VALDEZ, DENNIS BRYAN A.

Micro 2: WF 2:00-3:00 PM
BMLS 3D 5 February 2014
ASSIGNMENT: Opportunistic Fungi



I. CANDIDIASIS
Candidiasis is an acute-to-chronic fungal infection that can involve the mouth, vagina, skin, nails,
bronchi, lungs, alimentary tract, bloodstream, or urinary tract.

A. Clinical Findings
Transmission: As a member of the normal flora, it is already present on the skin and
mucous membranes. It is, therefore, not transmitted. The presence of C. albicans on the
skin predisposes to infections involving instruments that penetrate the skin, such as
needles (intravenous drug use) and indwelling catheters.
1. Cutaneous & Mucosal Candidiasis
o Thrush - can occur on the tongue, lips, gums, or palate. It is a patchy to confluent,
whitish pseudomembranous lesion composed of epithelial cells, yeasts, and
pseudohyphae.
o Vulvovaginitis - Yeast invasion of the vaginal mucosa l characterized by irritation,
pruritus, and vaginal discharge.
o Onychomycosis - Candidal invasion of the nails and around the nail plate. A painful,
erythematous swelling of the nail fold resembling a pyogenic paronychia, which may
eventually destroy the nail.
o Other forms of cutaneous candidiasis include invasion of the skin. The infected areas
become red and moist and may develop vesicles. Interdigital involvement between the
fingers follows repeated prolonged immersion in water.
2. Systemic Candidiasis
o Candidemia - can be caused by indwelling catheters, surgery, intravenous drug abuse,
aspiration, or damage to the skin or gastrointestinal tract.
o Candidal endocarditis - frequently associated with deposition and growth of the yeasts
and pseudohyphae on prosthetic heart valves or vegetations.
o Kidney infections are usually a systemic manifestation, whereas urinary tract infections
are often associated with Foley catheters, diabetes, pregnancy, and antibacterial
antibiotics.
3. Chronic Mucocutaneous Candidiasis
o Most forms of this rare disease have onset in early childhood, are associated with cellular
immunodeficiencies and endocrinopathies, and result in chronic superficial disfiguring
infections of any or all areas of skin or mucosa.

B. Laboratory Diagnosis
1. Specimen Sources
a. swabs and scrapings from superficial lesions,
b. blood
c. spinal fluid
d. tissue biopsies
e. urine
f. exudates
g. material from removed intravenous catheters.



VALDEZ, DENNIS BRYAN A. Micro 2: WF 2:00-3:00 PM
BMLS 3D 5 February 2014

2. Laboratory Methods
a. Direct Microscopic Exam
i. Tissue biopsies, centrifuged spinal fluid, and other specimens may be examined
in Gram-stained smears or histopathological slides for pseudohyphae and
budding cells
ii. . Skin or nail scrapings are first placed in a drop of 10% potassium hydroxide
(KOH) and calcofluor white.
o Blastoconidia (budding yeast cells) 2 to 4 microns in diameter and/or pseudohyphae
showing regular points of constriction, resembling links of sausage.
o True septate hyphae may also be produced.
o Blastoconidia, hyphae, and pseudohyphae are strongly gram positive.
b. Culture
Candida spp. grow well on standard mycologic media at 35C, such as
Sabouraud agar pH 5.6 with chloramphenicol and gentamicin, and on many
bacterial media, including blood agar, brain-heart infusion agar and tryptose agar.
Cycloheximide (Actidione) inhibits candidal growth and hence should not be
incorporated in the laboratory work-up for Candida. The time needed for species
identification can be shortened by using media that can differentiate Candida
spp. by colony color such as CHROMagar Candida , which inhibits bacterial
growth and provides presumptive identification of C. albicans, C. tropicalis, C.
dubliniensis, and C. krusei. Fongiscreen 4H also allows similar detection. Other
culture media that allow rapid identification of C. albicans include
CandiSelect, Sanofi Diagnostic Pasteur, France; Fluoroplate Candida, Merck,
Germany; Murex Candida albicans, Murex Diagnostic, USA; and Albicans
ID, BioMerieux, France. Alternatively, identification of C. albicans could rely
on this species ability to produce germ tubes in serum after 3 hours at 37C
(>90% of C. albicans isolates produce germ tubes).
1. All specimens are cultured on fungal or bacteriologic media at room
temperature or at 37C. Yeast colonies are examined for the presence of
pseudohyphae.
2. C. albicans is identified by the production of germ tubes or
chlamydospores.
3. Other Candida isolates are speciated with a battery of biochemical
reactions.
4. Contaminated Foley catheters may lead to "false-positive" urine cultures.
Positive blood cultures may reflect systemic candidiasis or transient
candidemia due to a contaminated intravenous line.
5. Cultures of skin lesions are confirmatory.
Note: Sputum cultures have no value because Candida species are part of the oral flora.


