Study of Fungi (Mycology

Mycology
Mycology - the study of fungi
Fungi - includes molds and yeasts.
Molds - exhibit filamentous type of growth.
Yeasts - exhibit pasty or mucoid form of fungal growth.
 50,000 + valid species
 Fungi stain gram positive, and require oxygen to survive.
 Fungi are eukaryotic, containing a nucleus bound by a
membrane, an endoplasmic reticulum, and mitochondria.
(Bacteria are prokaryotes and do not contain these)
 Fungi are heterotrophic like animals and most bacteria;
requiring organic nutrients as a source of energy.
(Plants are autotrophic)

Mycology
Fungi are dependent upon enzyme
systems to derive energy from organic
substrates.
 Saprophytes - live on dead organic matter.
 parasites - live on living organisms.
Fungi are essential in recycling of
elements, especially carbon.

Mycology
Role of fungi in the economy:
Industrial uses of fungi -
 Mushrooms. (Class Basidiomycetes)
 Truffles. (Class Ascomycetes)
 Natural food supply for wild animals.
 Yeast as food supplement, supplies vitamins.
 Penicillium - ripens cheese, adds flavor
(roquefort, etc.).
 Fungi used to alter texture, improve flavor of
natural and processed foods.

Mycology
Fermentation
 Fruit juices (ethyl alcohol).
 Saccharomyces cerevisiae - brewer's and
baker's yeast.
 Fermentation of industrial alcohol, fats, proteins,
acids, etc.
Antibiotics -
 First observed by Fleming; noted suppression of
bacteria by a contaminating fungus of a culture
plate.

Mycology
Plant pathology -
 Most plant diseases are caused by fungi.
Medical importance -
 50-100 species are recognized human pathogens.
 Most prefer to be free-living saprophytes; and only
accidentally become pathogens.
 To be pathogenic, they must tolerate the temperature of
the host site and possess an enzymatic system that
allows them to utilitize animal tissues.
 Increased incidence of fungal infections in recent times.

Mycology
Importance of Medical Mycology
 During the time period between 1941 - 1973, the number
of reported deaths in the U.S. due to scarlet fever,
typhoid, whooping cough, diphtheria, dysentery and
malaria decreased from 10,165 cases to 107; but
reported deaths due to mycoses during the same time
period, increased from 324 to 530.
 Increased mobility - We can travel to a geographical area
where a fungus exists as part of the commensal flora of
the local population, or is endemic to the area.
 We have an aging population.

Mycology
The Immunosuppressed Patient:
 AIDS
 Drugs - immunosuppressant drugs used
in organ transplant patients, cancer and
leukemia patients.

Mycology
Immunology of the Mycoses
Antibody mediated immunity (B-cell, humoral)
 Antibodies are often produced in response to a
fungal infection, but do not confer immunity.
 Serological tests for identification of fungal
diseases detect these antibodies.
Cellular mediated immunity (T-cell)
 T-cell immunity is effective in resistance to
fungal infections.

Mycology
Basic terms as they relate to mycology:
 Hypha (hyphae plural) - fundamental tube-
like structural units of fungi.
 Septate - divided by cross walls.
 Aseptate - lacking cross walls.
 Mycelium - a mass (mat) of hyphae
forming the vegetative portion of the
fungus.
 Aerial - growing or existing in the air.
 Vegetative - absorbs nutrients.
 Fertile - bears conidia (spores) for reproduction.

Mycology
Basic Terms (continued)
Sporulation & Spores - preferred terms when there is a
merging of nuclear material. Self-fertile are termed
homothallic. Mating types are termed heterothallic.
 Sexual spores - exhibit fusion of nuclei.
 Ascospore - spore formed in a sac-like cell known as
an ascus. Often eight (8) spores formed.
(Ascomycetes)
 Basidiospore - sexual spore produced on a
specialized club-shaped structure, called a basidium.
(Basidiomycetes)
 Zygospore - a thick-walled spore formed during
sexual reproduction (Phycomycetes)

Mycology
Sporulation & Spores (continued)
 Asexual spores - most common type.
 Conidia - asexual fungal spores borne externally in
various ways from a conidiophore; often referred to a
macroconidia (multicellular) and microconidia
(unicellular).
 Arthroconidium (Arthrospore) - special type of
asexual spore formed by disarticulation of the
mycelium.
 Blastoconidia (Blastospore) - asexual spore formed
from a budding process along the mycelium or from
another blastospore.

