Cryptococcus neoformans

One hundred years ago, Cryptococcus

neoformans was discovered when this
encapsulated yeast was isolated
independently from peach juice in Italy
and from the tibial lesion of a patient in
Germany
 it had been given a variety of names,
including Saccharomyces neoformans,
Blastomyces neoformans,
Cryptococcus hominis
• Reports of infection due to other species
of Cryptococcus (C. albidus and
C.laurentii) are extremely rare
• C. neoformans var. neoformans has long
been isolatable from soil and avian
habitats.
• Birds do not become infected, probably
because their relatively high body
temperature is inimical to C. neoformans.
• However, birds are likely to distribute the
yeasts in nature.
• There are two varieties of C. neoformans
and four major serotypes, based on
capsular epitopes.
• Nearly all infections of patients with
AIDS involve strains of a single variety
and serotype, although such strains vary
at the phenotypic and genotypic levels
• The 19 species within the genus
Cryptococcus are characterized as
variously encapsulated budding yeasts.
• Despite reports of other pathogenic
species, the major pathogen is the species
C. neoformans, an encapsulated, usually
spherical yeast of which two varieties are
recognized, C. neoformans var.
neoformans and C. neoformans var.
gattii.
• By using cross-absorbed rabbit polyclonal
antisera, the capsular serotypes of C.
neoformans were defined as serotypes A
through D and serotype AD.
• Rare isolates lack a capsule or possess a
capsule but cannot be serotyped.
• On routine laboratory media, colonies of
C. neoformans develop within 36 to 72 h.
• They are white to cream colored and
opaque and may be several millimeters in
diameter.
• Colonies may also develop sectors that
differ in pigmentation.
• The colonies are typically mucoid, and the
amount of capsule can be judged from the
degree of colonial mucosity.
• Highly encapsulated colonies may
coalesce and slowly trickle down a slant
to puddle in the bottom of the tube or drip
off the medium of inverted plates.
• Microscopically, most clinical isolates
appear as spherical, budding,
encapsulated yeast cells in both tissue
and culture.
• Rarely, short hyphal forms are seen, and
filamentous variants have been isolated
• Capsule size or thickness is determined
by the genetics of the strain as well as by
conditions of growth.
• For a given strain, capsule production in
vitro can be optimized by cultivation on
• solid or liquid medium in the presence of
glucose, thiamine, glutamate, neutral pH,
• temperature of 37 0 C, elevated carbon
dioxide, or decreased concentration of
iron.
• As judged from the ability to readily
isolate the major pathogenic variety of C.
neoformans from soil and avian habitats,
C. neoformans is ubiquitous in nature, yet
the incidence of cryptococcosis
is relatively low.
• Therefore, it is likely that many more
people inhale the yeast cells (or
basidiospores) than become ill.
• Indeed, an intact cell-mediated immunity
(CMI) and competent phagocytes provide
strong defenses against cryptococcosis.
• limited serological studies indicate that
most healthy individuals possess
antibodies to C. neoformans
Growth and Physiology
• C. neoformans has minimal growth
requirements of simple carbon and
nitrogen sources, and even vitamin
supplementation with thiamine may not
be required for growth.
• C. neoformans generally does not grow as
rapidly as yeasts such as Saccharomyces
cerevisiae and Candida albicans under
similar conditions.
• Unlike other species of Cryptococcus,
the ability of C. neoformans to use low-
molecular-weight nitrogenous
compounds such as creatinine may
partially explain its ecological niche in
avian guano.
• It is inhibited by alkaline pH and high
temperature.
• Unlike C. neoformans, most other species
of Cryptococcus are unable to grow at 370C
and are nonpathogenic.
• Indeed, the growth of C. neoformans is
tightly regulated by temperature, since in
vitro temperatures of 39 to 400C will
significantly slow its growth rate, and at
this temperature, the yeasts undergo
intracellular vacuolization and produce
aberrant budding patterns and
pseudohyphal structures.
• All species of Cryptococcus are
nonfermentative, hydrolyze starch,
assimilate inositol, and produce urease.
• These characteristics, as well as the
morphological features of the capsule
and the rare presence of pseudohyphae,
distinguish them from other clinically
important yeasts.
• most strains readily excrete large
amounts of urease, and its detection in
vitro has led to a rapid urease test for the
identification of C. neoformans
• A unique biochemical feature of C
neoformans is its ability to produce
diphenol oxidases , which may
• function as antioxidants and enhance
survival of the yeast in the host .
• The importance of temperature to
pathobiology is further emphasized by
temperature- sensitive mutants of C.
neoformans, which do not grow at 370C
and are avirulent in animals regardless of
their ability to form capsules or produce
melanin
• The presence of this enzyme, which
oxidizes a number of diphenolic
substrates and leads to the production
• of melanin, has been used for
identification .
Virulence Factors
• Pathogenicity requires the production of
phenol oxidase, growth at 370C, and the
presence of a capsule.
• The basis for the neurotropism of
C. neoformans is unclear but may involve
selective evasion of host defenses or
enrichment in the target tissues.
(e.g., utilization of catecholamines, which
are excellent substrates for phenol
oxidases and are abundant in the central
nervous system [CNS] and the adrenal
glands, another target organ).
• The capsule or CPS potentiates infection,
depresses inflammation, inhibit
phagocytosis and suppresses both cellular
and humoral immunity
Cryptococcosis
• Cryptococcosis is a chronic, subacute to
acute pulmonary, systemic or meningitic
disease, initiated by the inhalation of
basidiospores and/or desiccated yeast
cells of Cryptococcus neoformans.
• 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.
• Although C. neoformans is regarded as
the principle pathogenic species, C.
albidus and C. laurentii have on occasion
also been implicated in human infection.
• Pulmonary Cryptococcosis:

