Fungus Flashcards
Fungal cell wall
Membrane - ergosterol instead of cholesterol
(target of amphotericin)
Cell wall - unrelated to bacteria
- lots of cell mass, 90% polysaccharides
- inner - glucan and chitin - glycosilidic cross-link -> stability
- glucan also allows expansion and contraction (middle layer)
- glucan is key antigen for immune response
- outer - glycoproteins
Capsule - Cryptococcus only
- glucuronoxylomannan (GXM) polysacc -> shed during infection
Overview of fungi
Kingdom within eukaryotes (have nuclear membrane)
- > 3 Phyla -> 3 medical genera (by sexual structure)
- Ascomycota (asci contain ascospores)
- largest, most diverse -> Aspergillus, Candida, (Saccharomyces, Neurospora)
- Basidiomycota (basidium) - Cryptococcus neoformans
- Zygomycota (zygosporangium) - Mucos sp.
Non-motile (medically-important)
Digest food externally -> absorb
Both sexual and asexual reproduction
Limited anti-fungal rx dt similarities with mammalian cells
Antifungal overview
Fewer targets due to similarities with mammalian cells
Azoles -> p450 enzymes - ergosterol synthesis
Polyenes -> ergosterol
Echinocandins -> beta glucan synthesis
Resistance - usually genomic vs acquired elements
- increased expression
- alteration of targets
- can have high mutation rates (-> use multiple agents)
Polyene antifungals
Bind to sterols -> disrupt membrane permeability
- greater affinity for ergosterol (fungi) vs cholesterol (host)
Poorly absorbed from GI
High toxicity
Amphotericin B - used systemically but toxic, fungicidal
Nystatin - only used topically or oral (not absorbed)
- only fungistatic at attainable concentrations
Azoles antifungals
Target p450 enzyme for ergosterol synthesis
- > weird steroids in cell membrane
- > inhibit membrane/hyphae growth (static)
- also more susceptible to phagocytosis
Oral, systemic but hepatotoxicity in 0.01%
Thrush, systemic, chronic (ex AIDS)
Fluconazole, ketoconazole
Echinocandins
Inhibit synthesis of cell wall
- beta glucan synthase
- cidal
Caspofungin
Antimetabolite antifungals
Flucytosine
Uracil -> 5F-uracil -> inhibits protein synthesis
Thymidine -> 5F-deoxyuracil -> inhibits DNA synthesis
Can be cidal or static depending on organism
Overview of fungal growth
Exogenous (free-living) vs
Endogenous (must be in human or animal)
- ex Candida in GI
Absorptive heterotrophs - secrete enzymes, absorb smaller compounds
Acid tolerant (grow optimally at pH s agar
Secondary metabolites
Synthesized by non-ribosomal polypeptide or polyketide synthase
NOT necessary for fungal growth
Beneficial:
- lovastatin from Aspergillus
- equisetin (anti-viral) from Fusarium
- penicillin from Penicillium
Aflatoxins - produced by Aspergillus, Fusarium
- grain or peanuts contaminated
- mycotoxicoses, carcinogens (low amounts needed)
Yeast
One of two main forms (vs hyphae - different organisms)
Oval cell
Division = budding/fission
apical -> pinches off cell and nuclear membranes
Smooth pasty colonies
Hyphae
Elongated filament
Septa -> mononuclear segments - vs aseptate/coenocytic (division and growth without fission) Growth - exclusively apical - Spitzenkorper = special vesicles near tip - can extend into new substrate - penetrating due to turgor pressure - release polymer degrading enzymes - can be rapid (40 microns/s) - can also grow aerial -> spores
Spores
Reproductive propagule
- conidia = asexual - either micro (airborne) or macro (-> ID)
- arthrospore - fragmentation of hyphae
Usually form from aerial hyphae
- hyphae + spores = mycelium
Can be small (1 micron) -> lower respiratory
Overview of yeast growth
Often dimorphic - both yeast and hyphal depending on env’t
- elongated yeast = pseudohyphae = intermediate form
- beginning of hyphae from yeast = germ tube
Classical thermal
- soil (low temp) -> filamentous hyphae -> conidia
- body temp -> yeast
- unnatural, “dead-end” env’t (no spore production)
Candida albicans
- soil -> yeast; body -> hyphae
- find yeast, hyphae and pseudohyphae in infected tissue
Cutaneous mycoses pathogenesis
Dermatophytes - keratinase -> nutrient source
- filamentous septate hyphae
- invade hair from inside (endothrix) or outside (ectothrix)
-> produce micro and macroconidia
Epidermophyton
Trichophyton - anthrophilic - human-> human via skin fragments
Microsporum - zoophilic - pets
- fluoresces in UV light
Non-dermatophytes - some dimorphic
Cladosporium - geophilic - soil (dimorphic!)
