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Micro-2|Midterm

Agents of Systemic Mycoses


Dissemination of any fungal agent, yeast, or
bacteria-like fungus to involve any tissue or
organ. Agent must be dimorphic.
Histoplasma capsulatum
Coccidioides immitis
Paracoccidioides brasiliensis
Blastomyces dermatitidis
Histoplasma capsulatum
Histoplasmosis
o an infection of the
reticuloendothelial system resulting
in patchy bronchopneumonia
containing yeast-laden
phagocytic cells within alveolar
spaces.
o aka Reticuloendothelial
cytomycosis, Cave dse,
Spelunkers dse, Darling dse
Worldwide in distribution
o it is endemic in the Mississippi,
Missouri, St. Lawrence, and Ohio
River Valleys
Strong association with bird and bat
droppings.
Teleomorph: Emmonsiella capsulata
This organism resides in soil with high
nitrogen content.
H.capsulatum var. duboisii, endemic in
Central Africa, causes disease involving
the skin and bones.
H.capsulatum var. farciminosum,
causes epizootic lymphangitis in horses
and mules.
Heterothallic requires 2 mating strains
to produce sexual forms.
Macroscopic Morphology:
o slow growth
o white to dark tan with age
o woolly, cottony, or granular
Microscopic Morphology:
o Microconidia- Round to pyriform
o Macroconidia large echinulate
or tuberculate
Yeast form: Small, oval yeast in
histiocytes, phagocytes
o resemble C.glabrata;
differentiated by fluorescent
techniques or culture.
Therapy: Itraconazole, Amphotericin B
Coccidioides immitis
Coccidioidomycosis
o usually an asymptomatic or mildly
symptomatic self-limiting upper
RTI, but may become disseminated
and fatal.
San Joaquin Fever or Valley Fever

it is endemic in southwestern US,


esp. Arizona and California.
Caution must be taken with cultures;
o organism produces arthroconidia
which are very easily inhaled.
Probably the most virulent of all human
mycotic agents.
Allergic manifestations: toxic
erythema, erythema nodosum(desert
bumps), erythema multiforme (valley
fever), and arthritis(desert rheumatism)
Soil-dwelling mold;
Primary dse usually resolves w/o therapy
and confers a strong specific immunity to
reinfection.
Secondary coccidioidomycosis, which
can include nodules, cavitary lung dse,
and/or progressive pulmonary dse.
Filipinos and blacks run the highest risk
of dissemination w/ meningeal
involvement.
Reside in a narrow ecologic niche known
as the Lower Sonoran life zone,
characterized by rainfall and semiarid
conditions.
C.immitis San Joaquin Valley region of
California
C.posadasii found in the desert of
southwest of the US, Mexico and
S.America.
Macroscopic Morphology:
o Rapid growth
o white to tan to dark gray
o Young colonies tenacious
o Old colonies cottony
o Tend to grow in concentric rings
Microscopic Morphology:
o Alternating one-celled, barrelshaped arthroconidia w/ disjunctor
cells
Yeast form: Spherules containing
endospores
Progressive cleavage production of
endospores by rupture of the spherule
wall, w/c releases endospores into the
bloodstream and surrounding tissue.
Microscopic examination of culture:
fertile hyphae arising at right angles to the
vegetative hyphae, producing alternating
hyaline arthoconidia.
Therapy: Fluconazole, Amphotericin B
Paracoccidioides brasiliensis
Paracoccidioidomycosis
o aka South American
blastomycosis, Brazilian
blastomycosis, Lutz-Splendoreo

Micro-2|Midterm

Almeida dse, Paracoccidioidal


granuloma
o a chronic, progressive fungal dse
endemic to central and south
America.
P.brasiliensis is pulmonary, and infection is
usually unapparent and asymptomatic,
subsequent dissemination leads to
formation of ulcerative granulomatous
lesions of the buccal, nasal and GI
mucosa.

