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3 Domains of Life:
- Bacteria
- Archaea
- Eukarya - animals, fungi, ...
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Fungus (pl. fungi)
- eukaryotic
- unicellular to filamentous
- achlorophyllous
- absorptive nutrition
- reproduction is sexual, asexual, or both
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Significance of fungi
- Decomposition of organic matter
- Antibacterial agents, pharmaceuticals, food production, ecology of plant growth
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Drugs
- antibacterials - Penicillin and Cephalosporins
- antifungal agents
- Ergotamine (St. Anthony’s Fire)-facilitate delivery and/or relieve migraines
- cortisone and prednisone - Steroids in “the pill”
- Lovastatin (mevacor) - lowers cholesterol
- Compactin (mecastatin) - lowers cholesterol and treats heart disease and atherosclerosis
- Cyclosporin - immunosuppression
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Food
- Saccharomyces cerevisiae -brewer’s yeast, bread yeast
- Riboflavin (vitamin B2) - used in enriched flour
- Edible mushrooms
- Ripening of cheeses
- Citric acid for soda
- Wine production
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Enhanced plant growth
- Mycorrhizae - the association between fungi and plant roots
- 90% of plants species in nature have a mychorrizal association with a fungus
- The fungus receives sugars from the plant’s photosynthesis and provides the plant with increased
- absorption of water and mineral nutrients
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When pathogenic, fungi are primarily pathogens of _____ and less frequently of _______.
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Fungi were the first microbial agents to be recognized to cause disease.
- 1835: Bassi, silkworms affected by fungus, first recognized microorganism agent of an contagious animal disease
- 1839/1841: Schonlein/Gruby, fungal, transmissible human disease - Favus
- 1882: Koch discovered the “tubercle bacillus”
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Basic characteristics of the fungi
- Eukaryotic w/ true nucleus in a nuclear membrane, Difficult to find selective therapeutic agents to w/o harming the host
- nearly every ecological niche
- Thousands of known species
- Aerobic or facultative anaerobic
- chemoheterotrophs, never photosynthetic
- Most are decomposers
- Have a cell wall
- Ergosterol in the cell membrane
- Sexual and asexual cells, can differentiate into a spore producing cells
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Cell wall
- Animals - None
- Plants - Cellulose (glucose polymer)
- Bacteria - Peptidoglycan
- Fungi - Chitin
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Fungi have a cell wall
- Similar functions as that of bacteria, protection, structural stiffness, ...
- Represents a large part of the cell mass - 15-30%
- costs a large amount of energy in its synthesis
- All medically important fungi have a cell wall - Potential drug/therapeutic target
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Composition of Fungal cell wall
- Chitin: β(1,4) linked homopolymer of N-acetylglucosamine
- beta (1-3) glucans: Remove it, put the cell in water, and get lysis. Can be theraputic target.
- beta (1-6) glucans: cross-linking of glycoproteins occurs through the carbohydrate remnant of a GPI anchor and the b(1-6) glucan.
- Mannoproteins: N and O-linked glycoproteins with 15-90% mannose; antigenic; Outside of cells.
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Surface glycoproteins (mannoproteins) functions
- 1. Adherence - to host cells and other surfaces
- 2. Protection - of underlying glucan layer
- 3. Antigenic - elicit host antibody response; pathogen-associated molecular pattern (PAMP)
- 4. Potential drug target
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Fungal cells contain _______ in the cell membrane.
ergosterol
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Ergosterol is used instead of ______ in most fungi (except Pneumocystis). This is a major target for antifungal drugs , eg. _______ and _______.
