Mycology review

Main types of disease• Allergy • Hypersensitivity pneumonitis

– Occupational in many cases due to chronic exposure to antigens (fungi, actinomycetes, others)

– smallest spores get furthest into lungs– <5 m reach alveoli

• Toxicity– Mushrooms– Ergot – Mycotoxins

• Infections

Mushroom Toxins

• Main lethal toxins act more slowly than rapid non-lethal toxins

• In most cases of potentially lethal toxins, e.g. -amanitin , the main signs of disease in deep organs appear ~one day after ingestion

• Rapidly acting toxins, psilocybin, muscarine usually act with 2 hours typically.

-amanitin• Inhibits RNA polymerase II (mRNA synthesis)• Liver main target but also affects other sites• Toxin excreted in bile (enterohepatic circulation)

and in urine• Fulminant hepatitis can develop.• Depending on extent of liver damage,

transplantation may be needed• Typical disease is associated with feeling a little

unwell a few hours after ingestion, recovering, then becoming really sick the following day

Muscarine• Rarely serious• Cholinergic poisoning (muscarine receptor

binding)• Acts rapidly within an hour typically• Causes PSL (perspiration, lacrimation, salivation)• Atropine is antidote but only needed when very

serious poisoning occurs• Simple test to rule out fear is to check pupil

enlargement in a dark room. Poisoning is associated with retention of pin-point pupils

Ergot• Mainly associated with poisoning via bread

since gets into grain• Contains many toxins. Outbreaks associated

with blood vessel constriction in extremities caused tingling – St Anthony’s fire. Can lead to extremity loss. Other outbreaks associated with bizarre behavior

• Recent outbreaks occur in areas where not good control on food quality

Mycotoxins• Toxins, mainly formed in poorly stored, especially

not fully dried, food but also can form when grain is damaged in field conditions and molds get going before harvest

• Most toxins destroyed by heating (not aflatoxin B1)• In most cases, toxicity at low concentration inhibits

immune response and ,makes livestock less able to fight infection - failure to grow

• Trichothecenes are cytotoxic, have been explored as chemical warfare agents and as potential therapies for cancer

• Aflatoxin B1 is both clearly toxic, killing animals when eaten at high concn. Survivors or ones receiving small amounts develop cancer (especially liver)

Aflatoxin B1• Formed by Aspergillus flavus (and sibling species

Aspergillus oryzae) ONLY• I.e. NOT formed by A. fumigatus• Requires activation by liver enzymes to AFB2a• AFB2a forms adducts with DNA which are

associated with oncogenic activity• In persons infected with hepatitis B or hepatitis C

viruses, the ingestion of elevated aflatoxins in foods is linked to increased liver cancer

• Allowable levels in foods (grains, corn, nuts especially) strictly controlled

Infections

• Skin pathogens– tinea versicolor– dermatophytes

• Subcutaneous• Deep seated pathogens• Opportunists

– Those controlled mainly via T cell-mediated immunity– Those controlled mainly via neutrophils– Candidiasis (a mixture)

tinea versicolor• Areas of depigmentation, occasionally

hyperpigmentation, on skin. No inflammation

• Overgrowth of Malassezia spp. that are part of the normal flora of skin (yeasts that require lipid for growth)

• Yeasts not grow on regular Sabouraud agar

• Identified by KOH mount of scraping (clusters of round yeasts with filaments)

• Lesions fluoresce greenish yellow in Wood’s light

dermatophytosis

• Molds that infected keratinized tissues (hair, skin, nails)

• Many species (Trichophyton, Microsporum) + Epidermophyton floccosum but the 3 genera are closely related

• Grow on Sabouraud agar and produce spores that allow their identification. These spore types are not found in human tissues where septate hyphae, with or without arthrospores, are the only features produced

KOH mount - Dermatophyte in skin showing hypha breaking into arthrospores

Scrape at growing edge where mycelium is causing inflammation

Stained KOH MOUNT

tinea cruris

Cultivation on selective medium containing cycloheximide(dermatophytes less susceptible) and antibacterials

Spores developing in culture allow species identification

Sources for infection

Anthropophilic species: humansZoophilic species: animalsGeophilic species: soil

Anthropophilic species tend to cause less inflammation.

tinea pedis (athelete’s foot) typically anthropophilic

tinea capitis (hair)both zoophilic and anthropophilic (most commonly anthropophilic in USA at present)infection may be ectothrix (arthrospores in outer layer around air shaft) or endothrix (arthrospores massed within hair shaft)

tinea capitis

Ectothrix species Microsporum canis and M. audouinii

Usually fluoresce in Woods lightNot usual after puberty

tinea capitis Endothrix species

Doesn’t fluoresce in Woods light. Continues after puberty

Trichophyton tonsuransmost common world wide

Id reactions to fungal infection under foot. (No fungus seen or cultivatable from id)

