MYCOLOGY

 Mycology - specialized discipline in the field of

biology concerned with the study of fungi,
including their taxonomy, environmental
impact, and genetic and biochemical properties

Characteristics of Fungi  Molds

 eukaryotic; they possess a true nucleus,  have a fuzzy or woolly appearance

with a nuclear membrane and mitochondria.
 characterized by tubular branching cells
 lack chlorophyll and must absorb nutrients
which constitute a hypha
from the environment.
 fungal cell walls are made of chitin, whereas  hyphae are divided in most fungi by cross walls
those of plants contain cellulose called septa into multicellular hyphae
 Most fungi are obligate aerobes that grow  Some fungi do not develop septa and have
best at a neutral pH, although they tolerate nonseptate hyphae (also called coenocytic
a wide pH range. hyphae)
 Moisture is necessary for growth, but spores  Branching, intermingled and often fused or
and conidia survive in dry conditions for intercommunicating hyphae constitute
extended lengths of time. mycelium which forms the visible, usually dry
colony of mold observed on natural substrates
 Mycoses - refers to infections that are caused by
or on culture media.
fungi
A. Aerial mycelia- extend above the surface of the
 Further characterization of a fungal disease
colony and are responsible for the fuzzy appearance.
is made by the use of a prefix which is
derived either from the part of the body B. vegetative mycelia- extend downward into the
affected (e.g. nail-onchyomycosis, skin- medium to absorb nutrients
dermatomycosis, etc.) or from the name of
pathogen involved (e.g. coccidioidomycosis)
 Alternatively, the disease names are often
coined by addition of the suffix osis to the
generic name of the pathogen (e.g.
aspergillosis, histoplasmosis,
cryptococcosis, etc.).
 Sometimes the disease names bear a
geographical connotation as in the case of Types of hyphae based on microscopic appearance
South American Blastomycosis, North
American Blastomycosis, etc. 1. Antler hyphae - have swollen, branching tips
that resemble moose antlers.
General characteristics
2. Racquet hyphae- contain enlarged, club-shaped
2 forms: areas
 Yeasts 3. Spiral hyphae- are tightly coiled
 single vegetative cells that typically form a 4. Rhizoids - rootlike structures, might be seen in
smooth, creamy, bacterial-like colony some of the Zygomycetes, and their presence and
without aerial hyphae placement can assist with identification.
 identification is based primarily on
biochemical testing
 reproduce by budding or fission (involves
maturation of the bud to an independent
blastoconidium (daughter cell)
Types of Hyphae based on pigmentation:

1. Hyaline (moniliaceous) hyphae- nonpigmented

or lightly pigmented

2. Phaeoid (dematiaceous) hyphae- darkly

pigmented (pale to dark brown or almost black)
because of the presence of melanin in the cell wall.

 Fontana- Masson Stain - stains melanin,

causing phaeoid hyphae to appear brown,
whereas hyaline hyphae stain pink to red.

 Dimorphic fungi - include a mold phase and

a yeast phase.

 yeast or tissue state - seen in vivo or when

the organism is grown at 37° C with
increased CO2.

 mold phase - seen when the organism is

grown at room temperature (22° to 25° C)
in ambient air conditions.

Examples:

1. Blastomyces dermatitidis

2. Coccidioides immitis

3. Histoplasma capsulatum var. capsulatum,

4. Paracoccidioides brasiliensis

5. Sporothrix schenckii

6. Penicillium marneffei
  Polymorphic Fungi- have both yeast and
mold forms in the same culture (not been
described as agents of human mycoses)

 Yeast phase is typically observed initially,

followed by the mold phase as the colony
ages.

Example:
1. Exophiala spp.

Reproduction

 Asexual Reproduction (Imperfect, fungi

imperfecti)

 results in the formation of conidia (singular,

conidium) following mitosis

Conidiogenous cells:

1. phialides- vaselike structures that produce

Other:
phialoconidia
 Arthroconidium- formed by fragmentation
2. annellides- ringed structures that produce of fertile hyphae
annelloconidia.

 Chlamydoconidia (chlamydospores)
 round, thickwalled spores formed
directly from the differentiation of
hyphae in which there is a
concentration of protoplasm and
nutrient material
  requires the joining of two compatible nuclei,
followed by meiosis and subsequent
production of zygospore.
 A fungus that reproduces sexually is known
as a teleomorph.

Anamorph- the form when a teleomorph

occasionally reproduces asexually
intercalary (within the hyphae)
terminal (on the end of the hyphae).  If more than one anamorph is present for the
 Microconidia- are small, unicellular, round, same teleomorph, the anamorphic strains
elliptical, or pyriform in shape are termed synanamorphs
 Macroconidia- large, usually multiseptate,  Ex: Pseudallescheria boydii, has two
and club or spindle shaped anamorphs, Scedosporium boydii and
Graphium sp. These two anamorphs are
 Sporangiospores synanamorphs to each other.

