Medical Mycology

Introduction • Mycology - the study of fungi

• Fungi are eukaryotic, heterotrophic, spore-bearing microorganisms with rigid cell wall and
exist in different forms
o ranging from, unicellular to multicellular (filamentous forms)
• Classified according to means of reproduction
o sexual
o asexual
• there are between 100,000 to 200,000 species
• mostly found in the environment
• About 1% are presently known to be pathogenic for man
• are essential in recycling of elements, especially carbon
• Heterotrophic
o they require organic nutrients (from food) as a source of
energy
o similar to animals and most bacteria (opposite of plants -
autotrophic)
• require oxygen to survive
• dependent upon enzymes systems to derive energy from organic substrates
• Non-motile
• Cell membrane contains ergosterol
• Cell wall mostly made up of chitin, mannan & glucan
• do not contain chlorophyll
• Usually resistant to antibacterial antibiotics
• can be divided into:
o saprophytes - live on dead organic matter
o parasites - live on living organisms

Medical • Antibiotics
importance of o First observed by Fleming but did not proceed further
fungi • noted suppression of bacteria by a contaminating fungus of a culture plate
• Disease
o 50-100 species recognized human pathogens
o Most prefer to be free-living saprophytes
o usually become pathogens accidentally
o To be pathogenic, fungi must
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• tolerate the temperature of the host site
• possess enzymatic system that allows them to parasitize animal tissues
o Increased incidence of fungal infections in recent times
Emerging • Increased mobility
importance of o Travel to a geographical area where a fungus exists as part of the commensal flora
fungi of the local population, or is endemic to the area.
• The immunocompromised / immunosuppressed
o AIDS
o Drugs - tetracycline (acne); birth control pills; indiscriminate use of antibiotics;
immunosuppressant drugs used in organ transplant patients, cancer and leukemia
patients.
• Environmental factors
o air & water pollution eg “sick building syndrome”; over processed & "fast" foods;
fad diets etc.
• Ageing population

Mycoses

• most fungi are opportunistic pathogens

• only few fungi are primary pathogens
o Eg dimorphic fungi that cause systemic mycoses and
dermatophytes
• human body is a hostile environment for fungal invasion
• most fungi are saprophytes
o their enzymatic pathways function more efficiently at the redox
potential of non-living substrates
o less efficient at the relatively more reduced state of living
metabolizing tissue
• Some fungi are commensals that have adapted to human environment
o Eg Candida and Malasezzia
• Fungal disease results from the complex interplay between
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o fungal virulence factors
o host defence factors
• Infection depends on
o inoculum size
o general immunity of the host
Virulence factors 1) Ability to adhere to host cells by way of cell wall glycoproteins
2) resist phagocytosis - production capsules
3) suppress the production of complement – by producing cytokine called
granulocyte-macrophage colony-stimulating factor (GM-CSF) eg Candida
albicans
4) Ability to acquire iron from red blood cells as in Candida albicans
5) Ability to damage host by secreting enzymes such as keratinase, elastase,
collagenase
6) Ability to resist killing by phagocytes as in dimorphic fungi by changing
form
7) Ability to secrete mycotoxins
8) Having a unique enzymatic capacity
9) Exhibiting thermal dimorphism
10) Ability to block the cell-mediated immune defences of the host
11) Surface hydrophobicity
Fungal pathogenesis • the process by which fungi infect and cause disease in a host
• not all fungi are pathogens and have the ability for pathogenesis
(virulence)
• most fungi are opportunists
• similar to bacterial pathogenesis

Adherence
• yeasts in particular are able to colonize the mucosal surfaces of the
gastrointestinal and female genital tracts
o buccal and vaginal epithelial cells
• adherence is mediated by fungal adhesins and host cell receptors
o eg in C. albicans, mannoprotein is an adhesin, and fibronectin a receptor

Invasion
• must pass an initial surface barrier - skin, mucous membrane or respiratory
epithelium
• some are introduced through mechanical breaks
o eg Sporothrix schenckii infection typically follows a thorn prick or
other trauma
• fungi that infect the lung produce conidia small enough to be inhaled
o eg arthroconidia of Coccidioides immitis can remain suspended in
air before reaching the terminal bronchioles to initiate pulmonary
coccidioidomycosis
• dimorphic fungi undergo a metabolic shift that can change their
morphology to become more invasive
• direct invasion across mucosal barriers by the endogenous C. albicans is
associated with a morphologic change - formation of hyphae
o the new form is able to penetrate and spread
• extracellular enzymes eg, proteases, elastases are also associated with the
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hyphal form of Candida, as well as, other dimorphic and pathogenic fungi
o these enzymes contribute to invasion or spread - their role is
unknown

