Mycology: The study of fungi

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MYCOLOGY
Mycology The study of fungi Mycoses diseases
caused by fungi
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FUNGUS

• Widely distributed in nature (air, water, soil,
  decaying organic debris)
• 400,000 types
• Eukaryotic, highly developed cellular structure
• Facultatively anaerobic/strict aerobic
• Nonphotosynthetic

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Feature Fungi Bacteria
Size 4 µm (smallest) 1 µm Nucleus Yes
eukaryotes No prokaryotes Organelles Yes No
Memb. sterols Yes No (ex.
Mycoplasma) Cell wall content
Chitin Peptidoglycan Glucan Muramic
acid Mannan Teichoic acid Dimorphism
Yes No
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Single cells reproduce by budding.
Yeasts
Dimorphic fungi
Fungi
Moulds
Long filaments (hyphae) or a mat (mycelium)
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YEAST

• Unicellular
• Microscopic
• Oval to round (Dia 3-15 µm)
• Reproduce by budding
• Macroscopic
• Pasty colonies (resemble bacteria)

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MOULD

• Multicellular
• Microscopic
• Hyphae (dia 2-10 µm)
• Spores
• Macroscopic
• Surface texture
• Cottony/wooly/ velvety/ granular.
• Pigmentation

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Classification of Hyphae

• BASED ON
• Existence of septa
• - Septate
• - Nonseptate
• Shape and Morphology
• Racquet Spiral
• Nodular Root-like (rhizoid)
• Pectinate Chandler

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DIMORPHIC

• Capable of growing in mould or yeast form under
  different environmental conditions (temperature,
  CO2, nutrients)

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Subcellular Structure
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Subcellular Structure of Fungi

• Capsule (present only in some fungi)
• Cell wall
• Cell membrane
• Cytoplasm
• Nucleus, nuclear membrane, nucleolus, ER,
  mitochondria, vacuoles

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CAPSULE

• Structure Polysaccharide
• Functions
• Antiphagocytic
• Virulence factor
• Exist only in some fungi
• Cryptococcus neoformans (encapsulated yeast)

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CELL WALL

• Antigenic in nature
• Structure Multilayered
• polysaccharides (90) hexose and hexosamine
  polymers
• proteins and glycoproteins (10)
• Functions
• Provides shape, rigidity, strength and
  protection from osmotic shock

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Major polysaccharides of fungal cell wall

• POLYMER MONOMER
• Chitin N-acetyl glucosamine
• Chitosan D-Glucosamine
• Cellulose D-Glucose
• ?-Glucan D-Glucose
• ?-Glucan D-Glucose
• Mannan D-Mannose
• The type and amount of the polysaccharide vary
  from one fungal species to other.

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CELLULAR MEMBRANE

• Structure bilayered
• Phospholipids
• Sterols (ergosterol, zymosterol)
• Functions
• Protects cytoplasm
• regulates the intake and secretion of solutes
• facilitates capsule and cell wall synthesis

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FUNGAL SPORES
Spores function in reproduction of fungi. 1.
Sexual reproduction --Sexual spores 2. Asexual
reproduction--Asexual spores 3. Parasexual
reproduction--Genetic exchange
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LABORATORY DIAGNOSIS

• Direct microscopic examination
• Gram stain
• potassium hydroxide (KOH)
• calcofluor white, India ink
• Culture
• Sabouraud dextrose agar
• Mycobiotic agar
• Serology

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Fungal cell
Cell membrane and cell wall
Mannoproteins
b-(1,6)-glucan b-(1,3)-glucan
Chitin
Phospholipid bilayer of cell membrane
b-(1,3)-glucan synthase
Ergosterol
Ergosterol Synthesis Pathway
DNA/RNA Synthesis
Squalene
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Amphotericin B
Ergosterol
Cell membrane
Ca
Binding to ergosterol, Intercalation of cell
membrane
Ca
Na
Na
K
K
Leakage of intracellular cations and proteins
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Azole
Cell membrane
Ergosterol
Ergosterol Synthesis Pathway
Squalene
Accumulation of toxic sterols in cell membrane
Toxic sterols
Inhibition of 14-alpha-demethylase
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ß(1,3) glucan synthase
glucan synthase inhibitor
Depletion of ß(1,3) glucans in cell wall
Inhibition of ß(1,3) glucan synthase
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Cytosine permease
Cytosine deaminase
Phosphorylation
FdUMP
FdUMP
Conversion to deoxynucleosides
FUTP
Substitution for uracil
Inhibition of Protein Synthesis
Inhibition of thymidylate synthase
Inhibition of DNA synthesis
5-FC, 5-fluorocytosine 5-FU, 5-fluorouracil
FdUMP, 5-fluorodeoxyuridine FUMP,
5-fluorouridine monophosphate FUDP,
5-fluorouridine diphosphate FUTP,
5-fluorouridine triphosphate dUMP, deoxyuridine
monophosphate dTMP, deoxythymidine monophosphate
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ANTIFUNGAL DRUGS