Ascospores- sexual spores in a large, saclike

structure called an ascocarp

Taxonomy

 over 100,000 named fungal species

 Asexual structures characteristic of the
 Most causative agents of clinical infections
Order Mucorales; they are mitotic spores
are found in four groups of fungi:
produced within an enclosed sporangium,
often supported by one sporangiophore 1. Phylum Glomeromycota (order Mucorales)
 Sexual Reproduction 2. Phylum Ascomycota

3. Phylum Basidiomycota

4. Deuteromycota (division Fungi Imperfecti)

A. Mucorales

 rapidly growing organisms normally found in

the soil.
 often opportunistic pathogens in
immunocompromised hosts
  generally produce profuse, gray to white,
aerial mycelium characterized by the
presence of hyaline, sparsely septate hyphae
 Reproduction: production of
sporangiospores
 Examples:
1. Lichtheimia (formerly Absidia)
2. Mucor
3. Rhizomucor
4. Rhizopus. D. Fungi Imperfecti

 contains the largest number of organisms

that are causative agents of mycoses,
including cutaneous, subcutaneous, and
systemic disease.
 Lack a sexual reproductive cycle and are
characterized by their asexual reproductive
structures, primarily conidia

Identification Methods and Strategies

B. Ascomycota
 Characterized by the production of sexual I. Culture Media And Incubation Requirements
spores known as ascospores (formed within
a saclike structure known as an ascus)  Agar plates or screw-capped agar tubes are
 Microsporum spp., Trichophyton spp., and satisfactory for the recovery of fungi;
 Pseudallescheria boydii.
 screw-capped culture tubes - more easily
 Examples:
stored, require less space for incubation,
1. Microsporum spp.,
and are more easily handled, have a lower
2. Trichophyton spp., and
dehydration rate
3. Pseudallescheria boydii.
 Cotton-plugged tubes are unsatisfactory for
fungal cultures.

Incubation:

 room temperature, or preferably at 30°C, for

21 to 30 days, with 40- 50% humidity

Inhibitors:

0.5 μg/mL of cycloheximide – inhibits growth of

C. Basidiomycota
 Include mushrooms as well as pathogenic contaminating molds
species of Cryptococcus 16 μg/mL of chloramphenicol - inhibits growth of
 Sexual reproduction results in dikaryotic contaminating bacteria
hyphae and four progeny basidiospores that
are supported by a club-shaped basidium
 Characterized by the presence of clamp
connections that occur at the septations in
the vegetative hyphae
 Media Indications for use Media composition Mode of action

Primary Recovery Media

Brain-heart Primary recovery of Brain-heart infusion, The agar provides a rich

infusion agar saprobic and pathogenic enzymatic digest of medium for bacteria, yeast,
fungi animal tissue, and pathogenic fungi.
enzymatic digest of
casein; dextrose,
sodium chloride

Brain-heart Primary recovery of Brain-heart infusion, The agar provides a rich

infusion agar pathogenic fungi enzymatic digest of medium for yeast and
with antibiotics exclusive of animal tissue, pathogenic fungi.
dermatophytes enzymatic digest of
casein; dextrose,
sodium chloride,
antibiotics

Brain-heart Recovery of fungi from Brain-heart infusion, Enhances the recovery of

infusion blood peptone, glucose, yeasts in blood
biphasic blood disodium phosphate
culture bottles

Chromogenic Isolation and Chromopeptone Chromogen mix contains

agar presumptive Glucose substrates that react with
identification of yeast and Chromogen mix enzymes produced by
filamentous fungi Chloramphenicol different organisms that
result in the production of
characteristic color
changes.

Dermatophyte Primary recovery of Dextrose, Dermatophytes produce

test medium dermatophytes, cycloheximide, alkaline metabolites, which
recommended as gentamycin. raise the pH and change the
screening medium only chloramphenicol, medium from red to yellow.
phenol red

Inhibitory mold Primary recovery of Chloramphenicol, Examine plates for growth.

agar pathogenic fungi casein, dextrose, Chloramphenicol inhibits
exclusive of starch, sodium bacterial growth.
dermatophytes phosphate, magnesium
sulphate, sodium
chloride, manganese
sulphate

Potato flake Primary recovery of Potato flakes, glucose, Growth is enhanced by a

agar saprobic and pathogenic cycloheximide, pH alkaline reaction of
fungi chloramphenicol, fungus. Chloramphenicol
bromthymol blue and antibiotics inhibit the
growth of bacteria and
nonpathogenic fungi.

Mycosel Primary recovery of Cycloheximide, Inhibits bacteria and

dermatophytes chloramphenicol, saprophytic fungi
dextrose
SABHI agar Primary recovery of Sabourad dextrose, Isolates and enhances
saprobic and pathogenic brain-heart infusion growth of pathogenic fungi
fungi agar

Yeast-extract Primary recovery of Yeast extract, Enhances the recovery of

phosphate agar pathogenic fungi dipotassium Blastomyces dermatitidis
exclusive of phosphate, and Histoplasma
dermatophytes chloramphenicol capsulatum from
contaminated specimens

Differential Test Media

Acetate Detection of ascospores Potassium acetate, Potassium acetate is

Ascospore agar in ascosporogenous yeast extract, dextrose necessary, and yeast extract
yeasts (e.g., increases the sporulation of
Saccharomyces spp.) yeasts.

Christensen’s Identification of 2% urea, phenol red Produces urease and a

urea agar Cryptococcus, change in the pH.
Trichosporon, and
Rhodotorula spp.

Cornmeal agar Identification of Cornmeal, Tween 80, Addition of Tween 80

with Tween 80 Candida albicans by trypan blue enhances the production of
and trypan blue chlamydospore chlamydospores, and the
production; identification addition of trypan blue
of C. albicans by provides a contrasting
microscopic morphology background for observing
the morphologic features of
yeasts.

Cottonseed Conversion of the Cottonseed meal, Allows conversion to yeast

conversion agar dimorphic fungus glucose phase within 3 days.
Blastomyces spp. from
mold to yeast form

Czapek’s agar Differential identification Sodium nitrate, Produces characteristic

of Aspergillus spp. sucrose, yeast extract features of yeast and
fungus.

