disease is not clinically evident.
• Cryptococcal pneumonia: culture of bronchoalveolar lavage
washings.
• Direct microscopic examination of India ink preparation
Preliminary diagnosis of cryptococcal infection is made by direct
microscopic examination of India ink preparations of samples.
Isolation and Identification:
• Definitive diagnosis is confirmed by the
culture of specimens, often the
cerebrospinal fluid (CSF) or blood, and
sometimes in respiratory secretions.
• Cryptococcus neoformans and C. gattii
grow well at 37oC.
• On Sabouraud dextrose agar colonies
appear soft, creamy, opaque in 3-5
days, then colonies become mucoid
and creamy to tan.
Cryptococcus colonies on
Sabroud's dextrose agar
Demonstration of
brown colour effect
• Cryptococcus
colonies are brown
on bird seed agar,
modified tobacco
and Eucalyptus leave
extract agar as well
as on Pal ‫׳‬s medium.
• Other yeasts develop
white to creamy
colonies.
Colonies on bird seed agar
Differentiation of C. gattii
and C. neoformans on
canavanine glycine
bromothymol blue (CGB)
• On Canavanine Glycin
Bromthymol blue (CGB)
medium, Cryptococcus
neoformans develop noncoloured colonies and ,
while C. gattii develops
blue colonies.
Biochemical identification
• Cryptococcus neoformans
and C. gattii do not ferment
sugars, but assimilate
several sugars such as
glucose, galactose, sucrose,
maltose and inositol, but
not lactose or nitrate and
hydrolyses urea.
Serological diagnosis of cryptococcosis
• Cryptococcal antigen from cerebrospinal fluid is the best test
for diagnosis of cryptococcal meningitis in terms of sensitivity.
Rapid diagnostic methods to detect cryptococcal antigen by:
1. Cryptococcal antigen (CRAG) detection:
i. Rapid latex agglutination: either by polyclonal Ab as: CryptoLa; Myco-immune; CALAS or IgM based assay as: Murex
Cryptococcus and
ii. Cryptococcal antigen lateral flow assay (LFA):uses goldconjugated, monoclonal antibodies impregnated onto an
immunochromatographic test strip to detect cryptococcal
capsular polysaccharide glucuronoxylomannan antigen (CRAG)
for all 4 C. neoformans serotypes (A–D).
Serological diagnosis (contin’d)
iii. Cryptococcal Antigen enzyme immunoassay (CrAg EIA) :The
detection of Cryptococcus antigen by enzyme immunoassay is a
highly sensitive and specific test. It is extremely useful in cases
where microscopic techniques are inconclusive (when?).
2. Detection of Cryptococcus antibodies Cryptococcal
antibodies are not helpful in diagnosis because of their poor
sensitivity and specificity.
135
136
137
Diagnosis of cryptococcosis
(contin’d)
• Histopathology
• i) Hematoxylin and eosin stain shows lightly basophilic cell
wall surrounded by a clear zone.
• ii) Cryptococcus neoformans will stain with Periodic acid–
Schiff or silver methenamine.
• iii) Mucicarmine stains the capsule - shows clear zone
containing carminophilic material. Alcian blue also stains the
capsule
• iv) A combined Periodic acid–Schiff - alcian blue stain contrasts
the cell wall and capsule.
Capsule-deficient variety is demonstrated by:
• (a) Immunofluorescent antibody technique.
• (b) Fontana-Masson silver staining
Cryptococcal prostatitis. Left:Fontana-Masson stain demonstrates melanin in
the cell wall. Right: The mucoid capsule of the pathogen is stained with
mucicarmine.
Cryptococcus in the lung.
Methenamine silver stain (MSS)
Diagnosis of cryptococcosis
“Radiology”
Molecular Diagnosis of
Cryptococcus
Molecular typing techniques:
• 1. Multilocus enzyme electrophoresis (MLEE)
• 2. DNA fingerprinting
• 3. Random amplification of polymorphic DNA (RAPD)
• 4. PCR fingerprinting amplified fragment length polymorphism
(AFLP)
• 5. Restriction fragment length polymorphism (RFLP) of PLB1 gene
• 6. Restriction fragment length polymorphism (RFLP) of GEF1 gene
• 7. Restriction fragment length polymorphism (RFLP) of URA5 gene
• 8. Sequencing of ITS1-5.8S-ITS2 rDNA region
• 9. Sequencing of intergenic spacer region (IGS)
• 10. Sequencing of multilocus sequence typing (MLST)
• 11. Sequencing of multilocus microsatellite typing (MLMT)
• 12. Matrix-assisted laser desorption ionization-time of flight mass
spectrometry-based method (MALDI-TOF) analysis
Treatment and Prognosis
• TREATMENT: Give appropriate antifungal therapy .
• IMMUNIZATION: No immunization is currently available;
however, some vaccines are currently in clinical trials,
including GXM conjugated to tetanus toxoid vaccine, which
has been shown to be effective in immunocompetent
individuals in clinical trials.
• PROPHYLAXIS: HIV patients may receive antifungal therapy
such as fluconazole when no symptoms of infections are
present