- cholesterol
- Amphotericin B
- azoles
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Cells differentiate into a ____ producing cells.
spore
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Five phyla/taxonomic groups based on ____:
- the type of sexual spore produced
- 1. Ascomycetes (Ascomycota) - ascospores
- 2. Basidiomycetes (Basidiomycota) - basidiospores
- 3. Zygomycetes (Zygomycota) - zygospores
- 4. Chytridiomycetes or chytrids (Chytridiomycota) - oospores
- 5. Fungi Imperfecti or Deuteromycetes (Deuteromycota) - sexual form is unknown
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Asexual form = Imperfect
mostly the only form as medical pathogens
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Fungal Nutrition
- Absorptive heterotrophs - small molecules or breakdown complex organic molecules
- Secondary metabolites produced - Toxins, antibiotics, nutrients for other organisms
- Acid tolerant - more acid tolerant (optimal pH <=5);
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three major types of growth structures of Fungi
- Mushrooms - fruiting body, Largest organism
- Molds
- Yeasts
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Yeasts
- Reproduce asexually by budding or by fission
- Reproduce sexually through fusion of 2 mating types
- Grow as single celled organisms
- Smaller than RBC, larger than bacterium
- Form smooth colonies
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Molds
- Multicellular
- Produce hyphae: spores germinate, bud -> multi cellular organism (hypha); branch; penetrate substrate
- Interconnecting network of hyphae = mycelium
- “furry” or textured
- mycology - Study of molds
- Infections caused by fungi - “mycosis” or “mycotic infections”
- Medically important molds - Aspergillus, Penicillium
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Variations of hyphae
- Septate hyphae
- Coenocytic hyphae
- Septate hyphae w/ clamp connections
- Hyphae w/ arthroconidia and disjunctor cells
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Molds produce asexual spores:
conidia
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Some species are able to take on two forms:
- Dimorphism
- Pathogenic dimorphic fungi:
- • yeast-like at 37C = body temperature
- • mold-like at 25C = environment
- C. albicans has the reverse temperatures
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Innate immunity to fungi
- Toll-like receptors recognize molecules on pathogens
- Barrier epithelium - GI and respiratory mucosa; contains defensins, calprotectin, and other cationic peptides
- PMNs (polymorphyonuclear leukocytes) - WBC, professional phagocytes; Engulf and kill the cells
- Inflammation - PMN and macrophages are responsible
- Saliva - histatins
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Histatins
- histidine-rich cationic peptides in the serous fluid from Von Ebner's glands
- 24-38 amino acids length
- 10 of them
- humans and higher primates
- Enter cytoplasm -> bind and disrupt mitochondria -> Induces rapid cellular K and Mg ion imbalance and cellular volume loss
- Non-lytic loss of ATP from actively respiring cells
- disrupt cell cycle
- generation of reactive oxygen species
- -> Cell death (by ruining energy source)
- Future therapy
- Caveat - C. albicans has protease capable of degrading
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Adaptive immunity to fungi - Cell-mediated immunity:
- major protective role
- Activation of phagocytes, antigen-specific cytotoxic T-lymphocytes, and release of various cytokines
- T-lymphocytes (CD4) specific for fungi
- Cytokines activate PMNs and macrophages
- TH1 (T-helper 1) immunity is the most important
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Adaptive immunity to fungi - Humoral Immunity:
- Minor to mixed role
- Antibody response relatively minor
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Antifungal agents
- Antifungal agents that act on metabolic pathways in fungi might be toxic to host cells
- Systemic vs topical agent
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Why do we see an increase in the frequency of fungal infections?