Niger seed agar Identification of Guizotia abyssinica C. neoformans and C. gattii

(birdseed agar) Cryptococcus neoformans seed, dextrose, produce the enzyme phenol
and Cryptococcus gattii chloramphenicol oxidase, resulting in a
brown pigment through
metabolism of caffeic acid.

Nitrate Detection of nitrate Potassium nitrate, If the yeast produces nitrate

reduction reduction to confirm peptone, meal extract, reductase, a cherry red
medium Cryptococcus spp. sulfanilic acid, N, N- indicates a positive test
dimethyl-1- result.
naphthylamine
Potato dextrose Demonstration of Potato infusion, D(1) Carbohydrate and potato
agar pigment production by glucose Note: Some infusion promotes the
Trichophyton rubrum; laboratories use potato growth of yeasts and molds,
preparation of microslide flake agar, because it and the low pH partially
cultures and sporulation may be more stable. inhibits bacterial growth.
of dermatophytes

Rice medium Identification of White rice extract, Polysorbate 80 enhances

Microsporum audouinii polysorbate 80 chlamydospore formation
by C. albicans.
Differentiates Microsporum
canis, which grows well
with a yellow pigment, from
M. audouinii, which shows
no growth.

Trichophyton Identification of Casamino acids, Trichophyton spp. may be

agars 1-7 Trichophyton spp. dextrose, differentiated by their
monopotassium growth in the presence of
phosphate, magnesium different amino acids.
sulfate, amino acids
(e.g., inositol,
thiamine), ammonium
nitrate

Urea agar Detection of Peptone, dextrose, Urea provides a nitrogen

Cryptococcus spp.; sodium chloride, source for organisms
differentiate monopotassium producing urease. Urease
Trichophyton phosphate, urea, releases ammonia, which
mentagrophytes from phenol red increases the pH and is
Trichophyton rubrum; indicated by a color change
detection of Trichosporon from red to yellow.
spp.

Yeast Identification of yeasts by Yeast extract, peptone, Most yeasts produce acid,
fermentation determining fermentation bromcresol purple, and which is indicated by a
broth a specific carbohydrate change in the solution from
(e.g., dextrose, maltose, purple to yellow as a
sucrose) positive fermenter.

Yeast nitrogen Identification of yeasts by Ammonium sulfate, Assimilation of carbon by

base agar determining carbohydrate carbon source (e.g., yeast cells produces a
assimilation glucose, sucrose, positive result.
raffinose

Trichophyton Agar 2, 3 and 4 are used with medium 1 to determine whether an isolate requires
inositol, thiamine or both.
Trichophyton Agar 5, equivalent to Trichophyton Agar 1 with added nicotinic acid (2 mg/L), is used
with medium 1 to determine the requirement for nicotinic acid
Trichophyton 7 is used with medium 6 to determine the requirement for histidine.
II. Direct Microscopy

Method Use Time Advantages Disadvantages

required
Acid-fast stain Detection of 12 min Detects Nocardia spp.* Tissue homogenates
and partial acid- mycobacteria and some isolates of are difficult to observe
fast stain and Nocardia Blastomyces spp. because of
spp., background staining.
respectively
Auramine- Detection of 10 min Excellent screening tool; Not as specific for
rhodamine stain mycobacteria sensitive and affordable. acid-fast organisms
and Nocardia as Ziehl-Neelsen
spp., stain.
respectively
Calcofluor white Detection of 1 min Can be mixed with KOH; Requires use of a
stain fungi detects fungi rapidly fluorescence
because of bright microscope;
fluorescence background
fluorescence
prominent, but fungi
exhibit more intense
fluorescence; vaginal
secretions are
difficult to interpret.
Gram stain Detection of 3 min Commonly performed on Some fungi stain well,
bacteria most clinical specimens but others (e.g.,
submitted for Cryptococcus spp.)
bacteriology; detects most show only stippling
fungi. and stain weakly in
some instances; some
isolates of Nocardia
spp. fail to stain or
stain weakly.
India ink stain Detection of 1 min Diagnostic of meningitis Positive in fewer than
Cryptococcus when positive in CSF. 50% of cases of
spp. in CSF meningitis; not
sensitive in non–HIV-
infected patients.
Lactophenol Most widely 1 min Lactic acid preserves Mechanical treatment
cotton blue wet used method of structures; slides can be dislodges fungal
mount staining and made permanent. structures.
observing fungi
Potassium Clearing of 5 min; if Rapid detection of fungal Requires experience,
hydroxide specimen to clearing is elements. because background
make fungi not artifacts are often
more readily
complete, an confusing; clearing of
visible
additional 5- some specimens may
 10 min is require an extended
necessary time.
Masson-Fontana Examination of 1 hr, 10 min Aids differentiation of Difficult to interpret
stain melanin melanin and hemosiderin when only rare
pigment in pigments. granular staining is
fungal cell present.
walls
Methenamine Detection of 1 hr Best stain for detecting Requires a
silver stain fungi in fungal elements. specialized staining
histologic method that is not
usually readily
section
available to
microbiology
laboratories.
Papanicolaou Examination of 30 min Cytotechnologist can Fungal elements
stain secretions for detect fungal elements. stain pink to blue.
malignant cells
Periodic Detection of 20 min; 5 Stains fungal elements Nocardia spp. do not
acidSchiff (PAS) fungi min well; hyphae of molds and stain well.
stain additional if yeasts can be readily
counterstain distinguished.
is used
Saline wet Examination of 1 min Quickly performed and Specimen must be
mount fungal cost-effective. fresh; not all elements
elements are visible with this
preparation.
Wright’s stain Examination of 7 min Detects Histoplasma Most often used to
bone marrow or capsulatum and detect H. capsulatum
peripheral Cryptococcus spp. and Cryptococcus
blood smears spp. in disseminated
disease.
Identification of Molds in Culture

Growth rate
Slow-growers - form mature colonies in 11 to 21 days
intermediate-growers - form mature colonies in 6 to 10 days
Rapid-growers form mature colonies in 5 days or less.