Injury
• In response to fungus and to cell injury, cytokines are released
o fungal PAMPs binding to PRRs
o results in inflammatory response and extracellular killing by
phagocytes
• leads to further destruction of host tissues
• fungi secrete enzymes to digest cells eg proteases, phospholipases, and
elastases
• Many molds growing on grains, nuts and beans secrete mycotoxins
o if ingested, can cause loss of muscle coordination, weight loss, and
tremors
• Mycotoxin aflotoxins produced by some Aspergillus sp. are carcinogenic
• Stachybotrys chartarum produces mycotoxin associated with "sick
building syndrome"
• mycotoxin symptoms include dermatitis, inflammation of mucous
membranes, cough, fever, headache and fatigue
• circulating products of C. neoformans have been shown to downregulate
immune
functions
• the injury caused by some fungi seems to be due to the destructive aspects
of delayed-type hypersensitivity (DTH) responses
o resembles tuberculosis

Immunology of the Mycoses Host defence factors include:

1) Physical barriers, such as skin and mucus membranes
2) The fatty acid content of the skin
3) The pH of the skin, mucosal surfaces and body fluids
4) Epithelial cell turnover
5) Normal flora
6) Chemical barriers, such as secretions, serum factors
7) Most fungi are mesophilic and cannot grow at 37oC
8) Humoral and cell-mediated immunity
*Antibodies are important for fungal serodiagnosis

Immunology of the Mycoses Humoral immunity

• antibodies are produced against many fungi
• antibodies help in clearing fungal pathogens through opsonisation
o important against Candida and Cryptococcus
• Complement
o act as opsonins
o may even cause damage to the cells through complement activation

Cell mediated immunity

• More important in mycoses
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Factors predisposing to fungal
infections
Basic Structures
Vegetative structures
Medical Mycology
• non-specifically by
o effector cells (polymorphonuclear leucocytes)
o professional phagocytes (monocytes and macrophages)
• phagocytes are very important against Candida, Aspergillus and
Zygomycetes
• specifically by T lymphocytes which includes
o delayed-type hypersensitivity
o contact allergy
o chronic granulomatous reactions
1) Prolonged antibiotic therapy
2) Underlying disease
• HIV infection, cancer, diabetes, etc.
3) Age
4) Surgical procedures
5) Immunosuppressive drugs
6) Irradiation therapy
7) Indwelling catheters
8) Obesity
9) Drug addiction
10) Transplants
11)Occupation
• Hypha (hyphae plural)
o fundamental tube-like structural units of fungi.
o Septate - divided by cross walls
o Aseptate/Coenocytic - lacking cross walls
• Mycelium
o a mass / mat of hyphae forming the vegative portion of the fungus
o Vegetative hyphae - absorbs nutrients
o Reproductive hyphae - bears conidia or spores for reproduction
• Aerial - growing or existing in the air
• Spores
o Sporulation & Spores - preferred terms used when there is a
merging of nuclear material / genes combine
a) Septate hyphae
o if one hyphal strand is damaged, the pores can be plugged to
prevent death of the whole hyphal strand
o eg Basidiomycetes and Ascomycetes
b) Non-septate hyphae/coenocytic
o are considered primitive
o If a hyphal strand is damaged, then the entire strand will die
o eg Zygomycetes

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Growth of a hypha from a spore

Fungi morphology • Two forms of growth:

o Yeasts/spherules
o Molds
• Yeast
o unicellular
o Most yeast divide by budding (asexually)
o Yeasts form moist, pasty or mucoid form colonies similar but larger
than bacteria’s
o Eg: Cryptococcus neoformans, Saccharomyces cerviciae
• Yeast-like
o Similar to yeasts but produce pseudohyphae
o Eg: Candida albicans

• Molds
o multicellular
o exhibit filamentous type of growth
o hyphae/mycelium
o parts of the mycelium grows into the agar to provide nutrients to the
aerial hyphae forming fuzzy outgrowth or mold on the agar
o Eg: Aspergillus spp., Trichophyton rubrum
• Dimorphic fungi
o fungi that exists 2 different morphological forms at 2 different
environmental conditions
• yeasts in tissue and in vitro at 37oC
• molds in their natural habitat and in vitro at room temperature
o Eg: Histoplasma capsulatum, Blastomyces dermatidis, Coccidioides
immitis