Treatment of dermatophytes

Limited area of skin: topical agents

tinea capitis and large areas of involved skin or nails oral therapy (azoles, griseofulvin, allylamines)

When possible, confirm there is a dermatophyte via microscopy

Mycotic keratitisInfection of the eye

• Infection of the eye caused by many different fungi. • 2006 outbreak associated with Fusarium - a mold

growing in contact lens solution held for long periodsAnamorph shows sporulation Characteristic of Fusarium

Anamorphs produced in culture identify mold species causing keratitis

Subcutaneous infections

• Fungi grow in the environment and penetrate into the body through skin via trauma (thorns, splinters etc)

• Mycetoma

• Chromomycosis

• Sporotrichosis

Mycetoma and Chromomycosis• Both often chronic diseases developing slowly over several

years. Many different species form these diseases and actinomycetes also can cause mycetoma

• Mycetoma diagnosis - large granules of mycelium• Chromomycosis - pigmented fungal cells• Different species identified from anamorph in culture

Sporotrichosis

• Usually lymphocutaneous. Infection follows trauma injecting fungus (splinters, etc).

• Single etiologic agent, Sporothrix schenckii• Temperate regions• Dimorphic

– Cigar shaped yeast at 37 C (and in disease)– Mold at room 25 C

• Dissemination rare, seen in AIDS and severely immunosuppressed

Sporothrix dimorphism Room temperature vs 37 C

Lymphocutaneous sportrichosis• Non healing initial lesion that doesn’t respond to

antibacterials• New painful lesions appear along draining lymphatics• Diagnosis - Culture most successful, often too few

yeasts to be seen in tissue • Clinical signs (most often Sporothrix) but could be

Mycobacterium marinum or some other microbes

Lymphocutaneous diseaseSaturated potassium iodide KI works (large amount over several weeks)Azoles (e.g.; itraconazole)

Systemic diseaseamphotericin B, azoles. KI does not work

Treatment of sporotrichosis

Deep-seated true pathogens• Infect and can cause disease in immunocompetent persons

as well as immunosuppressed• Geographic limitation – predominate in specific regions.• Infect via lung: then can disseminate to many sites

including skin• Dimorphic - environmental form differs from parasitic• Originally considered rare, deadly diseases but, in most

cases, now recognized as common infection.• Severe clinical disease is quite rare: most infected recover

without significant illness. Evidence for infection without clinical disease comes from positive skin test reaction to Histoplasma and Coccidiodes antigens

Main pathogensMain foci in N. America

Histoplasma capsulatum Ohio River - Mississippi River Valley dominant (sporadic worldwide)

Coccidioides immitis (C. posadasii)Desert regions of the South West (Sonoran Life Zone also similar regions in Mexico and parts of S. and Central America)

Blastomyces dermatitidisSimilar to Histoplasma but reaching further north into Ontario. Some regions of Africa and India. Rare compared to others and more common in dogs than in humans

Note: distributions incorrectly labeled in Murray text p.768

• Histo = Histoplasma/histoplasmosis• Cocci = Coccidioides/coccidioidomycosis• Blasto = Blastomyces/blastomycosis

Resistance• Dominated by T cell-mediated immunity for Histo &

Cocci (but ? Blasto) so people with HIV or immunosuppression get worse infections

• Men more susceptible than women in Blasto and Cocci • BUT if pregnant & non-immune, susceptibility is far

greater for Cocci for which growth is stimulated by estradiol

• Sign of resistance– DTH skin test developing (Th1 response)

• Sign of continuing infection, – rising CF antibodies (Th2 response)

Typical Pathogenesis• Infection via lung by spores of environmental

form• Fungi converts to pathogenic form and

establishes local infection then disseminates to many sites often including skin/mucosa

• Diagnosis made by detecting the fungus in lesions using histology and culture. Serology can be helpful

Histoplasmosis• Ohio River-Mississippi River geographic region• Fungus thrives in bird guano enriched soils (especially

under starling roosts) and in bat guano• In environment, mold produces macroconidia and

microconidia (microcroconidia are spores that are small enough to reach alveoli)

• Inhalation establishes infection and germinating microconidia convert to yeast form

• In disease, histoplasma yeasts remain intracellular within macrophages (included fixed macrophages) except when the cell breaks open. Then rapidly picked up by other macrophages