- NOT because of Acquisition of new virulence factors
- altered hosts - more immunocompromised
- selective antifungal pressure - drug resistance
- sudden environment change - more exposed
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Identification of fungal pathogen:
- Direct microscopic examination - cornerstone of diagnosis; characteristic structures
- Culture
- Serology
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Direct methods
- Most fungi do not stain well with Gram stain
- 1. Saline wet mount - hyphae
- 2. NaOH/KOH mount - remove interwined host material by hydrolyzing the lipid
- 3. India Ink - id fungi with a capsule (Cryptococcus)
- 4. Histological stains vs. cell wall carbohydrate - fluorescent stain calcofluor-white binds to cellulose and chitin
- 5. Periodic acid Schiff (PAS) - polysaccharide; magenta
- 6. Gomori methenamine silver - carbohydrates; black
- 7. Molecular methods - Primers specific for fungal rRNA combined with PCR and/or FISH techniques
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Periodic acid Schiff (PAS)
- acid converts Glycol groups to reactive aldehydes
- Schiff’s reagent colors them bright magenta
- Polysaccharides are very strongly stained
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Identification of fungi from cultures
- Plate to appropriate agar or liquid media
- Colony morphology and color
- Growth at 37C - Most environmental fungi don't grow
- Characteristics of spores and conidia
- Carbohydrate utilization profiles (yeasts)
- Periodic acid Schiff (PAS)
- Molecular methods - rRNA-primer PCR and/or FISH
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Serological tests
- Determination of serum antibody (Ab) and/or antigen (Ag) titers
- • monitor disease progression and response to therapy
- • histoplasmosis and coccidioidomycosis only
- • not sensitive and specific for invasive infections
- Detection of cell wall and cytoplasmic Ag and metabolites in serum or other body fluids for diagnosis of invasive fungal infection
- • for C. neoformans and H. capsulatum polysaccharide
- • rapid diagnosis of cryptococcal meningitis and disseminated histoplasmosis
- • Immunoassays for Aspergillus and Candida polysaccharides
- Detection of 1,3-β-glucan in the serum
- • Candida and Aspergillus
- • indicates the presence of fungi but does not identify the genus
- • promising in certain highly selective patient populations
- The detection of fungal metabolites (d-arabinol an d-mannitol)
- • Candidiasis and aspergillosis
- • Not commercially available, problematic
- Molecular methods
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Types of fungal infections
- 1. Allergic - Fungal spores in air -> allergic reaction
- 2. Toxic - Mycotoxins and mycotoxicoses
- 3. Mycoses
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Mycotoxicoses
- disease caused by Secondary metabolites produced from filamentous fungi, mycotoxin.
- Acute or chronic disease
- Acquired by ingestion, inhalation, or direct contact
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Aflatoxin
- produced by Aspergillus sp.
- Small, highly mutagenic molecule
- Found in peanuts and cereal grains
- Manifests as chronic and acute hepatitis, liver failure
- Causes hepatic cancer
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Ergot alkaloids
- produced by Claviceps sp.
- Found in rye and cereal grains
- Causes “Ergotism” - either a gangrenous form (peripheral vasoconstriction, necrosis of the distal extremities, edema, pruritus, muscle pain) or convulsive/neurologic (muscle spasms, seizures, and hallucinations)
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Classification of Mycoses
- Site of the infection - Superficial, cutaneous, subcutaneous, or systemic (deep) infections depending on the type and degree of tissue involvement and the host response to the pathogen
- Route of acquisition of the pathogen - exogenous vs endogenous. Endogenous involves colonization by a member of the normal flora or reactivation of a previous infection.
- Type of virulence exhibited by the fungus - Primary vs. Opportunistic
- Epidemiology - primary - well-defined geographic ranges; opportunistic - ubiquitous.
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Superficial mycoses
- Involves the keratinized outermost layers of skin, cuticle, or hair shaft
- Spread by direct contact with person/animal/soil
- Nondestructive, cosmetic
- Easy to treat
- not recognized by host defenses - elicit little or no host immune response
- Host can take care of the presence of fungi
- Pityriasis/Tinea infections
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causes of tinea infections - Pityriasis versicolor
- • Caused by Malassezia furfur (yeast)
- • Causes dandruff and dermatitis
- • Person to person transmission; In certain tropical environments, may affect up to 60% population
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causes of tinea infections - Tinea nigra
- • Hortaea werneckii (mold)
- • Causes dark brown patches on the palms of the hands and feet.
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causes of tinea infections - White Piedra
- • Caused by Trichosporon sp (fungi)
- • hair disease - white piedra (stone in Spanish) on
- hairs of the groin
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causes of tinea infections - Black Piedra
- • Caused by Piedraia hortae (fungi)
- • black piedra on hairs of the scalp
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causes of tinea infections - Otitis externa
- • Aspergillus, Malassezia sp (fungi)
- • external ear infected
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causes of tinea infections - Superficial dermatophytosis of the hair, nail and skin
• Caused by Arthroconidia and Trichophyton (mold)
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Cutaneous mycoses
- keratinized tissues of the epidermis, hair, and nails; do not invade living tissue
- keratinophilic and keratinolytic; utilize keratin to grow
- Dermatophytic and non-dermatophytic causes
- Spread by direct contact
- May invoke an inflammatory response; get redness
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causes of cutaneous infections - Dermatophytosis or Ringworm
- • Caused by Mycosporium, Trichophyton and Epidermaphyton (mold)
- • circular skin lesions with slightly raised, red margins, and surrounded by reddish, itching skin.