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Spore

A unit of sexual or asexual reproductive structure that may

be adapted for dispersal and for survival
o often exists for extended periods of time
o formed under unfavourable conditions
• Mating types = heterothallic
• Self-fertile = homothallic
• Sexual spores
o less common
o two strains of fungi
o fusion of nuclei
• Asexual spores
o most common type
o Grow from fragments
Fungal reproduction • About a third of all fungi duplicate utilizing more than one method of
propagation
• Environmental situation determines developmental states that lead to the
creation of focused structures for sexual or asexual reproduction
o help reproduction by effectively dispersing spores or spore-
containing propagules
1. Asexual reproduction
o occurs via vegetative spores (conidia) or budding or through mycelial
fragmentation
o sustain clonal populations adapted to an exact niche, and permit
faster dispersal than sexual reproduction
2. Sexual reproduction
o occurs with meiosis

Asexual reproduction
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Conidia
• Asexual spores that are formed at the end or the side of the conidiophore
o Conidiophore - a specialized hyphal structure that produces the conidium
• generally defined as mitospore (asexual)
• formed as an exospore at a hypha in way leading to the spores being given
away to the outside

The microscopic structures of a septate fungus showing asexually produced

conidia that leave the fungus and germinate to produce a new mycelium.

Budding
• Occurs in yeast
• Buds may separate or remain attached
• Bud is called blastoconidia

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Mycelial fragmentation
• The parent body (vegetative mycelial cell) breaks into two or more fragment
o during some unfavourable conditions
• Each body fragment develops into a distinct mycelium
• eg Rhizopus

Sexual reproduction

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• occurs with meiosis

• Sexual reproduction is carried out by diffusion of compatable nuclei from
two parent at a definite state in the life cycle of fungi
• exists in all fungal phyla (with the exclusion of the Glomeromycota)
• It differs from sexual reproduction in animals or plants

Modern Taxonomy

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Classification of Fungi • Fungi can be classified into FOUR major subdivisions

‒ based on the method of spore production of the sexual state of the fungi
• They include:
1. Zygomycetes (bread or pin moulds)
2. Ascomycetes (common moulds)

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3. Basidiomycetes (mushrooms and toadstools)
4. Hyphomycetes (conidial moulds)
Zygomycetes (bread or • A fast growing terrestrial fungi
pin mould) • Cosmopolitan in distribution
• mostly non-septate
• Asexual spores are
o Chlamydoconidia
o conidia
o sporangiospores
• Sexual reproduction produce
thick-walled sexual resting spores, a zygopore

Ascomycetes (common • Usually are parasites of plants or lichen forming

moulds) • terrestrial
• cosmopolitan in distribution
• Hyphae septate with pores that allow both cytoplasmic and nuclear
migration
• Asexual reproduction is by conidia
• Sexual reproduction is by formation of ascospores, usually 8, in an ascus.
• Asci is usually confined in a fruiting body or ascocarp

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Basidiomycetes • Parasites of mainly plants

(mushrooms and • terrestrial
toadstools) • cosmopolitan in distribution
• Hyphae septate with complex pores called doliphores
o This pores allow cytoplasmic but not nuclear migration
o hyphae can be distinguished by the presence of clamp connection
• Sexual reproduction is by the formation of basidiospores
o usually 4 on a basidium

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Hyphomycetes (conidial • A class of mycelial moulds which reproduce asexually by conidia on hyphae or
moulds) aggregation of hyphae
• once referred to as Fungi imperfecti or Deuteromycetes
o however most are now assigned to the Ascomycota, on the basis of
phylogenetic analysis
• cosmopolitan in distribution
• The hyphae are septate
• pores resemble the Ascomycetes
o allow cytoplasmic and nuclear migration
• There are 2 types of hyphomycetes:
o dematiaceus
o hyaline hyphomycetes
• This class contains the majority of medically important fungi causing:
o Dermatophytosis
o Aspergillosis
o Hyalohypomycosis