C. Agent of Candidiasis
Candida albicans (Synonym: Candida stellatoidea)

1. Macroscopic (Colony) Morphology
o On Sabouraud's dextrose agar colonies are white to cream colored, smooth, glabrous and
yeast-like in appearance. Microscopic morphology shows spherical to subspherical
budding yeast-like cells or blastoconidia, 2.0-7.0 x 3.0-8.5 um in size.
o India Ink Preparation: Negative - no capsules present.
VALDEZ, DENNIS BRYAN A. Micro 2: WF 2:00-3:00 PM
BMLS 3D 5 February 2014
o Dalmau Plate Culture on Cornmeal and Tween 80 Agar: Pseudohyphae with
blastoconidia and terminal vesicles (chlamydoconidia).
o Physiological Tests:
Germ Tube test is Positive within 3 hours
Hydrolysis of Urea is Negative
Growth on Cycloheximide medium is Positive
Grow in 1-2 days at room temperature or at 37
o
C.; monomorphic.

o Colony morphology: slightly dry, white to cream bacteria-like colony.

2. Microscopic Morphology
Appearance in Corn Meal Tween 80 Agar: Elongate pseudohyphal cells with
large clusters of blastoconidia (4-6 microns, oval, elliptical or round) at junctures
between cells. Sessile, intercalary, and many terminal chlamydiophores





































VALDEZ, DENNIS BRYAN A. Micro 2: WF 2:00-3:00 PM
BMLS 3D 5 February 2014


II. Cryptococus neoformans


A. Human Infection
Cryptococcosis (Other Names: Torulosis, European Blastomycosis, Busse-Buschkes Disease)
Cryptococcosis is an infection caused by fungi that belong to the genus Cryptococcus.
Cryptococcus neoformans (C. neoformans) is a type of fungus that is found in the soil throughout the
world, usually in association with large amounts of bird droppings.
Cryptococcosis neoformans occurs in two variants, distinguished by serology: C.
neoformans var. neoformans (serotypes A, B, and AD) and C. neoformans var. gattii (serotypes B and C).


B. Geography
C. neoformans occurs worldwide. C. neoformans var. grubii is much more common than C.
neoformans var. neoformans in the environment, and it also accounts for most of the clinical cases. C.
neoformans var. neoformans is reported to be most common in Europe.


C. Reservoir
C. neoformans may be found in humans and various domestic and wild animals. Soil and
decaying vegetation is also a reservoir for serotypes A and D, while serotypes B and C are found in
trees. C. neoformans is associated with various environmental niches, especially avian guano.


D. Clinical Findings
Transmission: This yeast occurs widely in nature and grows abundantly in soil containing bird
(especially pigeon) droppings. The birds are not infected. Human infection results from inhalation
of the organism. There is no human-to-human transmission.
The major clinical manifestation is chronic meningitis, which can resemble a brain tumor, brain
abscess, degenerative central nervous system disease, or any mycobacterial or fungal meningitis.
Cerebrospinal fluid pressure and protein may be increased and the cell count elevated, whereas
the glucose is normal or low.
Patients may complain of headache, neck stiffness, and disorientation. In addition, there may be
lesions in skin, lungs, or other organs.
The course of cryptococcal meningitis may fluctuate over long periods, but all untreated cases are
ultimately fatal. About 58% of patients with AIDS develop cryptococcal meningitis. The
infection is not transmitted from person to person.


E. Laboratory Diagnosis

1. Specimens, Microscopic Examination, & Culture
Specimens include cerebrospinal fluid, tissue, exudates, sputum, blood, cutaneous scrapings, and
urine. Spinal fluid is centrifuged before microscopic examination and culture.
For direct microscopy, specimens are often examined in wet mounts, both directly and after
mixing with India ink, which delineates the capsule
Media: SDA and PDA
Incubation temperature: 25C-37C
VALDEZ, DENNIS BRYAN A. Micro 2: WF 2:00-3:00 PM
BMLS 3D 5 February 2014
C. albicans is an oval yeast with a single bud. In tissues it may appear as yeasts or as
pseudohyphae. Pseudohyphae are elongated yeasts that visually resemble hyphae but are not true
hyphae. The spherical budding yeast cells (510 m in diameter) are surrounded by a thick
nonstaining polysaccharide capsule. The capsular polysaccharides, regardless of serotype, have a
similar structure: They are long, unbranched polymers consisting of an alpha 1,3-linked
polymannose backbone with beta-linked monomeric branches of xylose and glucuronic acid.
Colonies develop within a few days on most media at room temperature or 37C. Media with
cycloheximide inhibit Cryptococcus and should be avoided. Cultures can be identified by growth
at 37C and detection of urease.
Alternatively, on an appropriate diphenolic substrate, the phenol oxidase (or laccase) of C
neoformans and C gattii produces melanin in the cell walls and colonies develop a brown
pigment.

2. SEROLOGY
Tests for capsular antigen can be performed on cerebrospinal fluid and serum. The latex slide
agglutination test for cryptococcal antigen is positive in 90% of patients with cryptococcal
meningitis.
With effective treatment, the antigen titer dropsexcept in AIDS patients, who often maintain
high antigen titers for long periods.




References:
Brooks, G. F. (2013). Jawetz, Melnick & Adelberg's medical microbiology (26th ed.). New York:
McGraw-Hill Medical
Fisher, F. W., & Cook, N. B. (1998).Fundamentals of diagnostic mycology. Philadelphia: W.B.
Saunders.
Forbes, B. A., Sahm, D. F., Weissfeld, A. S., & Bailey, W. R. (2007). Bailey & Scott's diagnostic
microbiology (12th ed.). St. Louis, Mo.: Elsevier Mosby.
Levinson, W. (2010). Review of medical microbiology and immunology (11th ed.). New York:
McGraw-Hill Medical.