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Cryptococcus neoformans
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Laboratory Culture @ 25°C or 37°C
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Tissue/Exudate Form
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Source:
Veterinary Pathology |
Cryptococcus laurentii
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Cryptococcus laurentii on Dalmau plate. Note the absence of
hyphae.
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- Colonies are white or greenish on birdseed agar; cream to
yellow, pink, orange, or tan on routine growth media
- Like C. neoformans, may grow at 37° C on Sabouraud.
dextrose agar
- Like other Cryptococcus species, urease positive
- Used within the laboratory as a control organism to test
purity of sugars within certain media
- Yeast ovoid to elongate, as single cells or in pairs or
occasionally in chains of 3 or more cells
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Source of Cryptococcus laurentii
information:
UCSF Medical Center website |
Description and Habitats
Cryptococcus
is an encapsulated yeast. Following its first identification in nature
from peach juice samples, the major environmental sources of
Cryptococcus neoformans have been shown to be either soil
contaminated with pigeon droppings (Cryptococcus neoformans var.
neoformans) or eucalyptus trees and decaying wood forming hollows
in living trees (Cryptococcus neoformans var. gattii).
Cryptococcus neoformans var. gattii was also isolated from
goats with pulmonary disease.
Species
The
genus Cryptococcus includes around 37 species. Cryptococcus neoformans is the only species
that is pathogenic based on the mycological information found on
www.doctorfungus.org.
Among
the other Cryptococcus are Cryptococcus albidus,
Cryptococcus laurentii, Cryptococcus terreus, Cryptococcus
uniguttulatus, Cryptococcus luteolus and
Cryptococcus
gastricus.
Health Effects
Mycosis:
CRYPTOCOCCOSIS
Cryptococcosis manifests itself
most commonly as meningitis but in recent years many cases of pulmonary
disease have been recognized. C. neoformans is a very distinctive
yeast. The cells which are spherical and 3-7 microns in diameter,
produce buds which characteristically are narrow-based and the organism
is surrounded by a polysaccharide capsule. There is evidence that the
capsule may suppress T-cell function and can be considered a virulence
factor. C. neoformans also produces an enzyme called
phenoloxidase which appears to be another virulence factor.
The
ecological niche of C. neoformans is pigeon and chicken
droppings. However, although this organism can be easily recovered from
pigeon droppings, a direct epidemiological link has yet to be
established between exposure to pigeon droppings and a specific human
infection. Infection and disease production is probably a property of
the host--not the organism.
The source of human infection is not clear.
This organism is ubiquitous, especially in areas like abandoned
buildings contaminated with pigeon droppings. The portal of entry is the
respiratory system. Evidence is developing which indicates that the
initial exposure may be many years prior to the manifestation of
disease. The organism can be sequestered for this time.
Infection may be subacute or chronic. The highly fatal meningoencephalitis caused by
C. neoformans has a prolonged evolution of several months. The
patients symptoms may begin with vision problems and headache, which
then progress to delirium,
nuchal rigidity leading to coma and death unless the physician is
thinking about cryptococcus and does a spinal tap for diagnosis and
institutes aggressive therapy. The CSF is examined for its
characteristic chemistry (elevated protein and decreased glucose), cells
(usually monocytes), and evidence of the organism. The latter is
measured by the visual demonstration of the organism (India Ink
preparation) or by a serologic assay for the antigen of C. neoformans.
The India Ink test, which demonstrates the capsule of this yeast, is
supplemented by the latex agglutination test for antigen which is more
sensitive and more specific. The Latex Agglutination test measures
antigen, NOT antibody. A decreasing titer indicates a good prognosis,
while an increasing titer has a poor prognosis.
When you consider Cryptococcosis, think of Capsules and CNS disease. In addition to
causing meningitis, C. neoformans may also infect lungs and skin.
The disease in the lungs and skin is characterized by the formation of a
granulomatous reaction with giant cells. As with other fungal diseases,
there has been an increase in the recognition of pulmonary infection.
The yeast may also form a mass in the
mediastinum called a cryptococcoma.
The geographical distribution of
this organism is world-wide. The clinical material sent to the lab is CSF, biopsy material, and urine (for some unexplained reason the
organism can be isolated from the urine in both the CNS and systemic
infections). This organism will grow overnight on bacterial or fungal
media at 37 C. but growth is a little slower at room temperature. In
culture the organism grows as creamy, white, mucoid (because of the
capsule) colonies. Growth in culture is usually visible in 24 to 48
hours. As the culture ages, it turns brown due to a melanin produced by
the phenoloxidase. The organism is a round, single cell, yeast
surrounded by a capsule. Identification is based on physiological
reactions. Pathologists use a mucicarmine stain, which stains the
capsule, to identify the organism in tissue sections. There is usually
little or no inflammatory response. The Direct Fluorescent Antibody test
identifies the organism in culture or tissue section specifically, by
causing the yeast cell wall to stain green. To test the patient's serum
there are 3 serologic tests: The Indirect Fluorescent Antibody test, the
Tube Agglutination test for antibody, and the Latex Agglutination test
for antigen. The latex agglutination test can be used as a prognostic
test. As the patient improves, the serum antigen titer will also
decrease.
The drugs of choice to treat cryptococcus infection are
amphotericin B and 5-Fluorocytosine (5-FC). 5-FC is an oral drug. If it
is given as the only treatment, there are relapses so most physicians
use both drugs simultaneously. Actually, these two drugs are
synergistic, and thus, their association is advantageous.