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Hyaline (clear) hyphae

Dematiaceus (dark) hyphae

Apergillus flavus conidia

Mycotic diseases 1. Hypersensitivity
• an allergic reaction to molds and spores
2. Mycotoxicoses
• poisoning of man and animals by feeds and food products
contaminated by fungi which produce toxins from the grain substrate
3. Mycetismus
• the ingestion of preformed toxin (mushroom poisoning)
4. Mycoses
• Fungal infections
*In this lecture, we shall be concerned mainly with no 4
Endemic mycoses • caused by pathogens that are restricted geographically
o considered true pathogens because can cause disease (systemic
infections) in
healthy individuals
• the most common endemic mycoses in the Asia-Pacific region are:
o histoplasmosis - Histoplasma capsulatum
• ecologic niche remains unclear
o penicilliosis - Penicillium marneffei
• restricted to Southeast and Eastern Asia
o sporotrichosis - Sporothrix schenckii

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• tropical areas of the Asia-Pacific region linked to environmental reservoirs
• before the advent of AIDS, histoplasmosis and penicilliosis were only
occasionally reported
• blastomycosis is not known to be endemic in the Asia-Pacific region
o very few indigenous cases have been reported
o mostly isolated cases from India
• coccidioidomycosis and paracoccidioidomycosis are not reported in the Asia-
Pacific region, except as imported mycoses
Types of fungal Skin Mycology
infections in human • Superficial Mycoses
• Cutaneous Mycoses
• Subcutaneous Mycoses
Infectious Disease Mycology
• Dimorphic Systemic Mycoses
• Opportunistic Systemic Mycoses
Superficial Mycoses • superficial cosmetic fungal infections of the skin
• No living tissue is invaded
• Confined to the outer keratinized tissues of the skin and hair
• No host cellular / inflammatory response due to organisms being remote from
living tissue.
o essentially no pathology
• Example: Pityriasis (tinea) versicolor
o Agent: Malassezia furfur
o Distribution: worldwide Malassezia furfur (scanning em, colonies on
Dixon agar, KOH stain)

Pityriasis versicolor causes depigmentation in dark skin (bottom) and lightly

reddish brown on very pale skin (top). It prevents the development of suntan.

Superficial mycoses

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Fungus Disease caused

Malassezia furfur Pityriasis versicolor (Tinea

versicolor)

Phaeoannellomyces werneckii Superficial phaeohyphomycosis

(formerlyCladosporium/Exophiala (Tinea nigra)
werneckii)

Piedraia hortae Black Piedra

Trichosporon beigelii White piedra

Cutaneous Mycoses • Superficial fungal infection

• No living tissue is invaded
• a variety of pathological changes occur because of the presence of the
infectious agent and its metabolic products
• Example: Dermatophytosis (Ringworm or tinea)
o has keratinase to utilize keratin
o organism adhere to the perspiration-moist skin
• Agent: eg Trichophyton rubrum

Tinea capitis

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Tinea manuum typically has a raised border with a clear area in the middle,
while dermatitis does not.

Cutaneous mycoses • Have particular affinity for the keratin of the skin, nails, and hair.
• Most cutaneous infections are caused by a closely related group of fungi,
called the dermatophytes
• These keratinophilic prefer the non-living cornified layers.
• The disease caused by these organisms is called Adermatophytosis@ or
"dermatomycosis“
• Host response is patchy scaling or eczema forming eruptions
o inflammation may occur
• they are classified according to the area of the body that is involved.
• Dermatophytoses: - infection of skin, hair & nails caused by :
o Microsporum
o Trichophyton
o Epidermophyton
Dermaphytoses / • a common infection of the skin and nails
Ringworm • causes an itchy, red, circular rash (raised edge & ‘quieter’ centre), hence called
ringworm
• The different types of ringworm are usually named for the location of the
infection on the body
• Approximately 40 different species of fungi can cause ringworm
• People at risks are those who use public showers or locker rooms, athletes,
wear tight shoes, have excessive sweating and have close contact with animals

Types of ringworm includes:

• Tinea pedis (feet)
o commonly called “athlete’s foot”
• Tinea cruris (groin, inner thighs, or buttocks)
o commonly called “jock itch”
• Tinea capitis (scalp)
• Tinea barbae (beard)
• Tinea manuum (hands)

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• Tinea unguium (toenails or fingernails)
o also called “onychomycosis”
• Tinea corporis (other parts of the body such as arms or legs)
Dermatophytes • Dermatophytes produce septate, hyaline mycelium
Microsporum