Cutaneous mycoses clinical
Most common fungal infections (25% prevalence)
Dermatophytes -> tinea (ex capitus, pedis, cruris)
Tinea versicolor
- Malassezia furfur (normal flora) -> dandruff, hypopigmented
- dimorphic yeast and hyphae
- lipophilic, can be in blood if IV lipids
Tinea nigra - Cladosporium werneckii -> produces melanin
Dx: skin scrape -> KOH mount with Calcofluor
- can do culture (Sabourad’s) -> microscopy
Tx: topical antifungal (azole)
- oral azole, terbinafine, griseofulvin
- ciclopirox/Penlac for nails
- limit moisture, may need ID
Subcutaneous mycoses
Rare!
Puncture or abrasion -> contamination with soil fungus
Sporotrichosis:
- Sporothrix shenkii - classical dimorphism (“cigar” yeast at 37C)
- puncture -> nodules (-> disseminated if immune compromise)
Chromoblastomycoses
- dematiaceous (multiple soil species) - produce melanin
- puncture -> wart-like crusty lesion with brown cells
- mycetoma - abcess with pus -> requires surgery
Dx: KOH mount, biopsy
Tx: oral azole or flucytosine
Prevention - protective clothing
Overview of fungal infections
Cutaneous -> keratinized skin, nails, hair
- direct contact -> inflammatory rx
Subcutaneous - trauma -> dermis, fascia, etc
Systemic - blood or organs, from GI, resp
Opportunistic - invasive in immune compromised
Candida epidemiology
Normal colonizer of oral, vagina, GI (from birth)
- DTH skin test (+) in all immunocompetent
Infections - common invasive life-threatening
- most common fungal BSI, 4th most common nosocomial BSI
- immunocompromised (liver transplant)
- invasive procedures (GI surgery, catheters)
- ICU (trauma, NICU)
Almost all are Candida albicans
400K/yr worldwide, most in developed
Candida pathogenesis
Source: normal endogenous (or nosocomial in ICU)
Non-classical dimorphism (polymorphic) - all forms found in tissue
Yeast - normal room temp
- overgrow if immunocompromised (esp cellular), antibiotics
-> germ tubes -> vegetative/invasive hyphae
-> also pseudohyphae
Hyphae
- vegetative - form on or below agar (NOT aerial)
- adhesins vs keratinocytes ->
- invasion -> white punctate lesions -> thrush/pseudomembrane
(usually limited to upper epithelial layers)
-> yeast spread to new sites (also stress resistant)
Bloodstream if heavy skin colonization, immune compromise
- or biofilm on catheter -> yeast
Candida clinical syndromes
Normal hosts:
- vaginitis common
- diaper rash (moist areas)
- oral erythema
Immunocompromised
- oral thrush - infants, HIV, chemo (poor cellular immunity), dry mouth
(one of earliest, most common after HIV)
-> pain, dysphagia, susceptibility to other infections
- esophageal - leukemia, lymphoma, suppressed - serious
Candida species
C albicans - most common (50-60%)
- positive rapid germ tube test (1 hour in 37C)
C glabrata - second most common
- normal commensal, also only in mammal hosts (not env’t)
- more resistant to azoles, amphotericin
- no germ tubes or pseudohyphae
- 15% of infections worldwide
C tropicalis, parapsilosis
- negative rapid germ tube, positive pseudohyphae
- similar presentation to albicans
Candida diagnosis
Mucous, skin
-> tissue - KOH mount, PAS, GMS
-> culture (blood, Sabourad’s) -> smooth white colonies
Deep/blood infections - difficult
- blood cultures (Wright’s) may be negative
- new molecular methods -> higher sensitivity?