• Found in liver, bone marrow, etc. Can be seen in blood monocytes when severe immunosuppression present

Histoplasma capsulatum environmental and parasitic forms

Mycelium bearing Yeast within macrophages

infectious microconidia (arrows) and macroconidia

Symptoms differ in non-immune and immune persons

Primary infection• Immunologically naïve• ~ 10-14 days before

immune response controls fungal growth

• Symptoms associated with inflammation (fever, pain, etc.) but not seen for ~ 2 weeks

Secondary infection• Rapid immune response. • Usually controls fungus

quickly with minimal/no symptoms

• Heavy exposure to spores can cause massive symptomatic inflammation peaking around 4 days but then fairly rapid resolution

Lab tests for diagnosis/Treatment of histoplasmosis

• Antibody detection can be very helpful in chronic infections

• Urine antigen in severe disseminated infection• Biopsy for histopathology and culture

• Treatment– Newer azoles (fluconazole, itraconazole, voriconazole,

posaconazole) replacing Amphotericin B– Definitely needed if immunodeficient (incl. AIDS) or

progressive disease not coming under control

CoccidioidomycosisPulmonary infection with dissemination to many sites including skin and CNS

1% infected healthy persons are symptomatic and can need medical care.

Coccidioides is the most virulent fungal pathogen. Found in desert soils and produces infectious arthrospores

Increased severity in AIDS

Coccidioides

Growth on Sabouraud agarin vitro at room temp and 37 C

Form found in desertsoils

Mycelium Arthrospores

Converts to parasitic form during infection(

• Post puberty females more resistant than males– Stronger response associated with erythema nodosum and

erythema multiforme. Lesions are hypersensitivity reactions do not contain fungi

• Pregnant very susceptible if not already immune– increasing risk of dissemination with infection at later stages of

pregnancy

Coccidioidomycosis - Dissemination

Treatment and Resistance

• Amphotericin B • Newer azoles

• CNS infections– lifelong if immunosuppressed, e.g. in AIDS

DTH skin test positive – Good prognosisRising CF antibodies (IgG) - poor prognosis

Th1 based resistance aimed at endospores

Blastomycosis• Rare. Pulmonary infection with dissemination• Environmental source not known. i• In Great Lakes region(USA and Canada), Atlantic region

USA, occasional overseas• Disease more common in dogs than humans• Central role of T cell-mediated immunity not clear.

Seems PMNs may be quite important too• 95% cases in males• No epidemiologic skin test survey available but

antibody production is a good marker of infection

Blastomyces dermatitidisYeast with broad base bud at 37 C and in infection

conidiophores at room temp

Pulmonary and systemicblastomycosis affecting bone

Infection is via the lung, pulmonary disease may be severe or very mild. Severe skin lesions and lesions in the skin, bones, and prostate are the dominant presentation in many patients.

Blastomycosis treatment

• Amphotericin B has largely been replaced by newer azoles

Opportunists

• Unusual cause of infection unless patient has a predisposing condition such as an immunodeficiency

• T cell deficiencies– Cryptococcosis, pneumocystosis, microsporidiosis– Mucosal =/- cutaneous candidiasis

• Neutropenia– Aspergillosis, invasive candidiasis, systemic zygomycosis

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CryptococcosisEtiologic agent: • Cryptococcus neoformans

var. neoformans (pigeon manure) throughout worldvar. gattii (Eucalyptus trees) more restricted to regions

where winter freezing is not found. But a recent hybrid is causing outbreak in British Columbia, Canada where it is assocaited with Douglas fir

Grows as an encapsulated, budding yeast in vitro and in vivo

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Virulence determinants

• Acidic capsular polysaccharide– Antiphagocytic and T-independent antigen– Readily observable in India Ink

• Phenoloxidase– oxidizes phenolics to form a deep pigment similar to

melanin. Appears to be valuable in invasion of CNS

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Bird seed agar: phenoloxidase production by Cr. neoformans but not by Candida albicans turns colony dark

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Infection

• Pulmonary initially, ± dissemination May cause severe lung disease on occasion but often disseminates without much sign of lung disease. Clinically, CNS meningitis and/or skin lesions often first sites that show an infection s present

• Susceptible groups include those where T cells are compromised– AIDS– High dose steroids– Sarcoid treatments– Persons with chemotherapy

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Rapid Diagnosis via Detection of AntigenLatex agglutination test for cryptococcal capsular

polysaccharide. (Latex coated with antibody)Particularly valuable for CSF from meningitis

where test is more sensitive than direct India Ink

No agglutination Agglutination

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Cryptococcosis

• Treatment– Amphotericin B + 5-FC combined– Azoles (used for long term therapy or following

initial treatment)

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Pneumocystis

• Major cause of pneumonia in AIDS. Much more common until prophylaxis was given routinely but still often the primary AIDS-defining infection.