(Source of Mycosis Information:
Mycology Online)
Macroscopic
Features
Colonies of Cryptococcus neoformans are fast growing, soft,
glistening to dull, smooth, usually mucoid, and cream to slightly pink
or yellowish brown in color. The growth rate is somewhat slower than Candida
and usually takes 48 to 72 h. It grows well at 25°C as well as 37°C.
Ability to grow at 37°C is one of the features that differentiates Cryptococcus
neoformans from other Cryptococcus spp. However,
temperature-sensitive mutants that fail to grow at 37°C in vitro may
also be observed.
At 39-40°C, the growth of Cryptococcus neoformans starts to slow
down.
Microscopic
Features
On cornmeal tween 80 agar, Cryptococcus neoformans produces
round, budding yeast cells. No true hyphae are visible. Pseudohyphae are
usually absent or rudimentary. The capsule is best visible in India ink
preparations. The thickness of the capsule is both strain-related and
varies depending on the environmental conditions. Upon growth in 1%
peptone solution, production of capsule is enhanced
Laboratory
Precautions
No special precautions other than general laboratory precautions
are required.
Susceptibility
Usual Susceptibility
Patterns for Cryptococcus spp
Susceptibility
testing of Cryptococcus neoformans using the NCCLS reference
method is difficult due to the poor growth of the fungus, the
related requirement for a relatively long incubation period (72 h), and
difficulties in identification of resistant isolates. The only species
for which there exists significant amount of data is Cryptococcus
neoformans. Broadly stated, isolates of this species generally
appear susceptible to amphotericin B, as well as fluconazole and
flucytosine. Nevertheless, this depends significantly on the approach to
measurement, and isolates which are apparently resistant to all of the
current drugs have been identified.
In Vitro Susceptibility
Amphotericin
B and other polyenes
Amphotericin B exerts a fungicidal
effect on Cryptococcus neoformans. As with Candida
spp., the NCCLS method has difficulty distinguishing amphotericin B-resistant isolates from the susceptible ones. However, modifications of
this method, such as the use of Antibiotic Medium 3 supplemented to 2%
glucose (AM3) instead of the reference RPMI 1640 medium appear useful.
Shaking the microplates during incubation also appears helpful.
There are also data indicating that
minimum fungicidal concentrations (MFCs) may be better predictors of
clinical outcome than MICs. However, these modifications still require
validation and standardization. Using E test method instead of NCCLS
microdilution methodology may also enhance the detection of amphotericin
B-resistant isolates, regardless of the test medium used. The other
method under investigation, flow cytometry, in general yields rapid and
correlated results with NCCLS reference method and for amphotericin B
and fluconazole. Variety-related differences
in susceptibility patterns have also been observed. Cryptococcus
neoformans var. gattii appears less susceptible to
amphotericin B and flucytosine than var. neoformans. Defects in
sterol isomerase and depletion of ergosterol content in fungal cell
membrane may result in emergence of resistance to amphotericin B.
The novel lipid nanosphere-encapsulated
amphotericin B formulation, NS-718, generates very low MICs. Similar to
amphotericin B, liposomal
nystatin and the parent compound nystatin also show favorable in
vitro activity against most Cryptococcus neoformans isolates.
Promisingly, amphotericin B
combined with fluconazole, itraconazole, or posaconazole yielded no
antagonism in vitro and appeared synergistic for some isolates. In vitro
synergy between amphotericin B and flucytosine or rifampin was observed
for Cryptococcus neoformans strains isolated from patients who
failed to respond to amphotericin B therapy.
Azoles
Azoles, particularly fluconazole
and itraconazole,
have been shown to have fungistatic effects against most Cryptococcus
neoformans isolates. Although most Cryptococcus neoformans
strains are susceptible to fluconazole, isolates with high MICs have
been detected. Alternative susceptibility testing methods may yield
results that differ sharply from the NCCLS reference method. Using Etest
instead of the reference microdilution method has resulted in
misclassification of a number of susceptible Cryptococcus neoformans
isolates as being resistant to fluconazole, itraconazole, and
flucytosine. Colorimetric methods, on the other hand, might interpret
some fluconazole- and flucytosine-resistant isolates as susceptible.
Similarly, most of the Cryptococcus
neoformans strains are susceptible to itraconazole in vitro. Among
the newer azoles, posaconazole
(SCH56592) appears most potent on a weight basis, when compared to voriconazole
, fluconazole, itraconazole, and ketoconazole.
Flucytosine
Flucytosine has long been used as part of
combination therapy of cryptococcal
infections. Resistance may develop during
monotherapy. Use of yeast nitrogen base as the test medium was reported
to be very effective in some investigators' hands in testing flucytosine
susceptibility.
Glucan
Synthesis Inhibitors
(Echinocandins)
These novel agents have essentially
no activity against Cryptococcus neoformans.
In vitro data suggest that activities of amphotericin B and fluconazole
against Cryptococcus neoformans may be enhanced by caspofungin].
In Vivo Efficacy
The most commonly used
agents for treatment of cryptococcal infections are amphotericin B,
flucytosine, and fluconazole, and particularly amphotericin B and
flucytosine in combination. Fluconazole prophylaxis is also in common
practice in patients who have recovered from cryptococcal infections.
However, clinical failure with fluconazole has been reported.
Fluconazole combined with flucytosine and triple therapy with
amphotericin B, flucytosine, and fluconazole have been reported as
effective. Itraconazole is less effective than fluconazole as
maintenance therapy.
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