Mycology
 Asexual Spores (continued)
 Chlamydospore - thick-walled asexual spore
formed by direct differentiation of the
mycelium (concentration of protoplasm and
nutrients).
 Sporangiospore - an asexual spore
contained in a sporangium at the end of a
sporangoiphore.
 Thallospore - asexual spore produced on a
thallus (hypha).

Mycology
Miscellanous terms:
 Ascus - sac-like structure containing (usually eight)
ascospores developed during sexual reproduction in
the Ascomycetes.
 Conidiophore - a specialized branch of hypha on which
conidia are developed.
 Dematiaceous - pigmented, dark in color, usually gray to
black.
 Hyaline - colorless, clear.
 Dermatophyte - fungus that causes superficial mycoses.

Mycology
Miscellanous terms: (continued)
 Diphasic (dimorphic) - the ability of some fungi to grow
as either yeast or filamentous stages, depending on
conditions.
 Ectothrixic - ability of the fungus to grow on the outside
of a hair shaft.
 Endothrixic - ability of the fungus to grow and penetrate
into the hair shaft.
 Germ Tube - small projections which arise from cells of
certain yeasts; indicates the onset of hyphal formation.

Mycology
Miscellanous terms: (continued)
 Pseudohyphae - a chain of elongated budding cells that
have failed to detach (not true hyphae).
 Rhizoids - root-like structures.
 Sporangiophore - a special aerial hypha or stalk bearing
a sporangium.
 Sporangium - a sac or cell containing spores produced
asexually.
 Sterigmata - a specialized structure that arises from a
basidium and supports basidiospores.

Mycology
Classifications of Fungi:
 Geographic grouping - where they exist.
 Epidemiologic grouping - how organism is
transmitted.
 Taxonomy grouping - according to morphologic
and cultural characteristics.
 Topographic Grouping - type of mycosis
produced.

Mycology
Topographic Grouping of Fungi: (most often used)
 Superficial - Confined to the outermost layers of the
skin and hair. No host cellular or inflammatory response
due to organisms being remote from living tissue.
Essentially no pathology; the disease is recognized
purely on cosmetic basis.
 Cutaneous - in the keratin of the skin, nails, and hair.
These organisms prefer non-living cornified layers.
The disease is called a dermatophytosis or
dermatomycosis. Host response is patchy scaling or
eczema eruptions. They are classified according to the
area of the body that is involved.

Mycology
Topographic Grouping of Fungi: (continued)
 Subcutaneous - Involve the deeper layers of skin
and often muscle tissue. Man is an accidental
host following inoculation of fungal spores via
some form of trauma. This type of infection is
often identified by the presence of a
characteristic tissue reaction or granule.
 Systemic - Attack the deep tissues and organ
systems; often creating symptoms that
resemble other diseases.

Mycology
Categories of systemic disease:
 Those caused by truly pathogenic fungi with the ability
to cause disease in the normal human host when the
inoculum is of sufficient size (Histoplasma capsulatum,
Blastomyces dermatitidis, Coccidioides immitis,
Paracoccidioides braziliensis).
 Those caused by opportunistic fungi, low virulence
organisms, which require the patient's defenses to be
lowered before the infection is established (Aspergillus
spp. Candida albicans, Cryptococcus neoformans).

Mycology
Saprophytes:
 Contaminates or opportunistic pathogens?
 Most are inhibited by cycloheximide.
 Grouped by type of mycelia produced.
 Septate vs. aseptate
 Hyaline vs. dematiaceous

Mycology
Saprophytes:
Hyaline Members -
Aspergillus spp.
 Growth rate varies, colors vary,
surface velvety to cottony.
 Mycelium - septate and hyaline with
unbranched condiophores
(compare to Syncephalastrum,
which appears similar, but is
aseptate).
 A. fumigatus is considered a
potential pathogen, especially if
from a pulmonary source.

