Fungal Agents Structure And Biology

Front 1

to be considered a potential pathogen what is the requirement for a fungus?

Back 1

growth capability at 37degrees

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Aflatoxicoses

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casused by aflatoxins fo aspergillus flavus

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Ergotism

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A vasoconstricting disease caused by ergot alkaloids
Mycotoxin produced by claviceps species
Hallucinations, vomiting, diarrhea, convulsions, gangrene and death in animals and humans

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Claviceps mycotoxin

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A vasoconstricting disease caused by ergot alkaloids
Mycotoxin produced by claviceps species
Ergotism
Hallucinations, vomiting, diarrhea, convulsions, gangrene and death in animals and humans

Front 5

Which group of mycoses is communicable?

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derpatophyte group
ringworm

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Candida opportunistic infections

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Chelitis
thrush
systemic candidiasis

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Fungal cell membrane target for chemotherapeutics

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Ergosterol

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Cell wall composition

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Cell walls are rigid and composed of chitin, glucan, mannan

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how do you identify mold?

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Molds-multicellular
–Do not possess chlorophyll (no photosynthesis)
–Can be identified based on their reproductive components

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Yeast Identify

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Oval shaped fungal cell that reproduces through budding

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Fungal cell wall molecules

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mannan
Chitin
PLM
B-glucan

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Mannan

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–Glycoprotein that is the a large percentage of the cell wall mass
–Used in diganostic tests for Candida infections

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PLM

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Glycolipid linked with fungal survival in macrophages

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Chitin

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Polysaccharide that gives rigidity to the cell wall

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B-glucan

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Polysaccharide of the cell wall
–Serves as a Pathogen Associated Molecular Pattern (PAMP) for fungal species

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Lipid bilayer main component

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ergosterol
–Very closely related in structure to cholesterol
–Target of polyene antifungals and azole antifungals

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Yeast structures

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germ tube, pseudohypha

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Mold structures

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mycelium hypha septum sporangiophore sporangium sporangiospore conidiophore conidia

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Hyphae

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Filamentous, cylindrical, branching
cells that are usually aerial in culture
and environmental sources

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Mycelium

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Mass of hyphae

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Chlamydoconidia

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Chlamydoconidia
–Results from swollen hyphae
–Exhibits a thick spore wall to protect the essential fungal elements
–Seen in Blastomyces

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micro/macroconidium

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Microconidium
–Unicellular
–Trichophyton
•Macroconidium
–Multicellular
–Microsporum

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Pseudohyphae

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Pseudohyphae form when blastoconidia fail to form complete daughter cells
–A result of inefficient cell division
–Have increased pathogenicity when compared to yeast cells

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Germ tubes

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Formed by Candida species
•Resemble pseudohyphae, but are biochemically differentiated appendages to function in invasiveness of the fungus
•Result from a variety of host organism signals
•Highly express:
–Adhesins-to allow Candida to adhere to host tissues
–Secreted Aspartyl Proteases (SAPs) to function in local tissue structure and allow the organism to invade

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Germ tubes highly express

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Highly express:
–Adhesins-to allow Candida to adhere to host tissues
–Secreted Aspartyl Proteases (SAPs) to function in local tissue structure and allow the organism to invade

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Germ tube adhesins

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Adhesins-to allow Candida to adhere to host tissues

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SAPs

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Germ tube secretion:
Secreted Aspartyl Proteases (SAPs) to function in local tissue structure and allow the organism to invade

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Mycoses Primary pathogenesis

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Found in very specific regions of the world (endemic areas)
•Can cause disease in any individual
•Frequently see granulation if organism is not cleared by the immune system (see following slides)
–Recurrent infections, chronic infections
•Infection is initiated when aerosolized conidia are inhaled or introduced to the host
–Blastomyces dermatitidis, Histoplasma capsulatum, Coccidioides spp, and Paracoccidioides brasiliensis

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Opportunistic mycoses

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Opportunistic Pathogens
•Typically found worldwide and can exist in any environment
•Can be a commensal organism of humans
•Require immunocompromised status of the host

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Determinants of pathogenicity (5)

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Thermotolerance
Adaptation to parasytic lifestyle
Specific features (adhesins, Saps, capsule)
Evasion of host defenses
Dimporphism

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Thermotolerance and pathogenicity

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Ability to grow at 37oC is a key feature to fungal pathogenicity-

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Specific Pathogenic features

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Specific features:
–Adhesins
–Products that attack host tissues
•Secreted Asparyl Proteases (SAPs)-destroy host tissue at the site of the infection
–Cell Wall molecules/Capsules
•Block the entrance of antifungals to the cell and assist in evading the host immune system (inhibit phagocytosis)
•Capsule found in Cryptococcus species

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Pathogenicity: Evasion of host defense

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Evasion of Host Defenses
–Organisms such as Histoplasma capsulatum can survive in the phagosome and block formation of the phagolysosome. This seerves to inhibit phagocystosis of the fungus.