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causes of cutaneous infections - Favus
- • Trichophyton schoenleinii (mold)
- • First fungal disease described!
- • Chronic skin infection of the scalp
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causes of cutaneous infections - Onychomycosis
- • Trichophyton, Scopulariopsis (mold) and yeast Candida.
- • Dermatophytic and nondermatophytic causes
- • A chronic nail infection
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Subcutaneous mycoses
- Involves the dermis, subcutaneous tissue, muscle, and fascia
- Usually associated with traumatic injury - eg. puncture with a thorn, rubbing against a bush,
- etc and then introduce soil saprophytes fungi
- a low degree of infectivity
- Non- life-threatening
- Provokes an immune response and leading to cysts or
- granuloma
- Generally slow, chronic diseases
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causes of subcutaneous infections - Chromoblastomycosis
- • Fonsecaea, Cladophialophora, Phialophora , and Rhinocladiella (mold)
- • Localized, slowly expanding lesions - Superficial, warty to cauliflower-like tumors
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causes of subcutaneous infections - Sporotrichosis
- • Sporothrix schenckii (mold)
- • Skin infection, can be along lymphatic drainage
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Systemic mycoses
- Infections in the blood and internal organs
- Acquired from the respiratory tract or intestinal flora
- Pathogens are classified as either Primary or Opportunistic pathogen
- Both cause more frequent and severe infections in compromised hosts
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Primary fungal pathogens
- Blastomyces dermatitidis
- Coccidiodes immitis
- Histoplasma capsulatum
- Penicillium marneffei
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Blastomycosis
- Caused by Blastomyces dermatitidis
- Sporadic occurrences associated with recreational activities
- A high proportion of infected individuals have symptoms - Dogs are very susceptible
- Spores inhaled, germinate into pathogenic yeast phase cells in the lung
- Acute pulmonary infection, asymptomatic or influenza like
- Potential for pulmonary Blastomycosis - a granulomatous pulmonary infection
- May resolve spontaneously or disseminate through out the body
- There is a tropism for skin and bone
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Blastomyces dermatitidis pathogenesis
- • The surface protein Blastomyces Adhesin (Bad1) is required for virulence - homologous to invasin genes of G(-) bacteria; promotes macrophages to uptake the fungi, which promotes its dissemination
- • B. dermatitidis can survive in inactivated macrophages
- • Cell mediated immunity activates macrophages and leads to clearance and protective immunity
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Coccidiodomycosis
- Caused by Coccidiodes immitis
- Spherule is diagnostic
- Valley fever - non-specific fever with bronchopneumonia, mostly the disease resolves spontaneously; Dissemination is often fatal
- Route of infection inhalation
- Environmental form is highly infectious and even a hazard to lab workers
- US southwest
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Coccidiomycosis is a spectrum of manifestations
- Chronic skin diseases - Ulcers or abscesses
- Joint and bones - Severe synovitis and effusion of knees, wrists, feet, ankles, pelvis; Lytic lesions
- Meningitis, with possible hydrocephalus - Feared complication
- Potential for other organ involvement
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Histoplasmosis
- Caused by Histoplasma capsulatum w/ tuberculate conidia
- bird and bat droppings
- Infects monocytes or macrophages
- Spores are inhaled and go to the alveoli
- Asymptomatic - 50-90%
- Acute and symptomatic - remains localized
- - Self-limiting flu-like-unrecognized
- - Acute pulmonary - diffuse or localized, “buckshot”
- - Acute pericarditis
- - Rheumatic manifestations
- Chronic Pulmonary - TB-like on X-ray
- Disseminated - taken up by macrophages and survives in the bloodstream; show up at other sites; frequently fatal
- US center/east
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Penicilliosis
- Caused by Penicillium marneffei
- Inhalation
- The mold multiplies in the monocytes and macrophages.