Colonial morphologic features

Color
Colony topography:

verrucose (furrowed or convoluted),

umbonate (slightly raised in the center),
rugose (furrows radiate out from the center)

Colony Texture:
cottony (loose, high aerial mycelium),
velvety (low aerial mycelium resembling a velvet cloth),
glabrous (smooth surface with no aerial mycelium),
granular (dense, powdery, resembling sugar granules),
wooly (high aerial mycelium that appears slightly matted down).

Microscopic morphologic features

shape,

method of reproduction

arrangement of spores;

Size of the hyphae

Mycoses

Mycoses (singular, mycosis) - diseases caused by fungi

categorized based on the site of the infection

1. superficial
2. Cutaneous
3. Subcutaneous
4. systemic mycoses- not involving the skin or deeper tissues
just under the skin

Classification based on type of infection

The Major Mycoses and Causative Fungi

Category Mycosis Causative Fungal Agents

Superficial Pityriasis versicolor Malassezia species

Tinea nigra Hortaea werneckii

White piedra Trichosporon species

Black piedra Piedraia hortae

Cutaneous Dermatophytosis Microsporum species, Trichophyton species, and

Epidermophyton floccosum

Candidiasis of skin, Candida albicans and other Candida species

mucosa, or nails

Subcutaneous Sporotrichosis Sporothrix schenckii

Chromoblastomycosis Phialophora verrucosa, Fonsecaea pedrosol, and

others
 Mycetoma Pseudallescheria boydii, Madurella mycetomatis,
and others

Phaeohyphomycosis Exophiala, Bipolaris, Exserohilum, and other

dematiaceous molds

Endemic Coccidioidomycosis Coccidioides posadasii and Coccidioides immitis

(primary,
systemic) Histoplasmosis Histoplasma capsulatum

Blastomycosis Blastomyces dermatitidis

Paracoccidioidomycosis Paracoccidioides brasiliensis

Opportunistic Systemic candidiasis Candida albicans and many other Candida species

Cryptococcosis Cryptococcus neoformans and Cryptococcus gattii

Aspergillosis Aspergillus fumigatus and other Aspergillus species

Hyalohyphomycosis Species of Fusarium, Paecilomyces, Trichosporon,

and other hyaline molds

Phaeohyphomycosis Cladophialophora bantiana; species of Alternaria,

Cladosporium, Bipolaris, Exserohilum, and
numerous other dematiaceous molds

Mucormycosis Species of Rhizopus, Lichtheimia, Cunninghamella,

(zygomycosis) and other members of the Order Mucorales

Pneumocystis pneumonia Pneumocystis jiroveci

Penicilliosis Penicillium marneffei

A. Superficial Mycoses
fungal diseases that affect only the cornified layers (stratum corneum) of the epidermis

Disease Agent Manifestations Lab

Tinea Malasezzia furfur patchy lesions or scaling ✓ KOH: budding yeasts,

versicolor of varying pigmentation approximately 4 to 8 um, along
with septate, sometimes branched,
hyphal elements (spaghetti and
meatballs fungus)

✓ Wood's lamp- yellow fluorescence

✓ Culture: do not grow in routine

fungal culture: yeastlike colonies
may be observed only after the
culture medium is overlaid with
olive oil
Black Piedraia hortae hard, dark brown to ✓KOH: Thick-walled rhomboid cells
piedra black gritty nodules that containing ascospores
are firmly attached to
the hair shaft ✓ Culture: brown colonies that
grow slowly on Sabouraud dextrose
agar at room temperature.

White Trichosporon spp. [T. larger, softer, yellowish ✓ KOH: Hyaline hyphae 2 to 4 um
Piedra ovoides, T. asteroides, T. nodules on the hairs wide and arthroconidia
cutaneum, and T. inkin
✓ Culture: straw to cream colored
and yeastlike
✓ Urease +.

Tinea Hortea werneckii brown to black nonscaly ✓ KOH: septate hyphal element
Nigra (Phaeoannellomyces macules that occur most and budding cells
werneckii, Exophiala often on the palms of the
werneckii) hands and soles of the Culture: shiny, moist, yeastlike
fee colonies that start with a brownish
coloration that eventually turns
olive to greenish black;
Annelloconidia (conidia containing
transverse rings) are seen in older
hyphal colonies.
B. Cutaneous Mycoses (dermatophytoses)

Trichophyton, Microsporum, and Epidermophyton

Species within these genera are keratinophilic; that is, they are adapted to grow on hair, nails, and
cutaneous layers of skin that contain the scleroprotein keratin.
typically form two sizes of reproductive cells, macroconidium or microconidium

Geophilic- primarily inhabit the soil

Zoophilic- typically adapted to live on animals and are not commonly found living freely in soil or on dead
organic substrates.