• A fungus (from genes such as for rRNA) that causes respiratory infection

• Human species differs from animal pneumocystis species. • It does not show significant growth in vitro • Does not respond to typical antifungals and used to be

thought to be a protozoan. • Main treatment TMP-SMZ (pentamidine a back-up)

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Pneumocystis• Genetic analysis suggests multiple reinfections. Little

evidence for chronic carriage (previously believed) • Immunocompetent people totally resistant to disease• AIDS, SCID associated with severe pneumonia

Massive interference with oxygen diffusion from alveoli

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Prophylaxis

• Instituted when CD4 count <200 l

• trimethoprim-sulfamethoxazole**** • if not tolerated

– dapsone – aerosolized pentamidine– others

Microsporidiosis

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Agents closely related to fungi that grow intracellularly

Microsporidiosis

Unusual opportunist. Found in AIDS. Related to fungi. May cause Severe GI disease similar to cryptosporidiosis in AIDSAlso can sometimes cause disease at other sites (can be rubbed in eye and infect conjuctiva

Very tiny “spores” within cells detectable with special stains including calcofluor white and the modified acid fast stain used for Cryptosporidium

Treatments are limited

A modified Gram stain used to show microsporidia in diarrhea occurring in AIDS

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Opportunists associated with neutrophil deficiencies

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Neutrophils (and macrophages) kill spores by phagocytosis

Extracellular killing by neutrophils E.g. invasive candidiasis and invasive aspergillosis

Successful attack on large structures

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• In USA, most commonly caused by Aspergillus fumigatus

• Other species occasional including voriconazole-resistant A. lentulus which looks like A. fumigatus

Aspergillosis

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Aspergillus fumigatus

• Grows very well at 45 C• Mold producing abundant blastoconidia on

composts and rotting plant materials• Generally infects via lung (unless injected

somehow: e.g. contaminated bandages on wounds, contaminated i.v. drugs)

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Pulmonary phagocytes fail to kill sporesin presence of high dose steroid treatment

conidia

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Hyphae branch (usually 45º) as mycelium expands and penetrates blood vessel walls

Septate branching hyphaethat are

angiotropic

Infarcts follow blood vessel wall penetration

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CT scan:Characteristic “air” cresent sign can indicate invasive aspergillosis in lung

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Treatment of Aspergillosis• Newer azoles (Voriconazole and Posaconazole) tend to be

replacing amphotericin B and liposomal Amphotericin B

• Disease progresses rapidly – treatment urgency: i.e. need to treat on suspicion.

• Diagnosis often via histology on biopsy (later confirmed by culture).

• Tests for fungal products in blood are available in some research hospitals but not clear as to value.

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Aspergillus diseases (not associated with neutropenia)

• Allergic bronchopulmonary aspergillosis– Spores germinate in bronchioles and begin to grow– Allergic mucus response leads to plugging of bronchioles.

Much antibody produced– Significantly reduced lung capacity

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Aspergilloma

• Fungal mycelium grows as a ball in pre-existing scarred cavity (T.B, sarcoid)

• Corrodes edge: danger eventually of hemoptysis

• Fungus is growing largely saprophytically in area outside reach of immune system

• Treatment usually needs surgery

Cavity with aspergilloma

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Zygomycosis

• Caused by Zygomycetes • Anamorphs have sporangia and

sporangiospores • Spores germinate to form hyphae and

mycelium. Generally hyphae are wide, often irregular, lack regular septa.

• Hyphae are angiotropic

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Zygomycosis (Mucormycosis)• Systemic disseminated zygomycosis

– Neutropenia is main predisposing factor.– Hyphae are angiotropic and usually irregular

compared to Aspergillus

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Rhinocerebral zygomycosis

Infection via nasal turbinates and sinuses into CNS

This type of zygomycosis is largely restricted to persons with uncontrolled diabetes where ketoacidosis is present

Damage around orbit can be seen

Candida and Candidiasis

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Candidiasis

Skin and mucosal infection with local invasion of mucosa - (T cell-mediated immunity is important for skin and mucosal resistance)

• Diabetes• T cell deficiency – severe thrush and esophagitis

often in AIDS• Various conditions (often temporary) such as

disruption of normal microbiota (vaginitis common)

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Skin and Mucosal resistance• Predominantly T cells

important for resistance

• Candida– AIDS defining in

HIV-positive

Release of cytokines fromTh1 cells stimulatesepidermal growth

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Severe esophageal candidiasis in AIDSulcerative erosions and barium leak

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In areas where skin remains wet

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Vaginitis: satellite lesions and cottage cheese-like discharge

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Routes for invasion of blood

• Normal flora of GI tract may penetrate through wall

• Indwelling catheters left for long term– Candida invades from skin and follows the outside

of the line to the catheter tip. Colonizes tip • Dissemination including occasional endocarditis

Candidiasis

Deep-seated infections• Neutrophils an essential defense.