Microsporum canis are typically spindle-shaped with 5-15 cells

• Infects hair, skin, rarely nails (frequently in children, rarely in adults)
• often spontaneous remission occurs.
• attached singly, has thick walls & mature forms are echinulate (spiny)
• M. canis, M. gypseum

Trichophyton

Trichophyton rubrum showing slender clavate microconidia and cigar-shaped

macroconidia, some with terminal appendages.
• Infects hair, skin & nails (In both children & adults)
• chronic
• attached singly, have smooth walls
• Ectothrix infection – hair shaft
• T. mentagrophytes, T. rubrum

Epidermophyton
• Infects skin, nails, rarely hair (In adults, rarely children)
• attached in multiples, moderately thick, smooth walls (beaver tails)
• Grows slowly.
Trichophyton rubrum • an anthropophilic fungus
• has become the most widely distributed dermatophyte of humans
• frequently causes chronic infections of skin, nails and rarely scalp
• Granulomatous lesions may sometimes occur
• Trichophyton rubrum is the most common cause of tinea pedis (athlete’s foot)
worldwide
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- 70% of the population will be infected with tinea pedis at some time
- can be treated with non-prescription antifungal creams eg Clotrimazole and
Miconazole

Onychomycosis is caused by 3 main classes of fungi: dermatophytes, yeasts,

and non-dermatophyte molds. Dermatophytes are by far the most common
cause of onychomycosis with T. rubrum accounting for 70% of the cases.

Trichophyton rubrum culture:

• downy strain with typical wine-red reverse
• Y variety with both yellow and red pigmentation
• var. flava with yellow pigmentation
• granular strain with red surface and reverse pigmentation
Epidermophyton • an anthropophilic dermatophyte
floccosum • has a worldwide distribution
• often causes tinea pedis (athlete’s foot), tinea cruris, tinea corporis and
onychomycosis
• not known to invade hair in vivo
• no specific growth requirements have been reported

Athlete's foot usually begins between the toes. It can cause a scaly rash that may
itch, sting or burn.

Culture of E. floccosum

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Macroconidia of E. floccosum

Subcutaneous Mycoses • chronic localized infections of the skin and subcutaneous tissue caused by
fungi
- Involve the deeper layers of skin and often muscle tissue
• Fungi are soil saprophytes but able to adapt to tissue environment
• Example: Mycetoma
• Distribution: worldwide (bare-footed populations)
• Agent: Madurella mycetomatis

Mycetoma foot
• Man is an accidental host following inoculation of fungal spores via some
form of trauma
‒ is often tentatively identified by the presence of a characteristic tissue
reaction or granule
Mycetoma • chronic, suppurative or granulomatous infections, often localized
o affects the subcutaneous tissues
• usually on an extremity (hands, feet)
o can extend through the lymphatics or form sinus tracts
• caused by certain types of bacteria (actinomycetoma) and fungi (eumycetoma)
found in soil and water
• Nodules are formed, and a collection of pus and formation of sinuses results.
• Actinomycotic mycetomas are different from eumycotic mycetomas - they
have different treatments
• Nocardia brasiliensis, is the most commonly implicated actinomycetes
• ratio of cases of actinomycetoma to eumycetoma in Mexico has been reported
to be 92:8

Actinomycotic • sometimes classify as systemic

mycetoma • Lumpy-jaw named by Bollinger in 1976 because of its appearance in tissue
o The initial nodules formed are firm and described as "woody" or
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"lumpy".
• Sulfur granules
o Bread crumb-like aggregates of microorganisms and cellular debris
which take on the appearance of a sulfur particle
o True granules will appear microscopically as lobulated tangled masses
of delicate gram positive branching filaments or short bacillary elements
• Etiological agents are actually bacteria, not fungi
o Nocardia asteroides
o Nocardia brasiliensis
o Streptomyces somaliensis (Africa, S. America, Arabia)
o Streptomyces paraguayensis (S. America)

Eumycotic Mycetoma • Sometime termed Amaduromycotic mycetomas

• Clinical picture similar to actinomycotic mycetomas
• Disease is slowly progressive, with patients frequently dying of secondary
infections
• 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
• Can become systemic
• Treatment
o consists of excising early, localized lesions (amputation may be
required for advanced infections)
o Anti-fungal drugs have little effect