Identification
- rapid germ tube - (+) for albicans
- chlamydospores on special media - not normally performed
- definitive = fermentation, assimilation of carbon sources
Candida treatment
Oral - prevent with nystatin, azole rinses etc
- tx with fluconazole, caspofungin (inc esophageal)
Vaginitis - topical azoles, fluconazole
Disseminated - use scoring system/algorithm
- usually fluconazole
- echinocandins, azoles, amphotericin
Immunity to fungi
Innate = essential!
- non-inducible (skin, microbiome)
- recognition via PAMP, lectin receptors
(cell wall beta glucan vs dectin-1 receptor)
- neutrophils
- macrophages
Adaptive
- mostly CD4 -> Th1 -> IFN-g -> macrophage -> granulomatous response -> can have reactivation
- some Th17 -> recruit neutrophils, induce epithelial protection at mucosal surfaces
- antibodies present but unclear fx
Overview of fungal dx
Histology
- KOH or NaOH - disolves tissue, leaves fungi intact (dt carbs)
- PAS, methanamine silver
- india ink -> Cryptococcus capsule
Culture - may be dangerous due to spores!
- yeast -> 48 hours -> differential media
- molds - slower, may be negative
-> wet mount with lactophenol cotton blue to ID spores
- pathogens can grow at 37C!!
DNA probes
Serology
Immune susceptibility to fungal
Helps determine likely/posible pathogens
Breach of non-inducible - microbiome (antibx), catheters
Can be receptor, signalling, recruitment, effector
Receptor - ex dectin-1 polymorphism -> Aspergillus
Effector - esp neutrophils, macrophages
- CD4 -> Th1 -> IL12, IFNg, TNFa = essential
- also CD8, Th2, Th17, Treg -> length/severity
Ex: Immune reconstitution inflammatory syndrome (IRIS)
-> increase in CD4 vs established pathogens (Cryptococcus, Pneumocystis jirovecii)
Fungal pre-dispositions
Neutropenia -> Candida, Aspergillus, Mucormycoses, Trichosporon, Scedosporium, Fusarium
T-cell (HIV) -> Cryptococcus, Histoplasma, Blastomyces, Coccidioides, Paracoccidioides, P marneffi, dermatophytes, Candida
Skin/mucosal trauma - Candida, Aspergillus, Fusarium
Chronic granulomatous - Aspergillus, Candida (disseminated)
TNFa inhibitors - Aspergillus, Histoplasma
Graft vs host - Aspergillus
Ketoacidosis, Deferoxamide - Mucormycoses
Overview of endemic fungal pathogens
Inherent/primary pathogens! (do not need immune compromise)
Geography - restricted region!