Mycology
Saprophytes:
Hyaline Members -
Paecilomyces spp.
 Rapid grower, colors vary.
 Brush-like conidiophores.
 Long, tapered sterigmata.

Mycology
Saprophytes:
Hyaline Members -
Penicillium spp.
 Commonly rapid growing;
white to bluish-green.
 Conidiophores
characteristically form a
brush-shaped structure.
 Sterigmata are flask shaped.

Mycology
Saprophytes:
Hyaline Members -
Scopulariopsis spp.
 Moderately slow growing.
White turning brown with age.
 Branched or unbranched
conidiophores.
 Sterigmata are coarsely
roughened.

Mycology
Saprophytes:
Hyaline Members -
Trichoderma spp.
 Moderately rapid growth.
 Flask-shaped
conidiophores.
 Conidia are clustered.

Mycology
Saprophytes:
Hyaline Members -
Fusarium spp.
 Rapid growth, white colonies may
be come brightly colored.
 Short conidiophores often
branched, have macro- and micro-
conidia, which are oval to sickle-
shaped.
 Has been reported to cause eye
infections.

Mycology
Saprophytes:
Dematiaceous Members -
Alternaria spp.
 Rapid growth; colonies become
very dark with age; may
become overgrown with looser
white to gray aerial mycelium.
 Conidiophores bear single or
branched chains of large,
brown conidia.

Mycology
Saprophytes:
Curvalaria spp.
 Rapid growth. Velvety
colonies vary in color from
grayish-brown to black.
 Spirally arranged brown
conidia are borne at the tips.
 Brown, septate, unbranched
conidiophores.

Mycology
Saprophytes:
Cladosporium spp.
 Rapid growth. Green
colonies, reverse is black.
 Septate, dematiaceous
mycelium.
 Conidia are borne in
chains.

Mycology
Saprophytes:
Aseptate Members -
 All are susceptible to cycloheximide.
 Rapid growers.
 Some have root-like structures termed
rhizoids.
 Spore bearing structures are called
sporangiophores.

Mycology
Saprophytes:
Aseptate Members -
Mucor spp.
 Very rapid growth; can fill a
culture tube in one day.
 Unbranched sporangiophores.
 No rhizoids

Mycology
Saprophytes:
Aseptate Members -
Rhizopus spp.
 Rhizoids are present.
 Sporangiophores nodal in
origin.

Mycology
Saprophytes:
Aseptate Members -
Syncephalastrum spp.
 Very rapid growth. White to dark
gray colonies with dense, cottony,
aerial mycelium.
 Aseptate, hyaline mycelium, with
short, branched sporangiophores,
terminating into tips.
 Many tubular sporangia containing
chains of spores.

Mycology
Yeasts:
Candidia albicans
 Cutaneous infections such as
oral thrush or vaginitis, but
can become systemic.
 ID by positive germ tube test,
or production of
chlamydospore on cornmeal
agar.

Mycology
Yeasts:
Cryptococcus neoformans
 Associated with pigeon feces.
 Has thick capsule.
 Grows at 37 degrees & produces
melanin-like pigment on caffeic
acid agar.
 ID in CSF by India ink stain for
capsule.

Mycology
Yeasts:
Geotrichum spp.
 Commonly present in GI
tract.
 Implicated in respiratory
infections; frequently as a
secondary invader.
 Causes disease in the
immunosuppressed.
 Produces arthrospores.

Mycology
Bacteria - Like Fungi:
 Some produce sulfur granules
(Actinomyces israeli)
 Often require special media,
stains, & conditions for
growth, i.e. anaerobic
(Actinomycetes spp).
 Some are partially acid-fast
(Nocardia spp.)

Mycology
Dimorphic Fungi:
Sporothrix schenkii
 Rose fever, gardeners
often affected.
 Infection via traumatic
implantation.

Mycology
Dimorphic Fungi:
Coccidioides immitis
 San Joaquin valley fever.
 Mycelial phase present in
culture.
 Yeast phase in tissue.