• Membrane disrupting agents
• Amphotericin B, nystatin
• Ergosterol synthesis inhibitors
• Azoles, allylamines, morpholine
• Nucleic acid inhibitor
• Flucytosine
• Anti-mitotic (spindle disruption)
• Griseofulvin

• Glucan synthesis
• inhibitors
• Echinocandins
• Chitin synthesis
• inhibitor
• Nikkomycin
• Protein synthesis inhibitors
• Sordarins, azasordarins

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FUNGI

• Most of fungi are ubiquitous in nature.
• Exception Candida part of endogenous flora
• Mouth
• GI tract
• Skin, etc.
• Opportunistic pathogens can cause infections
  only in patients with immunodeficiency or
  debilitated patients.
• Endemic mycoses often causes diseases in
  immunocompetent hosts within endemic areas.

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Systemic (deep) mycoses
The Usual Infection Pattern Saprophytic fungi
in soil or bird droppings
Inhalation of spores
Lungs
Spread
Other tissues
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FUNGI

• Yeasts
• Candida sp.
• Cryptococcus neoformans
• Moulds
• Aspergillus sp
• Rhizopus sp (agents causing mucormycoses)
• Fusarium sp
• Many others
• Dimorphic
• Histoplasma
• Blastomyces
• Coccidioides
• Sporothrix

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TRUE SYSTEMIC (ENDEMIC) MYCOSES

• Coccidioidomycosis
• Histoplasmosis
• Blastomycosis
• Paracoccidioidomycosis

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TRUE SYSTEMIC MYCOSES General features

• Causative agents thermally dimorphic fungi that
  exist in nature, soil
• Geographic distribution varies
• Inhalation ?pulmonary infection ? dissemination
• No evidence of transmission among humans or
  animals
• Otherwise healthy individuals are infected

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Systemic fungal infections are uncommon

• Infection requires a large inoculum and a
  susceptible host infection often occurs in
  endemic areas most infections are asymptomatic
  or self-limiting in immune-compromised hosts,
  infections are more often fatal

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Systemic fungal infections are uncommon

• Systemic fungal disease is most often associated
  with four organisms
• 1. Coccidioides immitis
• 2. Histoplasma capsulatum
• Blastomyces dermatitidis
• Paracoccidioides brasiliensis (S. America)

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COCCIDIOIDOMYCOSIS

• Etio Coccidioides immitis
• Location Confined to southwestern US, northern
  Mexico, Central and South America
• Microbiology
• Tissue (37C) Spherules filled with endospores
• 25C hyphae, barrel-shaped arthroconidia

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Coccidioidomycosis

• Coccidiodes immitis is considered to be the most
  virulent of fungal pathogens.
• Restricted to hot, semi-arid areas of SW USA and
  Mexico.
• Grows in the soil, but inhalation of a single
  spore can initiate infection.

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COCCIDIOIDOMYCOSISPathogenesis

• Inhalation of the infectious particle,
  arthroconidia and spherule formation in vivo
• Engulfment within phagosomes by alveolar
  macrophages
• Activation of macrophages ---phagosome-lysosome
  fusion ---killing
• Immune complex formation
• deposition leading to local inflammatory
  reactions
• immunosuppression resulting from the binding of
  complexes to cells bearing Fc receptors

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COCCIDIOIDOMYCOSIS Clinical findings

• PRIMARY INFECTION -
• Asymptomatic in most
• Fever, chest pain, cough, weight loss
• Nodular lesions in lungs
• SECONDARY (DISSEMINATED) INFECTION (1)
• Chronic / fulminant
• Infection of lungs, meninges, bones and skin

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Coccidioidomycosis
Conidia
In infected tissues, C. immitis appears as a
mixture of endospores and spherules.
Spherules
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COCCIDIOIDOMYCOSIS Diagnosis

• Histopathology
• spherules or endospores seen in sputum, exudates
  or tissue
• Culture danger, highly infectious!
• SDA Mould colonies at 25 C
• Spherule production in vitro by incubation in an
  enriched medium at 40C, 20 CO2
• Serology
• Complement fixation assay (in cerebrospinal
  fluid), particle agglutination assay
• Skin test (coccidioidin and spheruline antigens)
  - Negative result may rule out the diagnosis