Macroscopic morphology:
o slow growth
o white to beige
o colony glabrous, folded or velvety
Microscopic morphology:
o typical budding yeast with
multipolar budding at the
periphery, resembling a mariners
wheel.
o daughter cells are connected by a
narrow base.
o many buds of various sizes can
occur, or there may be only a few
buds giving the mickey mouse
cap to the yeast cell.
Yeast form: Multiple blastoconidia,
budding from single, large yeast
Blastomyces dermatitidis
Blastomycosis
o most prevalent in middle-aged
men, as are other systemic
mycoses, presumably because of
mens greater occupational and
recreational exposure to the soil.
o Primary infection flulike
symptoms
o Pulmonary dse cough, weight
loss, chest pain, and fever
o Progressive
Pulmonary/invasive disease
ulcerative lesions of the skin and
bone.
o aka Gilchrist dse, N.American
blastomycosis, Chicago dse.
Teleomorph: Ajellomyces dermatitidis
This teleomorph does not occur in routine
laboratory because the species is
heterothallic.
Macroscopic morphology:
o slow to moderate growth
o white to dark tan
o Young colonies tenacious

o Old colonies glabrous to woolly


Microscopic morphology:
o Oval, pyriform to globose smooth
conidia, lateral hypha-like
conidiophores
Yeast form: Large yeast, Blastoconidia
attached by broad base
Mold phase: conidia are borne on short
lateral branches that are ovoid to
dumbbell-shaped and vary in diameter
from 2-10 micrometer.
Penicillium marneffei
dimorphic
the only true pathogen in the genus.
common cause of systemic infection in
immunocompromised patients who have
visited the endemic region of Southeast
Asia.
Yeast form: oval to cylindric and may

have a cross wall

Mold form: sparse green aerial and


reddish brown vegetative hyphae and
production of a red diffusible pigment.
PCR identification confirmation
Agents of Opportunistic Mycoses
Zygomycetes/Mucorales
o Absidia
o Cunninghamella
o Mucor
o Rhizopus
o Syncephalastrum
Septate and Hyaline Saprophytes
o Aspergillus
o Beauveria
o Chrysosporium
o Fusarium
o Paecilomyces
o Penicillium
o Scopulariopsis
o Trichoderma
Septate and Dematiaceous/Phaeoid
Saprophytes
o Alternaria
o Aureobasidium
o Chaetomium
o Cladosporium
o Curvularia
o Phoma
o Pithomyces
o Ulocladium
Absidia
predilection for vascular invasion causing
thrombosis and necrosis of the tissues
most freq. found in diabetic patients
suffering from ketoacidosis

Micro-2|Midterm

presents as Rhinocerebral zygomycosis


o in the sinuses where conidia are
inhaled and take up residence
o from the sinuses, infection rapidly
spreads to the orbits, face, palate,
and brain.
Cunninghamella
recovered from the sinuses or other
organs from disseminated dse.
isolate is common in the environment
rapidly growing Zygomycetes that form a
cottony colony that is initially white but
becomes gray with age.
Mucor
Rhinocerebral zygomycosis
commonly isolated from the environment
worldwide
sporangiospores are formed in
sporangia on erect sporangiophores
rapidly growing Zygomycetes that form
cottony, dirty white colonies that become
mousy-brown to gray with age.
Rhizopus
most common zygomycete that causes
human disease
classically involved in diabetic patients
with ketoacidosis, presenting as
rhinocerebral zygomycosis
Rhizopus sp. is extremely refractory to
treatment
Erect sporangiophores terminated by
dark sporangia and sporangiospores.
Syncephalastrum
rarely implicated in human dse, but has
ben documented in cutaneous infections.
sometimes confused w/ Aspergillus on
initial examination
colonies are initially white and become
gray with age.
o growth rate is rapid, as with other
zygomycetes, with colonies
covering the entire surface of the
agar.
SEPTATE & HYALINE SAPROPHYTES
Aspergillus
Allergic bronchopulmonary
aspergillosis/asthma, allergies
(growing in mucous plugs in the lungs but
do not penetrate the lung tissue)
Fungus ball: free in preformed lung
cavities (surgical removal to reduce
coughing, w/c may induce pulmonary
hemorrhage)

