Staining cryptococcus

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From:"Kellar, Eric" <kellarec@MSX.UPMC.EDU>
To:histonet <>, "'Bonnie Whitaker'" <>
Date:Wed, 23 Jun 1999 09:16:05 -0400
Content-Type:text/plain; charset="iso-8859-1"

Kwon-Chung et al. (1981) demonstrated that the Fontana-Masson procedure for
melanin is useful for staining the cell wall of C. neoformans, which
contains silver reducing substances that are are melanin precursors. Melanin
stains are useful in those cases of cryptococcosis in which the invading
yeast forms do not have detectable capsules "dry variants" with mucicarmine
stains, and may therefore be confused with nonencapsulated yeasts of similar
size and shape. The Fontana-Masson procedure can be used to confirm the
presence of melanin or melanin precursors in the cell walls of lightly
pigmented agents of phaeohyphomycosis. (a group of superficial and deep
infections caused by fungi that form pigmented hyphae and yeastlike cells in
tissue, i.e., and dematiaceous fungal infections other than
chromoblastomycosis and mycetomas.  In humans, cats, horses, p. is caused by
Drechslera spicifera; in chickens and turkeys by Dactylaria gallopava).
[Wood and Russel-Bell, 1983].*

C. neoformans consists of two varieties: C. neoformans var. neoformans, with
serotypes A, D and AD and C. neoformans var. gattii with serotypes B and C.
The two varieties differ in geographic distribution and habitat. C.
neoformans var. neoformans has a worldwide distribution, whereas
C.neoformans var. gattii occurs predominantly in the tropics and southern
hemisphere. Virtually all U.S. AIDS-related C. neoformans infections are
caused by C. neoformans var. neoformans (with approximately 80% serotype A. 

Establishing a predominant ecological niche for Cryptococcus neoformans var.
neoformans may be important to strategies to control infection among AIDS
patients. While recent data suggest that C. neoformans may colonize the
respiratory epithelium of dogs and cats, the vast majority of isolates have
been obtained from the environment. The pathogen is found in high titers
from aged pigeon droppings and soil contaminated with avian excreta and
nearly one half of AIDS-associated cases of one study of tropical
Cryptococcosis in Burundi had an exposure to pigeon coops. In the restricted
region of the Maltese Islands, LoPassa et al (71) showed that two clinical
isolates resembled the majority of pigeon dropping environmental isolates by
means of pulsed-filed gel electrophoresis (PFGE) and randomly amplified
polymorphic DNA (RAPD) analysis. However, the role of pigeon exposure is
believed by others to have been overemphasized. The lack of a consistent
relationship between pigeon exposure and infection, either in endemic or
epidemic outbreaks as well as the isolation of C. neoformans var. neoformans
from other sources such as dairy products, soil, and rotting vegetables and
fruit, raising questions regarding the most predominant source of pathogenic
C. neoformans. 

The presence of melanin in C. neoformans may also have a role in determining
the environmental habitat and infectious origin of this organism since
essentially all pathogenic Cryptococci are pigmented. Melanin is found in
the lignolytic homo-basidiomycete Coriolus hirsutus which uses laccase to
breakdown polyphenolic compounds during wood rotting; rRNA analysis relate
C. neoformans closely to these lignolytic mushrooms. This role of laccase in
wood pulp lignin degradation suggests that pigmented C. neoformans may be
present as a tree-dweller and indeed, has been isolated from wood and more
recently from the moist hollowed-out rotting cavities of trees. In addition,
the closely related C. neoformans var. gatti which causes Cryptococcal
infections in non-AIDS patients in the Southern hemisphere, has been found
in the flowering debris of red gum tree Eucalyptus camaldulensis. It is
interesting that Cryptococcus is a common cause of meningitis in the
tree-dwelling Kuala bear in Australia, accounting for 1% of all deaths. In
addition, a recent outbreak of meningitis among tree shrews in the National
Zoo in Washington, D.C. was attributed to Cryptococcus neoformans. Thus,
while pigeon droppings may represent a focal source of high titers of the
fungus, an additional reservoir may be a lower density of wood-borne
organisms in wide distribution. 

In addition, it is plausible that melanin protects Cryptococcus in its
natural environment by its protective properties against U.V. irradiation or
from the oxidative stress of fungal aging. The anti-oxidative properties of
melanin may also protect the organism in its natural environment from
various fungus-eating free amoeboid organisms such as Acanthamoeba polyphaga
which are thought to kill ingested organisms by oxidative mechanisms.
Laccase and its product, melanin appears to have multiple roles in survival
of the organism within the environment and in susceptible hosts, maintaining
C. neoformans as as an important and continued threat to the immunologically
impaired host.

*Spicer, Samuel S.(1987), Histochemistry in Pathologic Diagnosis. 4: 81-82. 

Eric C. Kellar
University of Pittsburgh Medical Center

	From:  Bonnie Whitaker []
	Sent:  Tuesday, June 22, 1999 7:14 PM
	To:  histonet
	Subject:  stain for crypto

	Hi All!
	One of my pathologists has a lung biopsy that she suspects crypto
	however it did not stain with mucicarmine.  She read somewhere that
	Fontana-Masson would stain a strain of crypto that was muci
negative.... can
	one of you biochemistry wizards explain to us what would be staining
	indeed this is correct) and shed any light on any special problems
	techniques required here.
	Bonnie Whitaker

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