- 3-24,000 deaths/yr in US, mortality 1-75%
Exposure - inhalation of spores, rarely human-human
-> dimorphism (yeast at 37C)
- lab cultures can be dangerous
Immune = cell-mediated -> macrophage -> granuloma
- latency within granuloma
- disseminates in immune compromise
- much more severe in HIV+
- skin DTH to determine exposure
- highly varialble - can be short assymptomatic -> latent, lethal
Coccidiodes Histoplasma Blastomyces Paracoccidiodes Penicillium marneffei
Coccidiodes mycology
C immitis, posadasii
Barrel arthroconidia (2-6 micron) -> fragment -> airborne
- within alveoli -> large spherules (12-100 micron)
-> rupture -> release endospores -> new spherules
150K cases annually in US
southern CA, Arizona -> central Texas (dry, hot, alkaline)
- reportable, 2/3 cases in AZ
Coccidiodes presentation
Macrophages -> neutrophils (resistant to neutro) ->
Cell mediated immunity = protective
- 2-4 weeks, DTH (+)
Often asymptomatic - more sx in dark-skinned men (ex Filipino)
Pulmonary - 40% lower resp (sputum, CP, fever, anorexia)
- 2-6 weeks
- “Valley Fever”
Erythema nodosum (EN) - 10%
- non-septic nodules in lung
- indicate protective immunity (not disseminated)
Rare - disseminated (usu defective cellular immunity)
-> granulomatous skin lesions, osteolytic, meningitis
- more common in third trimester
Coccidiodes dx and tx
tissue -> KOH or stains -> spherules
Sabourad’s culture -> dimorphism, tissue form
- hazardous!!
Serology - IgM (2-4 weeks) -> IgG
- proportional to current burden (increase if disseminated)
Primary - no tx needed
Disseminated - azoles, amphotericin
Histoplasma mycology and pathogenesis
Branching septate hyphae ->
- tuberculate macroconidia (ID)
- microconidia -> inhaled -2-3d> ovoid yeast
- > intracellular growth within macrophages
- > migrate widely to nodes, spleen, liver
- 9-15d> cell-mediated immune response
Virulence = growth within macrophages
- produces bicarb, ammonia -> raises pH of phagosome
Can have extracellular yeast, infect epithelial cells
Histoplasmosis presentation (and geo)
Widely distributed
Ohio and Mississippi River Valleys (up to 90% DTH +)
Most assymptomatic
Acute pulmonary - self-limited, flu-like
- anatomic abnormalities -> colonize pulmonary recesses
Opportunistic -> chronic - similar to TB
Histoplasma dx
Disseminated - blood (Wright’s) + for intracellular yeast
Tissue stain
Culture on special media -> dimorphic with tubercular macroconidia
Antigen in urine
Serology - decreases when inactive
- cross-reacts with Blastomyces
Skin test only used for epi (disrupts serology)
Blastomyces mycology and geography
Mold -> pear conidia ->
Characteristic yeast (= diagnosis via KOH, stain)
- thick-walled, large with broad based single bud
Geography - similar to Histo? skin test cross-reacts…
- Ohio and Mississippi rivers, Carolinas
- recreation along wooded waterways
Clinical disease is more common
Dogs susceptible in endemic zones - frequently severe/lethal
Blastomyces pathogenesis
Conidia -> inhalation -> yeast -> multiply
-> disseminate via macrophages, blood, lymph
Bad1 = virulence - promotes uptake by macrophages
-> replicate and travel until activated by T cells
- homology with invasin of Gram (-)
Immunity - several weeks -> T cells, Ig, DTH+ -> protective
Presentation:
Asymptomatic or flu-like
Disseminates -> organs (not uncommon) - can have 60% mortality
Trophic for skin, bone -> ulcerative lesions
Paracoccidioidis
South America - forested, tropical, armadillo nests
Most common men (coffee plantation)
Large yeast with multiple blastoconidia -> “pilot wheel”
Often chronic, subclinical with latent periods
primary pulmonary -> mucosal lesions (50%) -> cutaneous (25%)
Penicillium marneffei
Only thermally dimorphic (pathogenic) Penicillium
SE Asia - 3rd most common infection with HIV regionally
Pulmonary -> disseminated -> often fatal