Invasive aspergillosis/severe neutropenia,


chronic granulomatous dse, cystic fibrosis,
burns
o invades tissues causing infarcts
and hemorrhage
o Nasal colonization
pneumonia/meningitis
o Cellulitis in burn patients may
also disseminate
Beauveria
a rare human isolate, uncommonly
associated w/ keratitis
a known insect pathogen and is found
worldwide on vegetation and in the soil
Abundant, single-celled, tear-shaped
sympoduloconidia are formed on
sympodulae, w/c taper extremely from a
rather swollen base.
Chrysosporium
rare cause of disease
have been recovered from nails and skin
lesions
found in the environment worldwide
simple, wide-based, single-celled conidia
are produced on nonspecialized cells.
Fusarium
frequently seen in mycotic keratitis (soft
contact lenses wearer and of contact lens
solution)
Bone marrow transplant patients,
mortality from infections caused by the
fusaria approaches 100%
are easily recovered in blood culture
systems
SEPTATE & DEMATIACEOUS SAPROPHYTES
Alternaria
they are primarily implicated in chronic
fungal sinusitis
patients are often misdiagnosed and
treated for an extended time for bacterial
sinusitis
Aureobasidium
rare but can be traced to contaminated
dialysis lines and other similar devices
may be recovered from blood, tissues,
and abscesses
recovered worldwide primarily in wet
conditions, such as shower tiles and
water lines
young cultures are off-white to pink, but
with age, they become black with the
production of darkly pigmented
arthrocinidia.
Chaetomium
infections have been reported in the
brains of patients with central nervous

Micro-2|Midterm
system dse (patients have been
identified as IV drug users)
found in the environment and have a
predilection for cellulose products
o they have been known to
devastate printed literature and
library holdings and have been
associated with indoor air quality
problems
Cladosporium
an infrequent cause of disease,
Cladosporium sp. are primarily recovered
as Laboratory contaminants
infections are typically confined to the
sinuses or following traumatic inoculation
ubiquitous in nature, this isolate can be
recovered from nearly any location in the
world.
Curvularia
most often implicated in chronic
sinusitis in immunocompetent patients
this fungus is frequently recovered from
grass, leaves, and decaying
vegetations
multicelled conidia are produced on
sympodial conidiophore
Agents of Yeast Infections
These are caused by fungi that usually do not
induce dse and occur almost exclusively in
immunocompromised patients who are often
on treatment with corticosteroids, cytotoxic
drugs, or other immunosuppressive drugs.
Candidiasis
Cryptococcus (Torulosis, European
blastomycosis)
Rhodotorula
Pneumocystis

Candida albicans
Yeast endogenous to our mucous
membrane normal flora
form germ tubes at 37C in serum
form pseudohyphae and true hyphae
when it invades tissues (nonpathogenic
Candida do not)
Diseases/Predisposing conditions:
o Perleche crevices of
mouth/malnutrition
o Oral thrush
o Esophagitis
o Septicemia
o Endocarditis
o Cutaneous infections
o Yeast vaginitis
o Chronic mucocutaneous candidiasis
Diagnosis:

KOH: pseudohyphae, true hyphae,


budding yeasts
o Septicemia: culture lab
identification: biochemical
tests/formation of germ tubes
Treatment:
o Topical imidazoles or oral
imidazoles; nystatin
o Disseminated: Amphotericin B or
Fluconazole
Cryptococcus neoformans
Encapsulated yeast (Monomorphic)
Teleomorph: Filobasidiella neoformans
Environmental source: Soil enriched
with pigeon droppings
Diseases/Predisposing conditions:
o Meningitis/Hodgkins, AIDS (the
dominant meningitis)
o Acute pulmonary (usually
asymptomatic)/pigeon breeders
Diagnosis of Meningitis: CSF
o detect capsular Ag in CSF (by
Latex agglutination or counter
immunoelectrophoresis)
o India ink mount (misses 50%) of
CSF sediment to find budding
yeasts with capsular halos
Cultures (urease positive yeast)
Treatment: AMB + 5FC until afebrile and
culture negative, then fluconazole
Pneumocystis carinii/jirovecii
Fungus (based on molecular techniques
like biotyping)
o obligate extracellular parasite
o Silver stained cysts in tissues
Disease: Interstitial pneumonia
Pneumonia in AIDS patients even with
prophylaxis (mean CD4+/mm3 of 26),
malnourished babies, premature
neonates, and some other IC adults and
kids
Symptoms: fever, cough, shortness of
breath, sputum nonproductive except in
smokers
Pneumocystis attached to and kills Type 1
pneumocytes, causing excess replication
of type 2 pneumocytes and damage to
alveolar epithelium.
o serum leaks into alveoli, producing
exudate with foamy or
honeycomb appearance in H&E
stain
o (Silver stain reveals the holes in
the exudate are actually cysts and
trophozoites, w/c do not stain
with H&E)
o