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COCCIDIOIDOMYCOSIS Treatment

• Symptomatic treatment only (primary infection)
• Effective antifungal agents
• Amphotericin B
• Itraconazole
• Fluconazole(particularly for meningitis)

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HISTOPLASMOSIS

• Etio Histoplasma capsulatum
• Natural reservoir soil, bat and avian habitats
• Location May be prevalent all over the world,
  but the incidence varies widely (most endemic in
  Ohio, Mississipi, Kentucky)
• Micr. Yeast cell in tissue (37C)
• Hyphae, microconidia and macroconidia
  (tuberculate chlamydospore) at 25 C
  

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Histoplasmosis

• Inhalation of conidia from the environment is
  source of infection.
• This is more likely in endemic areas
• the Atlantic Ocean to N. Dakota (500,000
  cases/year in U.S.), except New England
  Florida.
• Most cases occur in Ohio Valley and Mississippi
  Valley)

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HISTOPLASMOSISPathogenesis

• Inhalation of microconidia / primary cutaneous
  inoculation
• Conversion to budding yeast cells
• Phagocytosis by alveolar macrophages
• Restriction of growth or dissemination to RES by
  bloodstream
• Suppression of cell-mediated immunity

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HISTOPLASMOSISPathogenesis

• Immune response
• Cell-mediated responses are of primary importance
• Phagocytic activity of macrophage is considered
  an important component of resistance to drugs.
• Activated macrophage can kill yeast cells
• Evasion of host defense
• Survival in macrophageselevates pH of
  phagosomes
• Yeast cells absorb iron (siderophore) and calcium
  from host
• Alteration of cell surface

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HISTOPLASMOSISClinical findings

• PULMONARY INFECTION
• Asymptomatic (95)
• mild / moderate / severe/ chronic cavitary
• DISSEMINATED INFECTION (1/200)
• RES (liver, spleen, lymph nodes, bone marrow)
• mucocutaneous infection
• PRIMARY CUTANEOUS INFECTION -

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HISTOPLASMOSIS Diagnosis

• Histology
• Culture of blood or bone marrow
• Serology
• Serological testing for antibody and histoplama
  antigen in blood and urine.
• Antigen
• In HIV-infected patients with disseminated
  histoplasmosis, histoplasma antigen detection in
  serum and urine is at least 50, and 90
  sensitive, respectively.

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HISTOPLASMOSISTreatment

• Not required for several cases
• Effective antifungal
• Amphotericin B
• Itraconazole
• Surgical resection of pulmonary lesions

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BLASTOMYCOSIS

• Etio Blastomyces dermatitidis
• Location America, Africa, Asia
• Microbiology
• Yeasts at 37C--bud is attached to the parent
  cell by a broad base
• Hyphae and conidia at 25 C

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Blastomyces dermatitidis

• Most cases are in southern, central, and
  southeastern USA. Infection is by inhalation of
  spores.
• Risk Factors
• Occupational contact with soil
• owning a dog.
• Living in endemic area.

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Blastomycosis

• Granulomatous mycotic infection
• lungs
• Skin
• but can spread to other organs.

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BLASTOMYCOSISPathogenesis

• Inhalation of infectious particles
• Primary cutaneous inoculation
• Infiltration of macrophages and neutrophils and
  granuloma formation
• Oxidative killing mechanisms of neutrophils and
  fungicidal activity of macrophages

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BLASTOMYCOSISPathogenesis

• Defense system
• Alveolar macrophage provide a modest first line
  of defense.
• T-cell stimulated PMNs kill Blastomyces cells by
  oxidative mechanisms.
• Conidia are more sensitive to killing by PMNs
  because yeast are too big.
• TH-1 response of primary importance
• Evasion of Defenses
• Escapes phagocytosis by neutrophils and monocytes
  by shedding its surface antigen after infection
• .

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BLASTOMYCOSIS Clinical findings

• ASYMPTOMATIC INFECTION
• PRIMARY CUTANEOUS INFECTION
• PULMONARY INFECTION
• CHRONIC CUTANEOUS INF.
• Subcutaneous nodule, ulceration
• DISSEMINATED INF.
• Skin, bone, GUT, CNS, spleen

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BLASTOMYCOSIS Diagnosis

• Direct microscopic exam- KOH, HE
• Culture
• Serology
• Immunodiffusion test
• ELISA to detect antibodies to exoantigen A
• Skin test (Blastomycin antigen) Limited/no
  diagnostic value

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BLASTOMYCOSIS Treatment

• Amphotericin B
• Itraconazole
• Fluconazole
• Corrective surgery