Mycology
Dimorphic Fungi:
Histoplasma capsulatum
 Mississippi Valley fever.
 Associated with bird
droppings.
 Disease mimics tuberculosis.
 Found frequently in
reticuloendothelial cells as
extracellular inclusions.
H. capsulatum mold phase
H. capsulatum yeast phase

Mycology
Dimorphic Fungi:
Blastomyces dermatitis
 North American
Blastomycosis (Gilcrest
disease).
 Large yeast cells with
single bud.

Mycology
Dimorphic Fungi:
Paracoccidioides braziliensis
 South American
Blastomycosis.
 Large yeast cells with
multiple buds (mariner’s
wheel).

Mycology
Superficial mycoses:
Malassezia furfur
 Cause of pityriasis versicolor
(tinea versicolor).
Phaeoannellomyces werneckii
 Cause of superficial
phaeohyphomycosis (tinea
nigra).

Mycology
Superficial mycoses:
Trichosporon beigelii
 Cause of white piedra.
Piedraia hortae
 Cause of black piedra.

Mycology
Cutaneous mycoses:
Microsporum spp. - in hair, skin, rarely nails;
frequently in children, rarely in adults;
often spontaneous remission occurs
(ringworm).
Trichophyton spp. - hair, skin & nails; in
both children & adults (athlete’s foot).
Epidermophyton spp. - skin, nails, rarely hair;
in adults, rarely in children (ringworm).

Mycology
Cutaneous mycoses:
Microsporum spp.
 Macroconidia are attached
singly.
 Thick walled.
 Mature forms are echinulate
(spiny).

Mycology
Cutaneous mycoses:
Trichophyton spp.
 Macroconidia are attached
singly.
 Have smooth walls.

Mycology
Cutaneous mycoses:
Epidermophyton spp.
 Macroconidia are attached in
multiples.
 Smooth walls (beaver tails).
 Grows slowly.

Mycology
Subcutaneous mycoses:
Chronic, supperative or granulomatous infections of the
subcutaneous tissues, usually on an extremity (hands,
feet); can extend through the lymphatics or form sinus
tracts. Caused by a variety of fungi and bacteria-like fungi
that live in the soil.
Chromoblastomycosis:
 Non-contagious skin diseases characterized by the
development of a warty lesion that has a cauliflower
appearance. Occurrence is usually on the legs or feet.
Caused by Fonsecaea pedrosoi, Cladosporium spp., and
Phialophora spp. Fonsecaea pedrosoi is the most
common etiologic agent of chromoblastomycosis

Mycology
Mycetoma:
 Localized, tumorous lesions in cutaneous and
subcutaneous tissues usually the foot. Nodules
are formed, and a collection of pus and
formation of sinuses results.
 Actinomycotic mycetomas must be
differentiated from Eumycotic (true fungi)
mycetomas and as they have greatly differing
treatments.

Mycology
Mycetoma:
 Actinomycotic mycetoma -
 “Lumpy-jaw” - The initial nodules formed are firm and
described as "woody" or "lumpy.“
 Sulfur granules - Bread crumb-like aggregates of
microorganisms and cellular debris which take on the
appearance of a sulfur particle.
 Caused by Nocardia spp. & Streptomyces spp.
 Advanced cases often require amputation due to
physical impairment.

Mycology
Mycetoma:
 Eumycotic Mycetoma -
 Clinical picture similar to actinomycotic mycetomas. Disease is
slowly progressive, with patients frequently dying of secondary
infections.
 Treatment consists of excising early, localized lesions
(amputation may be required for advanced infections). Anti-
fungal drugs have little effect.
 Body tries to wall off the offending fungus. Invasion of deeper
tissue may follow, with bone involvement, draining sinus tracts,
or progression through the lymphatics.

Mycology
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

Mycology
Systemic mycoses:
 Histoplasma capsulatum -
 Histoplasmosis - an infection of the
reticuloendothelial system resulting in patchy
bronchopneumonia containing yeast-laden
phagocytic cells within alveolar spaces.
 Worldwide in distribution, it is endemic in the
Mississippi, Missouri, St Lawrence, and Ohio river
valleys.
 Strong association with bird and bat droppings.