Eumycetoma caused by Madurella mycetomatis with sinuses draining black

grains and pus

Madurella mycetomatis culture on Sabouraud agar

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Tissue section stained with Grocott; Madurella mycetomatis hyphae (black) are
inside the fungal grain
Chromoblastomycosis • a chronic subcutaneous mycosis
• non-contagious skin diseases
• cauliflower appearance
o characterized by the development of a papule at the site of infection
that spreads to form a warty or tumor-like lesion
• usually occurs on the legs or feet but can involve deeper tissues
• Extensive fibrosis in deeper tissue may block the lymphatics and cause an
elephantiasis syndrome
• Complete cure is rare, but the condition usually remains localized
• In the lab, crusts and exudates from lesions mounted in 10% KOH will
reveal brown-pigmented, branching, septate hyphae
• Biopsy material and pus may reveal thick-walled, round, brown cells called
Asclerotic bodies
• Causative agents: Fonsecaea pedrosoi, Cladosporium spp., and
Phialophora spp.
• Prevention: wear shoes, avoid trauma that would inoculate organisms, and use
good hygiene.
• Can be found throughout the world, but more in tropical areas.
Systemic mycoses • Dissemination of any fungal agent, yeast, or bacteria-like fungus to involve
any tissue or organ
• Attack the deep tissues and organ systems
• often create symptoms that resemble other diseases.

Two categories of systemic disease:

1. 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 brasiliensis (Dimorphic Fungal Pathogens)
2. Opportunistic fungi, low virulence organisms, require the patient's defenses
to be lowered before the infection is established.
• Aspergillus sp., Candida albicans, Cryptococcus neoformans

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Dimorphic and
opportunistic
fungi

Dimorphic Systemic • Fungal infections caused by dimorphic fungal pathogens

Mycoses ‒ can overcome the host physiological and cellular defenses by changing form
• Example: Histoplasmosis
• Distribution: worldwide
• Agent: Histoplasma capsulatum
‒ from chicken and bats excreta

Opportunistic Systemic • occur in patients whose normal defense mechanisms is impaired

Mycoses • Mycoses of different fungus can occur especially in AIDS patients
• Among the infections include:
‒ Oral candidiasis
‒ oesophageal candidiasis
‒ vaginal candidiasis
‒ Cryptococcosis
‒ histoplasmosis
‒ Dermatophytosis

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Oral and Oesophageal candidiasis (thrush)

Cryptococcus

Candidiasis • Candida albicans is a normal gut flora.

• Normal flora of the gut and mouth of a healthy person
• It become pathogenic in immunocompromised person
• eg HIV positive, AIDS patient, diabetic, antibiotic therapy
Candidiasis in women
• white discharge (like cottage cheese), which doesn't usually smell
• itching and irritation around the vagina
• soreness and stinging during sex or urination
Candidiasis in men
• irritation, burning and redness around the head of the penis and
under the foreskin
• a white discharge (like cottage cheese)
• an unpleasant smell
• difficulty pulling back the foreskin
• Candidiasis can affect other areas of skin, such as the armpits, groin and
between the fingers. This usually causes a red, itchy or painful rash that scales
over with white or yellow discharge. The rash may not be so obvious on darker
skin.
Cryptococcus • C. neoformans is a fungus that lives in the environment throughout the world
neoformans • Infection can occur after breathing in the microscopic fungus
• Infection with the fungus Cryptococcus (either C. neoformans or C. gattii) is
called cryptococcosis
o Sporadic infection
oCan be transmitted by inhaling spores from pigeon faecal matter
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o Meningitis form is fatal

Cryptococcus • species comprises two varieties:

neoformans o C. neoformans var. grubii (serotype A)
• worldwide distribution
o C. neoformans var. neoformans (serotype D)
• more restricted distribution with infections being more prevalent in Europe
• Cryptococcosis usually affects the lungs or the central nervous system but can
also affect other parts of the body
• Brain infection due to Cryptococcus are called cryptococcal meningitis
• infections are rare in healthy individuals people
• most cases of C. neoformans infection occur in people with weakened immune
systems, particularly those who have advanced HIV/AIDS

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Aspergillus spp. • an ubiquitous, soil-dwelling, filamentous fungus that grows on soil, food,
dead leaves, household dust, etc
o Grows best at 37°C, & the small spores are easily inhaled & deposited
deep in the lungs
• The most common pathogens are Aspergillus fumigatus, A. flavus, A. niger &
A. terreus
• Aspergillus fumigatus is the most frequent cause of invasive fungal infection in
immunosuppressed individuals
• In immunocompetent persons, aspergillosis can occur in previously damaged
tissue or induce allergic responses
• in immunocompromised individuals, it is extremely invasive
o septate acutely branching hyphae in nearly every tissue