Anthropophilic- adapted exclusively to human hosts

Various Forms of Dermatophytoses and the Respective Affective Sites

Type of Ringworm Site Affected

Tinea capitis Head

Tinea favosa Head (distinctive pathology)

Tinea barbae Beard

Tinea corporis Body (glabrous skin)

Tinea manuum Hand

Tinea unguium Nails

Tinea cruris Groin

Tinea pedis Feet

Tinea imbricate Body (distinctive lesion)

Dermatophyte Colonial Morphology Microscopic Identification Morphology of

microconidia and/or
macroconidia

Microsporum Downy white to salmon- Sterile hyphae; terminal

audouinii pink colony: reverse tan to chlamydoconidia, favic
salmon-pink chandeliers, and pectinate
bodies; macroconidia rarely
seen (bizarre shaped if seen);
microconidia rare or absent

Microsporum Colony usually Thick-walled, spindle-shaped,

canis membranous with feathery multiseptate, rough-walled
periphery: center of colony macroconidia, some with a
white to buff over orange- curved tip; microconidia rarely
yellow; lemon yellow or seen
yellow-orange apron and
reverse

Microsporum Cinnamon-colored, Thick-walled, rough, elliptical,

gypseum powdery colony: reverse multiseptate macroconidia:
light tan microconidia few or absent
 Dermatophyte Colonial Morphology Microscopic Identification Morphology of
microconidia
and/or
macroconidia

Epidermophyton Center of colony tends to Macroconidia: large, smooth

floccosum be folded and is khaki walled, multiseptate, clavate,
green; periphery is and borne singly or in clusters of
yellow; reverse yellowish two or three: microconidia not
brown with observable formed by this species
folds

Trichophyton Different colonial types; Many round to globose

mentagrophytes white, granular, and microconidia, most commonly
fluffy varieties; borne in grapelike clusters or
occasional light yellow laterally along the hyphae; spiral
periphery in younger hyphae in 30% of isolates:
cultures; reverse buff to macroconidia are thin walled,
reddish brown smooth, club shaped, and
multiseptate; numerous or rare,
depending upon strain
 Trichophyton Colonial types vary from Microconidia usually teardrop-
rubrum white downy to pink shaped, most commonly borne
granular: rugal folds are along sides of the hyphae:
common; reverse yellow macroconidia usually absent but
when colony is young, when present are smooth, thin
but wine/ red color walled, and pencil shaped
commonly develops with
age

Dermatophyte Colonial Morphology Microscopic Identification Morphology of

microconidia
and/or
macroconidia

Trichophyton White, tan to yellow or Microconidia are teardrop or club

tonsurans rust, Suede like to shaped with flat bottoms; vary in
powdery: wrinkled with size but usually larger than other
heaped or sunken dermatophytes; macroconidia rare
center; reverse yellow (balloon forms found when present)
to tan to rust red

Trichophyton Irregularly heaped, Hyphae usually sterile; many antler-

schoenleinii smooth, white to cream type hyphae seen (favic
colony with radiating chandeliers)
grooves; reverse white
Trichophyton Port wine to deep violet Branched, tortuous. sterile hyphae;
violaceum colony, may be heaped chlamydoconidia commonly aligned
or flat with waxy in chains
glabrous surface;
pigment may be lost on
subculture

Trichophyton Glabrous to velvety Microconidia rare, large and

verrucosum white colonies; rare teardrop shaped when seen:
strains produce macroconidia extremely rare but
yellowbrown color; form characteristic rat tail types
rugal folds with when seen: many chlamydoconidia
tendency to skin into seen in chains. particularly when
agar surface colony is incubated at 37°C

Misc. Lab:

1. Hair Perforation Test.

In the hair perforation tests, sterile 5- to 10-mm hair fragments are floated on sterile water supplemented
with a few drops of sterile 10% yeast extract. Conidia or hyphae from the dermatophyte in question are
inoculated onto the water surface. Hair shafts are removed and microscopically examined in LPCB at
weekly intervals for up to 1 month.

T. rubrum- usually causes only surface erosion of hair shafts

T. mentagrophytes - typically forms perpendicular penetration pegs in the hair shafts

Microsporum canis – penetration capable

M. equinum- not penetrate hair.

2. 5-day urease test

Tubes of Christensen urea agar are very lightly inoculated with the dermatophyte and held for 5 days at
room temperature.

T. mentagrophytes - demonstrate urease production, resulting in a color change of the medium from
peach to bright fuchsia,

T. rubrum isolates are negative or require more than 5 days to give a positive reaction.

C. Subcutaneous Mycoses

fungal diseases that affect subcutaneous tissue.

usually the result of the traumatic implantation of foreign objects into the deep layers of the skin,
permitting the fungus to gain entry into the host.
organisms commonly found in soil or on decaying vegetation; therefore, agricultural workers are
most often affected.
some are moniliaceous (hyaline or light-colored), many are phaeoid, producing darkly pigmented
colonies and containing melanin in their cell walls.
infections are commonly chronic and usually incite the development of lesions at the site of trauma.

Disease Agent Manifestations Lab

Chromoblastomycosis ✓ Fonsecaea chronic mycosis of the ✓ identified on the basis of

(verrucous dermatitidis compactum, skin and subcutaneous characteristic structures,
and chromomycosis) ✓ F. pedrosoi tissue characterized by such as arrangement of
✓ Phialophora a lesion (verrucous conidia and the manner in
verrucosa, nodules that may which conidia are borne
 ✓ Cladophialophora become ulcerated and ✓ darkly pigmented or
carrionii crusted.) phaeoid molds
✓ Rhinocladiella ✓ colonies are velvety to
aquaspersa wooly and gray-brown to
olivaceous black

Eumycotic Mycetoma ✓ Pseudallescheria characterized by ✓ Pseudallescheria boydii -

boydii swelling, with formation of cleistothecia
✓ Acremonium characteristic exudate containing ascospores
falciforme draining to the skin
✓ Madurella surface through sinus ✓ Culture: white to dark
mycetomatis tracts. gray colonies on potato
✓ M. grisea dextrose agar at 22° C and
✓ Exophiala sp 35° C.