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Dimorphism

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•Dimorphism
–Depending on growth conditions, some fungi are capable of growing as either a yeast or a mold
–This ability typically provides an advantage for the survival of the organism
•Example:
–Germ tubes of Candida albicans
–Exist as a yeast at 25oC, at 37oC, the cell develops germ tubes (hyphae)

Front 35

Which species are obligate aerobes?

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Most species:
take NOTE of the ones that aren't if you find any.

Front 36

Methods of fungus direct detection

Back 36

wet mount
KOH prep
Fluorecent brighteners
Stains
Culture Isolation

Front 37

Fungal stains

Back 37

gram
india Ink
PAS
Mucicarmine Staining

Front 38

Mucicarmine Stain

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–Mucicarmine staining
•Stains nuclei black, host tissue yellow and fungal capsular polysaccharides pink
•Very useful for identification for Cryptococcosis.

Front 39

PAS

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Periodic acid Schiff staining (PAS) is used for visualizing tissue invasion by fungi
•Stains fungal cell walls red/pink

Front 40

Fungal Gram Stain

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–Gram stains-although fungi do not possess peptidoglycan in their cell wall, Gram stain results can be helpful in diagnosis

Front 41

India Ink Stain

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India ink-a negative stain that will be excluded from encapsulated organisms (Cryptococcus spp)

Front 42

Fungus and immunohistochemical tests

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Immunohistochemical tests are effective, but not many options readily available

Front 43

lab: mold vs yeasts

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Yeasts can be handled on the bench, but molds should be manipulated in biosafety cabinets

Front 44

Lab: isolation

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Sabouraud and potato dextrose agars are common isolation media
–These do not support bacterial growth
•After recovery of the fungus in pure culture, the following tests are performed:
•Culture morphology
•Sporogenesis tests
•Biochemical tests
–Carbohydrate assimilation tests
•Many molds take weeks to grow in culture (contamination, treatment)
•Serological tests and nucleic acid approaches are gaining in prominence for diagnosis of fungal infections

Front 45

Four Antifungal approaches

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Prophylactic
Preemptive
Empiric
Definitive

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Prophylactic therapy

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Prophylactic therapy-used to prevent infections in high risk patients
-Fluconazole (lifelong) for HIV positive patients

Front 47

Preemptive therapy

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Preemptive therapy-used in patients who have a positive diagnostic test result, but have yet to manifest disease
-Development of non-culture based tests has made this approach more readily used

Front 48

empiric therapy

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Empiric therapy-used in patients who exhibit signs and symptoms suggestive of a fungal infection, with no definite diagnosis

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Definitive therapy

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Definitive therapy-treatment of a defined disease.
-Antifungal susceptibility test results are in hand

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Treatment of fungal infection is influenced by:

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•Ability to define the pathogen (and in a timely manner)
•Immune status of the patient
–May lead to more empiric therapies in fungal cases

Front 51

Antifungal targets

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Target fungal processes:
–DNA synthesis
–Protein synthesis
–Spindle pole formation (cell division)
–Cell wall assembly
–Cell membrane function (MOST antifungals target ergosterol in the cell membrane)

Front 52

Antifungal: Natural Products

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Polyenes
Echonocandins

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Polyenes

Back 53

Polyenes
•Amphotericin B, Nystatin
•These target embedded ergosterol in the fungal cell membrane
•Result in the distortion of the cell membrane and dysregulation of cell membrane function

Front 54

Echinocandins

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Echinocandins
•Echinocandin B, caspofungin
•Target the fungal cell wall by inhibiting the formation of beta-glucan molecules
•Work very well against invasive candidiases and aspergilloses
•Little toxicity, but very expensive

Front 55

Antifungals: chemically synthesized compounds

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Terbinafine
Azoles

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Terbinafine

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Terbinafine
•Allylamine drug that blocks ergosterol synthesis in dermatophytes

Front 57

Azoles

Back 57

Azoles
•First Generation
–Miconazole, fluconazole, itraconazole
•Second Generation
–Have increased activity against molds
–Voriconazole, Posaconazole,and ravuconazole (experimental)

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Aspergillus

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Candida invading lung

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chlamydoconidia

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conidia

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Conidophores

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Germ tubes

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Hyphae septa

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micro macroconidia

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pseudohyphae

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pseudohyphae

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sporangium

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Systemic Coccidiomycosis- Coccidioides immitis

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Valley Fever

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Valley Fever