Neutropenia is a major predisposing factor

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Routes for invasion of blood

• Normal flora of GI tract may penetrate through wall

• Indwelling catheters left for long term– Candida invades from skin and follows the outside

of the line to the catheter tip. Colonizes tip • Dissemination including occasional endocarditis

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Disseminated candidiasis in neutropenic patients

Often see skin lesions

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Dissemination not uncommonly includes eye and vitreous fluid

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Diagnosis of Candida infected tissues Both yeasts and filaments present = Candida

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Direct smear from urine with pseudohyphae and yeasts

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Chronic mucocutaneous candidiasis

• Rare• Candida on dry skin

and nails. Masses of antibodies

• Susceptibility is multifactorialT cell (anergy may be

restricted to Candida)EndocrinopathiesZinc deficiency

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Candida• Main species is C. albicans (normal flora of mucosal

surfaces in humans and majority of vertebrate animals)

• Other species identified by different pattern of sugar assimilations. Species identification can be important for treatment choices, especially when serious disease present.

• Some species resistant to fluconazole or other azoles• Increasing variety of species being seen

97Yeast colonies Yeast cells

Candida species and basic growth form on Sabouraud agar (a high glucose medium)

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Special test for C. albicans

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Candida albicans

• Highly flexible morphology with filamentous forms binding different human proteins than do yeast forms. Filaments appear more invasive

• Phenotypic switching enhances capacity to change with environment

• Serious skin and mucosal infections do not cause disseminated disease unless PMNs become dysfunctional

Antifungal agents:

best target is only in fungi not in humans.

Fungitoxic drugs cause fungal death

Fungistatic drugs prevent further growth (gives immune system time to catch up)

5-fluorocytosine (5-FC) fungicidal

enters via cytosine permease

deaminated to 5-fluorouracil (5-FU)(cytosine deaminase absent in human cells)

permease

5-FC 5-FCInside fungal cell

5-FU

deaminase

RNA translation

DNAsynthesisinhibition

Fungal sterols as a target

• Fungal sterols are generally C28 sterols; especially ergosterol.

• (Humans cells have C27 sterols i.e.; cholesterol

Allylamines

(terbinafine = Lamisil®, naftidine)

Inhibit squalene epoxidase .fungistatic

Fungistatic/toxic

Accumulate in stratum corneum. High activity for ringworm infections

Acetyl CoA

Squalene

Squalene epoxide

Lanosterol

Ergosterol

Azoles

Inhibit lanosterol demethylase

Fungistatic

MiconazoleKetoconazoleFluconazoleItraconazoleVoriconazolePosaconazole

Acetyl CoA

Squalene

Squalene epoxide

Lanosterol

Ergosterol

Azole resistance in Candida albicans

Several different types of resistance

• Mutation in target (lanosterol demethylase)• Upregulation of pumps exporting drug

Different drugs affected differently by pumps.

• Different yeast species that are inherently resistant to azoles are appearing as pathogens

Polyenes

First fungitoxic drugs

Amphotericin B (AmB)

Nystatin (oral, not absorbed)

Bind to ergosterol and form ion channels in fungal membrane

Reduced nephrotoxicity of AmB if given as lipid complex or in liposomes

Resistance uncommon (often sterols changed)

Acetyl CoA

Squalene

Squalene epoxide

Lanosterol

Ergosterol

Echinocandins (caspofungin, 2001: micafungin, 2005)

Inhibit (1-3) glucan synthetase involved in forming carbohydrate polymers in hyphal walls.

Approved for invasive aspergillosis and invasive and serious mucosal candidiasis

Resistance when occurs has been linked to mutations in -glucan synthetase

Metabolism is cytochrome P450-independent

Griseofulvin:

Accumulates in stratum corneum

First effective oral therapy for dermatophytes (only fungi responding)

Interferes with microtubules and spindle formation during mitosis

Numerous other drugs are topical agents. May be caustic and impossible to use as systemic therapy

E.g. Whitfield ointment

salicylic acid, benzoic acid (weak acids, not ionized at lower pH)

HA

HA H+A-

acidification