✓ Scedosporium boydii -
produces oval conidia singly
at the tips

✓ Acremonium falciforme
produces mucoid clusters of
single- or two-celled slightly
curved conidia borne from
phialides at the tips of long,
unbranched, multiseptate
conidiophores

✓ Madurella produce
conidia from the tips of
phialides

✓ Culture: initially white,

and becomes yellow,
olivaceous, or brown, with a
characteristic diffusible
brown pigment with age.

Microscopic Morphology of Fungi Causing Chromoblastomycosis

Organism Microscopic Morphology

Phialophora Conidiogenous cells, phaeoid, flask-shaped phialides, with collarettes
verrucosa
Conidia oval, one-celled, occur in balls at tips of phialides

Fonsecaea pedrosoi Primary one-celled conidia formed on sympodial conidiophores

Primary conidia function as conidiogenous cells to form secondary one-celled

conidia.

Some conidia are similar to those seen in Cladosporium sp., Some are similar
to those in Rhinocladiella sp., and some are similar to those in Phialophora sp.

Fonsecaea Similar to F pedrosoi but with more compact conidial heads

compactum
Conidia are subglobose rather than ovoid

Cladophialophora Erect conidiophores bearing branched chains of one-celled, brown

carrionil blastoconidia

Conidium close to tip of conidiophore, termed shield cell

Fragile chains

Rhinocladiella Conidiophores erect, dark, bearing conidia only on upper portion near the tip
aquaspersa
Conidia elliptic, one-celled, produced sympodially

Disease Agent Manifestations Lab

Phaeohyphomycosis Exophiala mycotic disease caused by Most Exophiala spp- conidia

spp. such as darkly pigmented fungi or are borne from annellides,
E. fungi that have melanin in with conidia aggregating in
dermatitidis their cell walls. masses at the tips of the
subcutaneous nodules brain conidiophore
abscess or disseminated
infections E. dermatitidis- forms
conidia at the tips of
phialides
 Culture: olivaceous to black
colonies

Lymphocutaneous, Sporothrix Lymphocutaneoys chronic Direct examination: small,

fixed cutaneous, schenckii infection is characterized by cigar-shaped yeast
mucocutaneous nodular and ulcerative
sporotrichosis, lesions along the lymph Culture: grows well on most
channels that drain the culture media. including
Pulmonary primary site of inoculation. those containing
sporotrichosis cycloheximide
extracutaneous and
disseminated @ 22° C-initially white,
glabrous, and yeastlike,
turning darker and more
mycelial as they mature thin,
delicate hyphae bearing
conidia developing in a
rosette pattern at the ends
of delicate conidiophores.
@ 37° C-portion of the colony
will develop yeastlike cells.
D. Systemic Fungi

Organisms have historically been categorized together because they share several characteristics,
such as mode of transmission, dimorphism, and systemic dissemination.
diseases are contracted generally by the inhalation of infectious conidia.

Summary of Systemic Mycoses

Fungus Ecology Clinical Disease Tissue Form

Blastomyces Mississippi and Ohio River Primary lung Large yeast (8-12 μm)
dermatitidis valleys Chronic skin-bone Broad-based bud

Systemic, multiorgan

Histoplasma Ohio, Missouri, and Primary lung Small, oval yeast (2-5
capsulatum* Mississippi River valleys Asymptomatic μm) in histiocytes,
Immunodeficient hosts phagocytes
Bird and bat guano prone to disseminated
Alkaline soil disease

Coccidioides Semiarid regions- Primary lung Spherules (30-60 μm)

immitis, C posadasii southwest United States, Asymptomatic containing endospores
Mexico, Central and South Secondary cavitary
America Progressive pulmonary
Multisystem
In soil

Paracoccidioides Central and South Primary lung Thick-walled yeasts

brasiliensis America Granulomatous (15-30 µm) Multiple
In soil buds, "Mariner's
Ulcerative nasal and wheel"
buccal lesions

Lymph node involvement

Adrenals

Agent Recommended morphologic features of Confirmatory tests for

tissue form identification

Blastomyces 8-15 um, broad-based budding cells ith 1. Specific nucleic acid
dermatitidis double contoured walls are seen; probe
cytoplasmic granulation is often obvious 2. Broad-based budding
cells may be seen after in
vitro conversion on
cottonseed agar
 3. Exoantigen test

Histoplasma 2-5 um, small, oval to spherical budding 1. Specific nucleic acid
capsulatum cells often seen inside of mononuclear cells probe
2. Exoantigen test

Paracoccidioides 10-25 um, multiple budding cells (buds 1-2 Exoantigen test
brasiliensis um), resembling a Mariner's wheel, may be
present; buds are attached to the parent
cell by a narrow neck

Coccidioidomycosis Round spherules 30-60 um in diameter 1. Specific nucleic acid

containing 2-5 um endospores are probe
characteristic; empty spherules are 2. Exoantigen test
commonly seen
 Opportunistic Mycoses
1. Mucorales

Cunninghamella

common in the environment; can caused disseminated

disease and can be recovered in sinuses and other
organs
Sporangiophores are erect, branching into several vesicles
that bear sporangioles, and can be covered with long, fine
spine