Overview of opportunistic fungi
Geographically widespread
Almost always affect immunocompromised
- 100% lethal if untreated (still high if treated)
Grow well at 37C
90% of disease from 4 genera: Cryptococcus Aspergillus Mucor Pneumocystis
Cryptococcus epidemiology
Almost all cases associated with AIDS or other compromise
Most life threatening infections of any fungus
- most meningo-encephalitis
Est 1 million worldwide - 620K deaths/yr in sub-Saharan
- mortality rate 15-20% US treated, 55-70% South America, Africa
C neoformans - widespread in soil, pigeon shit -> compromised
- 2 different serotypes
C gatti - trees, Canada -> Pacific NW
- both compromised and healthy
Cryptococcus pathogenesis
Basidiomycetes - grows as yeast in agar and tissue
- urease (+), grows well at 37C
-> inhalation of spores or dessicated yeast
Capsule - glucuronoxylomannan (GXM)
- essential for virulence
- extracellular - protects within phagolysosome
-> ID via India Ink stain
Phenol oxidases - DA -> melanin - protects from oxidative damage
Lungs -> systemic -> preferential to CNS -> meningo-enceph
- either microcapillaries -> BBB or via macrophages
Cryptococcus clinical
Meningo-encephalitis most common
- insidious onset -> h/a, stiff neck after high fungal burden
Pulmonary -> cough, SOB, encapsulated nodules
Subclinical urinary, resp
Dx:
Capsule antigen in urine, plasma, CSF = definitive
- latex agglutination assay
Can visualize capsule - India ink, mucicarmine, PAS, methanamine
- less sensitive and specific than antigen
Culture - CGB agar: gatti turns yellow -> blue vs neoformans
- bird seed culture, L-dopa
Aspergillosis mycology and pathogenesis
A flavus, fumigatus (most common), terreus (resistant)
- only hyphae, not dimorphic
Hyphae - septate, aerial -> conidiophore -> brush conidiospore -> Airborne -> germ tubes -> colonize cavities -> septate, branched
Often fills cavities from trauma, TB, etc
Penetrating -> disseminated
Requires immediate and aggressive antifungal therapy
Ubiquitous -> 200K infections/yr
Aspergillosis clinical presentations
Immune compromise -> invasive (acute) or chronic
- neutrophils, organ transplant, steroids, COPD
-> fever, cough, CP, etc
- aspergilloma - “fungus ball” - fills cavity from TB, trauma
- dx via hyphal morphology
- serum test for galactomannan - only sensitive if neutropenic without antifungal therapy
Allergic bronchopulm Asp - ABPA -
- A fumigatus -> Th2 mediated disease
- prevalence 20% CF, 5-15% asthma
- dx via eosinophilia, IgE
Mucormyces
Zygomycetes - Mucor, Rhizopus - many similarities to Aspergillus
- hyphae/mycelium only, not dimorphic
- BUT not septate -> coenocytic with sporangiophores
- right angle branching, “folded”
- faster, more virulent than Asp -> increasing
Rhinocerebral (50%): DKA -> rhino -> sinus -> cerebral
Pulmonary: most in hematologic, neutropenic
Deferoxamine therapy predisposes
Other sx: cutaneous, pulmonary, disseminated
Difficult to isolate/ID organism - half are Rhizopus
Growing resistance dt azole prophylaxis therapy
10K infections/yr
Pneumocystis mycology
Specific species for each mammal host - co-evolution
C jiroveci = human - CANNOT BE CULTURED
- ubiquitous (70% Ig +) -> 400K cases/yr
Cyst -> rupture -> sporozoites -> pleiomorphic trophozoite
? Is it a parasite?
- morphology similar to Apicomplexa
- susceptible to pentamidine, not amphotericin
- cholesterol vs ergosterol in membrane
Evidence for fungus:
- DNA, elongation factors
- thymidylate synthase, dihydrofolate reductase (diploid)
- cell wall = chitin, glucan, mannoproteins
Pneumocystis clinical
Susceptibility
- low CD4 (AIDS, inflammation -> plasma cells -> Ig -> frothy exudate
- diffuse interstitial pneumonia -> impaired O2 exchange
- sudden onset, high mortality
Dx: methanamine silver or fluorescent specific antibodies
- PCR may be useful
Tx: does not respond to classic antifungals