Mycology
Systemic mycoses:
 Coccidioides immitis -
 Coccidioidomycosis - usually an asymptomatic or
mildly symptomatic self-limiting upper respiratory
tract infection, 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; organism
produces arthroconidia which are very easily
inhaled.

Mycology
Systemic mycoses:
 Paracoccidioides brasiliensis -
 Paracoccidioidomycosis - also called South
American blastomycosis.
 Blastomyces dermatitidis -
 Blastomycosis - also called North American
blastomycosis or Gilcrest disease.

Laboratory Methods in Medical Mycology
Collection, Handling and Processing of Specimens
 Skin - cleaned with 70% alcohol to remove dirt, oil and
surface saprophytes
 Nails - cleaned same as for skin. Usually clipped; need to be
finely minced before inoculating to media
 Hair - obtained from edge of infected area of scalp. Use a
Wood's lamp (fluorescence) to help locate infected hair. Hair
can be obtained by plucking, brushing, or with a sticky tape
 Body fluids - normal sterile collection procedures

Preparation of Specimens for Transport to Laboratory
 Hair & nails sent in a dry envelope, inside proper container
 Other specimens are usually sent frozen or on dry ice
 Packaging - biohazard regulations. Any growing cultures
must be on tube media (not plates). Aluminum screw-
capped inner tube with outer cardboard mailing tube
 Inside labeling information: Patient ID, specimen source,
suspected organism
 Outside labeling information - must state:
 WARNING: POTENTIAL PATHOGEN

Appropriate Processing of Specimen to Recover Fungus
 Skin, nails, & hair - direct exam following KOH preparation
 CSF - centrifuged; examine sediment microscopically,
inoculate media
 Pleural fluid, sputum, and bronchial aspiration - specimen
must be fresh as saprophytes would overgrow pathogens
such as H. capsulatum. Specimens may be refrigerated up
to 2 hours
 Gastric washings - same as for pleural fluids
 Genito-urinary specimens - first morning specimen preferred

Appropriate processing of specimen to recover fungus
(continued)
 Blood/bone marrow - generally inoculated directly to BHI
broth and BHI slant.
 Wound abscess or drainage - should be cultured
anaerobically, especially if actinomycosis is suspected
 Tissue specimens - examine for pus, caseous material or
granules; mince aseptically, can use small amount of sterile
saline and the supernatant inoculated

Direct Examination of Specimens
 Direct microscopic exam is required on any material sent to
lab for fungus culture. Look for spores, hyphae, mycelial
elements, budding yeast, mycotic granules
 Wet mount prep - good for yeast; examination is done in
natural environment, so loss of fragile structure is minimized
 KOH prep - done on skin scrapings, hail, nails, sputum,
vaginal specimens, etc. The KOH clears the specimen’s
tissue cells, mucous, etc., so fungal elements can be seen

Stains Used in Mycology
 Lactophenol Cotton Blue (LPCB) - very popular for quick
evaluation of fungal structures; will stain the chitin in cell walls of
fungi
 Periodic Acid - Schiff Stain (PAS) - stains certain polysaccharide
in the cell walls of fungi. Fungi stain pink-red with blue nuclei
 Gomori Methenamine Silver Stain - outlines fungi in black due to
the silver precipitating on the fungi cell wall. Internal structures
are deep rose to black; background is light green
 Gridley Stain - Hyphae and yeast stain dark blue or rose.
Tissues stain deep blue and background is yellow
 Mayer Mucicarmine Stain - will stain capsules of Cryptococcus
neoformans deep rose

Stains Used in Mycology
 Fluorescent Antibody Stain - simple, sensitive, and specific. Applications
for many different fungal organisms
 Papanicolaou Stain - good for initial differentiation of dimorphic fungi.
Works well on sputum smears
 Gram Stain - most fungi are gram positive; Actinomyces and Nocardia
are gram variable
 Modified Acid-Fast Stain - differentiates acid-fast Nocardia from other
aerobic Actinomyces
 Giemsa Stain – use on blood and bone marrow specimens.
 India Ink - demonstrates the capsule of Cryptococcus neoformans in CSF
specimens