Lichtheimia

have a predilection for vascular invasion, causing thrombosis

and necrosis of the tissues.
usually found in diabetic patients suffering from ketoacidosis
rhinocerebral zygomycosis- infection to the orbits, face,
palate, and brain
hyphae are broad and ribbon-like, with few septations
Erect sporangiophores, solitary or in groups (slightly
branched), terminate in an apophysis surrounded by a
sporangium.
 Internodal rhizoids are present

Mucor

have been implicated in rhinocerebral zygomycosis in

addition to disseminated disease.
Sporangiospores are formed in sporangia on erect
sporangiophores
sporangia frequently remain intact
Rhizoids, typical of some Mucorales, are absent in Mucor
spp

Rhizopus

most common Mucorales causing human disease

typically involved in diabetic patients with ketoacidosis,
presenting as rhinocerebral mucormycosis
have erect sporangiophores terminated by dark sporangia
and sporangiospores
base of the sporangiophores are brown rhizoids
sporangia are typically fragile and are not easily retained
when making slide culture preparations, resulting in an
umbrella-shaped structure at the end of the conidiophores

4. Syncephalastrum

rarely implicated in human disease but has been

documented in cutaneous infections
Sporangiophore has a large columella on which
merosporangia, containing stacks of sporangiospores, are
formed
sometimes confused with Aspergillus

Septate and Hyaline Saprophytes

Aspergilllus

second most isolated fungus after Candida spp

Aspergillus fumigatus is the species most commonly isolated;
other pathogenic species include A. flavus, A. terreus, and A. niger
Mortality is frequently above 90% in the immunocompromised host
most frequent cause of disease in bone marrow transplant recipients in addition to other cancer and
transplantation patients.
multiorgan system involvement, including the brain, liver, heart, and bone
frequent presentation is so-called fungus balls in the lungs of agricultural workers
A. fumigatus was linked to at least one case following the use of injectable steroids
Uniseriate species - phialides attach directly to the vesicle at the end of the conidiophore.
 Biseriate species - possess a supporting structure called a metula attached directly to the vesicle,
and attached to each of the metula are phialides producing conidia

Beauveria

Beauveria bassiana - rare human isolate, uncommonly

associated with keratitis
Known insect pathogen and is found worldwide on
vegetation and in the soil.
Abundant, single-celled, tear-shaped sympoduloconidia
are formed on sympodulae, which taper extremely from a
rather swollen base

Chrysosporium

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.

Caused multistate outbreak involving more than 100
individuals wearing soft contact lenses reported in 2006
In bone marrow transplant patients, mortality from
infections caused by the fusaria approaches 100%.
abundant macroconidia with fewer microconidia are
produced on vegetative hyphae
Macroconidia are banana- or canoe-shaped and are formed
singly, in small clusters, or clustered together in mats
termed sporodochia
Geotrichum

Has been implicated in pulmonary disease in

immunocompromised patients.
abundant arthroconidia formed from the vegetative hypha
that occurs singly or may be branched
Colonies are white to cream, yeastlike, and can be confused
with Trichosporon spp. Occasionally, aerial mycelium form,
producing colonies that resemble those of C. immitis.

Paecilomyces.

Paecilomyces lilacinus is extremely refractory to treatment

and was recovered in a hospital outbreak, with high
associated mortality.
Phialides of Paecilomyces are generally longer than that of
Peniciullium and more obviously tapered, and they may be
singly formed or arranged in a verticillate pattern, on which
long chains of spindle-shaped or somewhat cylindric conidia
are formed

Penicillium

rarely cause infections; most reports of disease involve

chronic fungal sinusitis.
Conidiophores are erect, sometimes branched, with metulae
bearing one or several phialides on which oval to ovoid conidia
are produced in long, loose chains

Scopulariopsis

commonly isolated from nail specimens and have been

implicated in pulmonary disease in immunocompromised
patients.
Conidiophores occur singularly or can be in clusters
Conidia are formed from annellides, which increase in length as
conidia are formed. The truncate-based conidia tend to remain
in chains on the annellides.

Trichoderma

are an emerging pathogen in the immunocompromised host

that can cause a range of infections, including pulmonary and
skin infections.
Characterized by yellow-green to green patches of conidia
formed on clusters of tapering phialides
Conidia may remain clustered in balls at the phialide tips.
Septate and Phaeoid Saprophytes
Alternaria

primarily implicated in chronic fungal sinusitis.

short conidiophores bearing conidia in chains that lengthen in
an acropetal fashion
Multicelled conidia have angular cross walls and taper toward
the distal end.

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.
Have hyaline hyphae giving rise to hyaline conidia directly from the vegetative hyphae.
With age, phaeoid hyphae develop and break up into arthroconidia, which do not bear hyaline
conidia.

Chaetomium

Have been reported in the brains of patients with central

nervous system disease. Several of these patients have been
identified as intravenous drug abusers.
found in the environment and have a predilection for cellulose
products.
known to devastate printed literature and library holdings
and have been associated with indoor air quality problems
numerous perithecia (pineapple-shaped ornamented with
straight or curled hairs or setae) are typically seen
at maturity the pigmented, lemon-shaped ascospores are
released within the perithecium.
Cladosporium

infrequent cause of disease, primarily recovered as laboratory

contaminants.
Infections are typically confined to the sinuses or following
traumatic inoculation..
Cladosporium spp. form brown to olive to black hyphae and
conidia
Conidiophores are erect and can branch into several
conidiogenous cells.
Spherical to ovoid conidia form blastically on the end of each
previously formed conidium. Branched conidium-bearing cells may dislodge, and the three scars on
each of these cells give them the appearance of a shield.