- Prevalent in HIV-positive individuals
- Fatal if untreated
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Systemic mycoses caused by opportunistic pathogens
- Most important is Candida albicans
- People at risk are:
- • Those undergoing blood, marrow, or organ transplantation or major surgery
- • HIV and immunosuppressed patients
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Aspergillosis
- Caused by ubiquitous Aspergillus flavus or A. fumigatus.
- Aspergilli grow as branched, septate hyphae that produce conidial heads when exposed to air in culture and in tissue.
- Wide range of diseases
- • 90% of clinical deep organ/tissue mycotic infections
- • Allergic manifestation-asthma and pulmonary infection
- • Sinusitis
- High mortality rate for untreated invasive aspergillosis
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Mucormycosis
- aka zygomycosis
- Caused by Mucorales sp, Rhizopus sp, and others
- Infection of the sinuses, brain, or lungs
- Invasive growth in the walls and lumina of blood vessel causing blood clots
- Diabetics are at particular risk for invasive infection
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Pneumocystosis
- Caused by Pneumocystis jiroveci - previously thought to be a protozoan parasite
- Airborne person to person
- One of the major causes of opportunistic mycoses in
- immunocompromised
- Causes pneumonia
- Human form cannot be cultured!
- • Only direct microscopy to diagnose
- • Foamy eosinophils
- Linked to the “mysterious” new disease AIDS in the 1980s
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Cryptococcus neoformans
- the only one known Encapsulated yeast
- Uni-nucleated budding yeast
- Nearly always yeast form in both environment and host
- Capsule (antiphagocytic) and melanin (ROS protection) are important virulence factors
- Found in pigeon droppings or eucalyptus trees
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Cryptococcosis
- Inhaled transmission
- Invades tissues when immunity weakens
- Often leads to chronic meningitis which is the most common disease
- Usually sub-acute or chronic
- Can also involve the skin, lungs, prostate gland, urinary tract, eyes, myocardium, bones, and joints
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Candida albicans
- Dimorphic yeast - grow via budding or like a mold (hyphae)
- Can be seen as hyphae in tissue or 37C
- A commensal, natural member of the GI tract, commonly present on human mucosa
- Acquired at birth
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Candidiasis
- Caused by Candida albicans, or lesser by C. tropicalis and C. glabrata.
- Causes generally mild mucosal infections, vaginal, oral, and lung infections
- Becomes invasive when immunity weakens
- In immunocompromised HIV patients, organ transplant recipients, etc, C. albicans can get into blood -> systemic infections
- Disseminated Candidiasis untreated can be fatal
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Most common cutaneous mucosal infections - C. albicans (yeast)
- No necessarily caused by weakened immune system
- Intertrigo - infection of humid folds of the skin, Diaper Rash
- Vulvovaginitis - inflammation or infection of the vulva and vagina
- Thrush - yeast infection of the mucus membrane lining the mouth and tongue
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Factors Predisposing to Oral Candidiasis
- Xerostomia
- Denture wearing
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Denture Stomatitis
- Red mucous membranes - Infection caused by Candida cells forming biofilm on denture material
- Risk factors - Older age, malnutrition, iron deficiency high carbohydrate diet
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Virulence factors of C. albicans
- Hydrolytic enzymes:
- - Aspartyl proteinases (10 proteins)
- - Phospholipase B (or C?)
- - Lipases
- Virulence correlates with level protease activity, facilitating adherence to host tissue
- Cause tissue damage, facilitate tissue penetration, destroy immunoglobulins, involved in nutrient acquisition
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The transition from yeast to hyphal growth facilitates virulence of C. albicans
- • Hyphae penetrate deeper
- • Hyphae cause higher level of damage
- • Breakdown of tissue by hyphae may facilitate growth from hyphal septa thereby extending the area containing yeast
- • Epithelial cells from the mucosa appear to be stitched together by the hyphae
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Host mechanisms against C. albicans
- Non-specific resistance mechanisms
- • Barrier epithelium
- • Contain defensins, calprotectin, and other cationic peptides
- • Saliva with histatins
- • PMNs
- Adaptive immunity
- • Cell-mediated immunity plays a major protective role
- • CD4+ T lymphocytes specific for fungi
- • fungal antigens -> cytokine production -> PMNs and macrophages activation
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