Fungal Culturing
 Tubed media is used rather than plated media because:
 there is less chance for spore release into the environment
 less chance for dehydration
 ease of storage
 The agar in a tube is inoculated in a straight line.
 The agar on plates is Inoculated like a large "S" so that rapid growing fungi
can removed
 Incubation should be aerobic (and anaerobic if Actinomycetes are
suspected)
 Incubate cultures at room temperature & also at 37C if dimorphic fungi are
suspected
 Cultures are kept for 4 weeks & should be examined every other day
 Systemic pathogens often require 10 days to 2 weeks

Media Used for Isolation of Fungi
 Sabouraud's dextrose agar (Sab-Dex) - classic medium, recommended
for most studies
 Sabouraud's dextrose agar with chloramphenicol - chloramphenicol
inhibits bacterial growth
 Mycosel agar - commercially available. Contains chloramphenicol to
inhibit bacterial growth; cycloheximide to inhibit saprophytic fungi and
some yeasts (including C. neoformans)
 Brain heart infusion slant (BHI) - more enriched than Sab-Dex. Used to
recover H. capsulatum
 Potato-dextrose agar (PDA) and Corn-meal agar - used in slide cultures;
these induce spore formation, which greatly aids in identification

Special Applications Media
 Caffeic Acid Agar - Cryptococcus neoformans will produce melanin
resulting in black colonies (protect media from light during incubation)
 Birdseed Agar - used to isolate Cryptococcus neoformans from
contaminated cultures
 KT Medium & Kelley Agar - used to convert dimorphic fungus
Blastomyces dermatitidis from mycelial to yeast form
 Modified Converse Liquid Medium (Levine's) - used to promote spherule
production by Coccidioides immitis
 Christensen's urea agar - urea is hydrolyzed by some yeast to form
ammonia. The pH increases turning the media from yellow to dark pink

Colony Morphology (macroscopic features)
 Surface topography - some fungal colonies may cover the entire surface
of agar; others grow in a more restricted manner
 Surface texture examples: cottony or wooly (floccose), granular, chalky,
velvety, powdery, silky, glabrous (smooth, creamy), waxy
 Pigmentation - fungi may be colorless or brightly colored. Color may be
on fungus itself, on its sporulating apparatus, on the agar, or on the
bottom of the colony (reverse pigmentation). Pigment color is due to the
color of the sporulating apparatus. Pigment can diffuse into the agar. It is
important to note the top pigment (obverse); the underside pigment
(reverse); and any discoloration of the medium

Microscopic Evaluation of Fungi
 Teased Preparations – part of the colony is removed to a slide
with inoculating needle. Lactophenol cotton blue stain often
used
 Slide Culture Techniques - provides a view of undisturbed
microscopic morphology
 Transparent Tape Preparation – clear cellophane tape used to
“lift” parts of a colony to a microscope slide

Biochemical Studies Used to ID Yeast and Yeast-like Organisms
 Carbohydrate assimilation & fermentation
 Measures utilization of a carbohydrate under anaerobic conditions as
determined by acid and gas production
 Specimen is inoculated beneath broth. Bromcresol purple is the indicator.
Acid production turns purple broth to yellow. Gas is detected by appearance
of bubbles trapped in a fermentation tube. Observe every 48 hours for 14
days
 Nitrogen assimilation
 Utilizes 3 tubes with different sources of nitrogen. Bromthymol blue is the
indicator (blue to yellow is positive).

Other Tests of Importance
 Germ tube - Candidia albicans & Candidia stellatoidea produce
germ tubes when incubated in a protein medium
 Demonstration of chlamydospores - yeast is inoculated by
jabbing the appropriate agar (Cornmeal agar with tween 80).
Cultures are observed every 24 hours for 3 days looking for
chlamydospore production

Hypersensitivity: Skin Tests and Serological Tests
 Skin tests - demonstrate T-cell immunity (cellular) to a fungus
 Serological tests - demonstrate B-cell (humoral) immunity to a fungus
(acute & convalescent specimens are required)
 Complement fixation
 Agglutination tests
 Precipitin tests
 Immunofluorescence
 Immunodiffusion techniques
 Counterimmunoelectrophoresis

Study of Fungi (Mycology

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