Curvularia

usually implicated in chronic sinusitis in immunocompetent

patients.
frequently recovered from grass, leaves, and decaying
vegetation.
Multicelled conidia are produced on sympodial conidiophores
frequently seen crescent-shaped conidia with three to five cells
of unequal size and an enlarged central cell.

Phoma.
Disease is usually secondar to traumatic inoculation.
Phoma spp. produce pycnidia, which appear as black fruiting
bodies that are globose and lined inside with short
conidiophores

Pithomyces.

Disease caused by Pithomyces spp. is usually secondary to

traumatic inoculation.
Conidia are somewhat barrelshaped, formed singly on simple
short conidiophores; have both transverse and longitudinal cross
walls and are often echinulate.

Ulocladium

sometimes implicated in subcutaneous infections, usually

following traumatic inoculation.
Conidiophores bear dark, multicelled conidia on sympodial
conidiophores; have angular cross walls and, in some species,
echinulate surfaces.
Yeast Infections

Yeast

eukaryotic, unicellular organisms that ar round to oval and range in size from 2 to 60 μm
classified into one of two groups
Important morphologic characteristics that are useful in differentiating yeasts include the size of the
yeasts, the presence or absence of a capsule, and broad-based or narrownecked budding
A. true yeasts- reproduce sexually, either by forming ascospores or basidiospores
B. yeastlike fungi- not capable of sexual reproduction or whose sexual state has not yet been
discovered (reproduce asexually by blastoconidia formation or budding)

Candida

commonly present as normal biota of the mucosa, skin, and digestive tract, and they are also the
most notorious agents of yeast infection
disease ranges from superficial skin infections to disseminated disease.
C. albicans
currently reigns as the premier cause of yeast infection in the world.
normal biota from a variety of sites, including skin, oral mucosa, and vagina
capable of causing disease in almost any site when host conditions are altered

Species Infections

C. Albicans oroesophageal candidiasis, intertriginous candidiasis (in which skin folds are involved),
paronychia, onychomycosis, perlèche respiratory infections, vulvovaginitis, thrush,
pulmonary infection, eye infection, endocarditis, meningitis, fungemia or candidemia, or
disseminated infection.

C. Glabrata Fungal UTI, genital infections

Infections associated with C. glabrata tend to be aggressive and difficult to treat with
traditional antifungal therapy

C. been shown to be prevalent in patients with hematologic malignancies, especially those

Tropicalis who are neutropenic

C. primary cause of fungemia in the neonatal intensive care unit (NICU)

Parapsilosis
Also the second most frequently isolated Candida spp. in positive blood cultures; this
could be due partly to its known selective growth in hyperalimentation solutions and
also to its ability to grow on intravascular catheter

Method Characteristics

Direct Budding yeast cells C. glabrata blastoconidia are notably

Microscopic (blastoconidia) 2 to 4 um in smaller
Examination diameter and/or pseudohyphae C. albicans - identified by the
smaller showing regular points production of germ tubes or
of constriction, resembling links chlamydoconidia (cornmeal agar
of sausage containing 1% Tween 80 and trypan
 blastoconidia, hyphae, and blue incubated at room temperature
pseudohyphae are strongly for 24 to 48 hours)
gram positive

Cultivation Smooth, creamy white colonies, but C. Albicans on BAP: colonies are star-like
some produce dry, wrinkled, dull or possess feet-like projections on agar.
colonies

Germ tube Production of germ tubes from when placed in a liquid nutrient Positive:
test environment and incubated at 35°C for 3 hours C. Albicans
C.
Dubliniensis
Cryptococcus neoformans

Causes cryptococcosis (acute, subacute, or chronic fungal infection that has several manifestations)
can present initially as a chronic or subacute pulmonary infection, eventually makes its way to the
central nervous system, where the yeast can cause cryptococcal meningitis
Disseminated disease with meningitis is commonly seen in immunocompromised patients.
strongly associated with such debilitating diseases as leukemia and lymphoma and the

Direct Microscopy Spherical, single or multiple

budding, thick-walled yeast 2
to 15 um in diameter usually
surrounded by a wide,
refractile polysaccharide
capsule

India Ink Reveals the large

polysaccharide capsule

Antigen Crytococcal antigen detection (CAD)

detection test for C. Neoformans may be
performed on CSF or serum

Cultivation Easily cultured on routine fungul

culture media without cycloheximide
Beginsas a smooth, white to tan colony
that may be mucoid to creamy

Inhibitory mold agar - C. Neoformans

appears as a golden yellow, nonmucoid
colony
Rapid Urease Test Principle
Hydrolysis of urea by the enzyme
urease produces ammonia and carbon
dioxide. The ammonia produces
alkaline conditions in the medium and
changes the indicator (phenol red)
from yellow to pink.

Medium: Urea broth,

Christenson’s agar

Atypical Yeast

Pneumocystis jiroveci

originally was thought to be a trypanosome (morphology is similar to that of microbes and protozoa,
and clinically it responds to antiprotozoal drugs but not to antifungal drugs

exists as three forms in its life cycle: trophozoite, precyst (sporocyte), and cyst (the latter is the
diagnostic form).

cell membrane contains cholesterol rather than ergosterol

contains only one or two copies of the small ribosomal subunit gene, whereas most other fungi
contain numerous copies of this gene

most commonly presents as pneumonia in an immunocompromised host.

most common opportunistic infection among those with HIV or AIDS in the United States
Calcofluor white, methenamine firm-walled cystic form
silver, and immunofluorescent spherical to concave,
staining uniform in size (4 to 7 um in
diameter), do not bud, and
contain distinctive
intracystic bodies.