Aspergillosis • a disease caused by Aspergillus, a common mold (a type of fungus) found

indoors and outdoors
• Most people breathe in Aspergillus spores every day without getting sick
• However, people with weakened immune systems or lung diseases are at a
higher risk of developing health problems due to Aspergillus
• Allergic aspergillosis that doesn’t cause infection include
o Allergic bronchopulmonary aspergillosis (ABPA)
o Allergic Aspergillus sinusitis
• Chronic pulmonary aspergillosis is a long-term (> 3 months) condition in
which Aspergillus can cause cavities in the lungs
• Invasive aspergillosis is a serious infection affecting people who have
weakened immune systems eg people who have had an organ or a stem cell
transplant
• Cutaneous (skin) aspergillosis causes infection in people with weakened
immune systems where Aspergillus enters the body through a break in the skin
(eg after surgery or a burn wound)
Histoplasma capsulatum • Agent of Histoplasmosis
o an infection of the reticuloendothelial system resulting in patchy
bronchopneumonia containing yeast-laden phagocytic cells within
alveolar spaces
o The yeast cells multiply within the giant cells produced and can
disseminate to other tissues.
o T cells & activated macrophage are important in histoplasmosis
immunity
• worldwide distribution but endemic in parts of the US
• Strong association with bird and bat droppings
• a severe biohazard, Class II Biological Safety Cabinet (BSCII) must be used

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Pneumocystis jirovecii • Causes a serious infection called pneumocystis pneumonia

(PCP)
• symptoms include fever, cough, difficulty breathing, chest pain,
chills and fatigue
• Most people who get PCP have a weakened immune system eg
HIV/AIDS patient or those who take medicines that lower the
body’s immune system
• PCP spreads from person to person through the air
• Some healthy adults can carry the Pneumocystis fungus in their
lungs without having symptoms
• PCP is diagnosed using a sample from a patient’s sputum or
lungs (either bronchoalveolar lavage or lung tissue)
• The patient’s sample can be examined under a microscope
• PCR can also be used to detect Pneumocystis DNA
• A blood test to detect β-D-glucan (a part of the cell wall of many different
types of fungi) can also help diagnose PCP
Fungal diseases

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Collection of fungal • Skin - cleaned with 70% alcohol to remove dirt, oil and surface saprophytes
specimen • Nails - cleaned same as for skin. Usually clipped; need to be finely minced
before innoculating 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
Laboratory diagnosis • In the past, only a positive culture could truly define a fungal infection
• Currently, confirmation is also possible with serological and molecular (eg
PCR) testing
• Lab tests include:
- Antigen testing
- Antibody testing
- Fungal Culture
- Histopathology
- Microscopy of fungi
- Molecular mycology
- Beta 1,3-D-glucan
Key factors influencing • Getting a high quality sample
good laboratory results o eg nail and respiratory samples are not large enough
o saliva is submitted instead of sputum
• Testing before antifungal therapy is given
o Some tests are more likely to be negative if antifungals have been
given
• Blood culture volume
o eg at least 20mL of blood is needed for Candida bloodstream
infections
• Sample processing in the laboratory
o Cultures are more often positive when specific fungal media is used
o Temperature of cultures is a major factor in yield.
• False positive
o eg false positive Aspergillus antigen tests are possible with some
antibiotics and foods
Direct examination of • required on any biological material sent to lab for fungus culture
specimens o Look for spores, hyphae, mycelial elements, budding yeast, mycotic
granules
• Wet mount prep
o good for yeast
o examination is done in natural environment, so loss of fragile structure
is minimal
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• Potassium hydroxide (KOH) prep
o done on skin scrapings, hair, nails, sputum, vaginal specimens, etc
o KOH digests and clears the specimen’s tissue cells, mucous, etc., so
fungal elements can be seen
Microscopic evaluation Methods
• Teased Preparation
• Slide Culture Techniques - best as it gives undisturbed microscopic
morphology
• Transparent Tape Preparation
Observations
• Hyphae structure. Hyphae (plural); hypha (singular)
o Septate vs. non-septate (aseptate)
o Dematiaceous vs. hyaline
• Spore bearing structures
• Spores - Many terms addressing reproduction
Stains • 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 certainpolysaccharide in the cell
walls of fungi. Fungi stain pink-red with blue nuclei.
• Gomori Methenamine Silver (GMS) Stain - silver nitrate outlines fungi in
black due to the silver precipitating on the fungi cell wall. The internal parts of
hyphae are deep rose to black, and the
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.
• Fluorescent Antibody Stain - simple, sensitive, and extremely
specific method of detecting fungi in tissues or fluids. Applications
for many different fungal organisms.
• Papanicolaou Stain - good for initial differentiation of dimorphic
fungi. Works well on sputum smears.
• Gram Stain - generally fungi are gram positive; Actinomyces and
Nocardia are gram variable.
• Modified Acid-Fast Stain - used to differentiate the acid-fast
Nocardia from other aerobic Actinomyces.
• Giemsa Stain - used for blood and bone marrow specimens.
Histoplasma capsulatum is an intracellular organism, which
appears as small oval to pear-shaped yeast-like cells with crescent
shaped red-stained protoplasm surrounded by clear halo in
segmented neutrophils.
• India Ink - demonstrates the capsule of Cryptococcus neoformans
in CSF specimens
• Calcofluor white stain – useful for diagnosing onychomycosis, use
fluorescence microscope