• Asymptomatic carriage of Cryptococcus

has been reported from the respiratory
tract.
• patients with chronic lung disease, such
as bronchitis and bronchiectasis, may
also have asymptomatic colonization,
with Cryptococcus being isolated from
their sputum over many years
• Subclinical cryptococcosis may result of
environmental exposure, normal
individuals may experience a self-limiting
pneumonia with accompanying
sensitization.
• Most primary infections of this type have
no diagnostic symptoms and are usually
discovered only by routine chest x-ray.
When present, symptoms include cough,
low-grade fever and pleuritic pain.
• Chronic pulmonary cryptococcosis also
increases the risk of dissemination to the
central nervous system.
Central Nervous System:

• Dissemination to the brain and meninges

is the most common clinical
manifestation of cryptococcosis and
includes meningitis, meningoencephalitis
or expanding cryptococcoma.
• Meningitis is the most common clinical
form.
• Cryptococcoma is a rare entity.
• Cutaneous Cryptococcosis:

• Primary cutaneous cryptococcosis in the

form of ulcerated lesions or cellulitis
occasionally occurs, especially in
immunosuppressed patients.
• These lesions may resolve spontaneously
or with systemic antifungal treatment.
However, all patients with skin lesions
should be monitored carefully for
possible dissemination to the central
nervous system.
Cryptococcosis of Bone:
• Osseous cryptococcosis occurs in up to
10% of disseminated cases and may
involve bony prominences, cranial bones
and vertebrae.
• The lesions are lytic without periosteal
reaction and symptoms of dull pain on
movement are reported.
• Occasional cases of arthritis have also
been reported, mostly involving the knee
joint.
Ocular Cryptococcosis:

• Ocular manifestations of cryptococcosis

most commonly include papilledema and
optic atrophy, due to raised intracranial
pressure.
• Other ocular signs of cryptococcosis are
uncommon and usually occur as a result
of dissemination.
Laboratory diagnosis:

1. Clinical material:
Cerebrospinal fluid (CSF), biopsy tissue,
sputum, bronchial washings, pus, blood
and urine.
2. Direct Microscopy:
(a) 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.
• (b) For tissue sections use PAS digest,
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.
• The demonstration of encapsulated yeast
cells in CSF, biopsy tissue, blood or urine
should be considered significant, even in
the absence of clinical symptoms.
• Positive sputum specimens should be
considered potentially significant, even
though Cryptococcus may also occur in
respiratory secretions as a saprophyte.
• Basically, all patients with a positive
microscopy for cryptococci, from any site
should be investigated for disseminated
disease, especially by culture and antigen
detection
India ink preparation of CSF showing a
typical yeast cell of C. neoformans
surrounded by a characteristic wide
gelatinous capsule.
Tissue section showing from a skin
biopsy showing typical yeast cells
of C. neoformans.
Tissue section of lung showing
showing atypical non-encapsulated
yeast cells of C. neoformans.
3. Culture:
Inoculate specimens onto primary isolation
media, like Sabouraud's dextrose agar.
Look for translucent, smooth gelatinous
colonies, later becoming very mucoid and
cream in color.
• When nonsterile specimens are to be
cultured, media containing cycloheximide
should not be used because the organism is
susceptible to this agent.
• Phenol oxidase leads to the formation of
melanin, which can be demonstrated by
the acquisition of a brown to black
pigment when C. neoformans is grown on
Staib’s birdseed agar or caffeic acid
medium
Bird seed agar plate showing the
typical brown colour effect seen
with C. neoformans.
Mucoid colonies of C. neoformans.
4. Serology:
• The detection of cryptococcal capsular
polysaccharide antigen in spinal fluid is
now the method of choice for diagnosing
patients with cryptococcal meningitis.
• In AIDS patients, cryptococcal antigen
can be detected in the serum in nearly
100% of cases.
• However, in non-AIDS patients antigen
detection in serum is less sensitive with
only about 60% of patients with
cryptococcosis reported as being positive.
 5. Identification:
• The genus Cryptococcus is characterized
by globose to elongate yeast-like cells or
blastoconidia that reproduce by
multilateral budding. Pseudohyphae are
absent or rudimentary.
• On solid media the cultures are generally
mucoid or slimy in appearance. Red,
orange or yellow carotenoid pigments
may be produced, but young colonies of
most species are usually non-pigmented,
and are cream in color.
• Most strains have encapsulated cells with
the extent of capsule formation
depending on the medium. Under certain
conditions of growth the capsule may
contain starch-like compounds which are
released into the medium by many
strains.
• Within the genus Cryptococcus,
fermentation of sugars is negative,
assimilation of nitrate is variable and
assimilation of inositol is positive.