Micro-2|Midterm
X-ray: patchy infiltrative (ground
glass appearance), the lower
lobe periphery may be spared.
COLLECTION, TRANSPORT, AND CULTURING
OF CLINICAL SPECIMENS
Cultivation
Requirements:
1. A battery of media must be used with the
ff. recommendations:
a. Media w/ or w/out blood
enrichment
b. Media w/ or w/out cycloheximide
2. Cultures should be incubated at 30C at
40-50% relative humidity for 30 days
before discarding as negative.
Fungal Culture Media: Primary Recovery
Media
a. BHIA 25C, systemic, yeast phase
b. BHIA w/ antibiotics for dermatophytes
c. BHIA w/ biphasic blood culture bottles
yeast phase, systemic fungi, 35C
d. Dermatophyte Test Medium (DTM)
screening medium for dermatophytes (red
color); indicator: phenol red
e. Inhibitory Mold Agar (IMA) primary
recovery of fungi; dermatophytes
f. Mycosel or Mycobiotic Agar primary
recovery of fungi; dermatophytes
a. Chloramphenicol inhibit
bacteria
b. Cycloheximide inhibit saprobes
g. SABHI agar (Saborauds Dextrose &
Brain Heart Infusion Agar) for
B.dermatitidis and H.capsulatum
h. Saborauds Dextrose Agar (SDA or
SAB) has thiamine, maintenance of
fungal culture
i. Yeast-Extract Phosphate Agar for
pathogenic fungi; H.capsulatum,
B.dermatitidis and C.immitis, except
dermatophytes
Fungal Culture Media: Special Purpose
Media
a. Ascospore Agar determine ascospores
b. Cornmeal Agar Chlamydospores
(C.albicans)
a. 1% glucose differentiate
T.rubrum and T.mentagrophytes
c. Cornmeal Agar w/ Tween 80 and
Trypan blue C.albicans (Chlamydospore
production)
d. Cottonseed Conversion Agar convert
dimorphic fungus from mold to yeast form
o

e. Czapeks agar Isolation and


differentiation of Aspergillus
f. Niger seed agar (Birdseed agar or
Staibs medium) C.neoformans
a. Guizotia abyssinica thistle
seeds
b. brown to black (phenol)
g. Nitrate reduction medium
h. Potato Dextrose Agar Pigment
production of T.rubrum
i. Rice medium M.canis (+), M.audouinii
(-)
j. Trichophyton Agar
DIRECT MICROSCOPIC EXAMINATION:
Temporary Stains
1. KOH 10-20%; breaks down keratin and
skin layers to see fungi present more
easily; skin, hair, nails, and tissue
2. Calcofluor White binds to
polysaccharides present in chitin of the
fungus or to cellulose; fluoresce apple
green or blue-white
3. India Ink aka Nigrosin preparations
can be used to examine CSF for presence
of encapsulated C.neoformans; Budding
yeast surrounded by a large clear area
against a dark background is
presumptive evidence of C.neoformans.
4. Lactophenol Cotton Blue Dye mounting fluid for examining cultures
microscopically, to enhance visualization
of fungi
a. Lactic acid kills bacteria
b. Phenol preservative
c. Glycerine Stain
Permanent Stains
1. PAS tissue specimens; magenta/red or
purple/red
2. Grams stain
a. Classic method
b. Hackers method: uses Lugols
iodine instead of Grams iodine;
uses acetone; no water/washing in
between
c. Philadelphia General Hospital (PGH)
Modification
3. Acid Fast Stain
4. Giemsa. Wight stain
5. Papanicolaou stain
6. Gomori methenamine-silver nitrate
7. Acridine Orange