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Onychomycosis / tinea
unguium most commonly
caused by Trichophyton
rubrum

Fungal growth • Temperature - ambient temperature (25-30oC ) for most fungi.

requirements oNocardia sp. and some dimorphic organisms grow best at 37oC.
oAny fungus capable of growing at 37oC, should be considered potentially
pathogenic.
• Atmosphere - True fungi are aerobic; there are a few anaerobes
among the bacteria-like fungi.
• Time - Some yeasts grow overnight. Saprophytes are fast growers
(several days). Generally cultures are held at least 4 weeks

Media • Sabouraud's dextrose agar (Sab-Dex/SDA) - classic medium, used to

cultivate dermatophytes and other types of fungi
• Sabouraud's dextrose agar with chloramphenicol - chloramphenicol
inhibits bacterial growth.
• Brain heart infusion slant (BHI) - more enriched than Sab-Dex. Used in
recovery of H. capsulatum.
• Mycosel agar - commercially produced agar containing chloramphenicol to
inhibit bacterial growth, and cycloheximide to inhibit saprophytic fungi and
some yeasts (including C. neoformans).
• Bird Seed Agar - a selective and differential medium for the isolation of
Cryptococcus neoformans from clinical specimens
• Potato-dextrose agar (PDA) and Corn-meal agar - are used in slide
cultures; as they induce spore formation, which greatly aids in identification.

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Colony Morphology • Surface topography - Some fungal colonies may be free growing, covering
(macroscopic features) the entire surface of agar in a particular manner; others grow in a restricted
manner.
• Surface texture - examples: cottony or wooly (floccose), granular, chalky,
velvety, powdery, silky, glabrous (smooth, creamy), waxy, etc.
• 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). The pigment can be diffused into the agar. It is
important to note the top pigment (obverse) and the discoloration of the agar
medium (reverse).
• Mycelium a. Vegetative mycelium - provides nutrition b. Aerial mycelium -
reproductive
Treatment of Mycoses • Nystatin
o used to treat Candida infections of the skin including diaper rash,
thrush, esophageal candidiasis, and vaginal yeast infections
o may also be used to prevent candidiasis in those who are at high risk
o may be used by mouth (orally), in the vagina, or applied to the skin
• Natamycin (Pimaricin)
o used to treat fungal infections around the eye including eyelids,
conjunctiva, and cornea
o used as eyedrops
• Amphotericin B
o polyene class of medication
o works in part by interfering with the cell membrane of the fungus
o used to treat most fungal infections especially dermatophyte
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infections such as aspergillosis, blastomycosis, candidiasis,
coccidioidomycosis, and cryptococcosis
• Flucytosine (5-fluorocytosine)
o effective against serious Cryptococcus and Candida infection
especially used together with amphotericin B
• Imidazoles eg miconazole
o used to treat ring worm, pityriasis versicolor, and yeast infections of
the skin or vagina
o used for ring worm of the body, groin (jock itch), and feet (athlete's
foot)
o applied to the skin or vagina as a cream or ointment
o oral form is also available

~End😊

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