Mycology review

Main types of disease Allergy Hypersensitivity pneumonitis Toxicity Occupational in many cases due to chronic exposure to antigens (fungi, actinomycetes, others) smallest spores get furthest into lungs <5 mm 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. a-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.

a-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 Subcutaneous Deep seated 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

Mycology. PF Lehmann October 14, 2008 142. Hyphal elements breaking up into arthroconidia ramifying through skin scales mounted in 10% KOH and Parker ink solution. KOH mount - Dermatophyte in skin showing hypha breaking into arthrospores

Scrape at growing edge where mycelium is causing inflammation Mycology. PF Lehmann October 14, 2008 tinea cruris Scrape at growing edge where mycelium is causing inflammation 147. Tinea of the groin (tinea cruris) showing typical circular, erythematous lesions with raised advancing margins Stained KOH MOUNT

Cultivation on selective medium containing cycloheximide Mycology. PF Lehmann October 14, 2008 Cultivation on selective medium containing cycloheximide (dermatophytes less susceptible) and antibacterials 226. Hydrolysis of urea. (A) T. rubrum negative after 7 days; (B) T. mentagrophytes positive in less than 7 days; and (C) uninoculated control negative after 7 days.

Spores developing in culture allow species identification Mycology. PF Lehmann October 14, 2008 Spores developing in culture allow species identification 222. Microscopic morphology of T. mentagrophytes var. interdigitale. Cultures usually produce numerous subspherical to pyriform microconidia, occasional spiral hyphae and spherical chlamydoconidia, the latter being more abundant in older cultures. Occasional slender clavate, smooth-walled multiseptate macroconidia may also be present in some cultures.

Anthropophilic species: humans Zoophilic species: animals Sources for infection Anthropophilic species: humans Zoophilic species: animals Geophilic 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) Mycology. PF Lehmann October 14, 2008

Ectothrix species Microsporum canis and M. audouinii Mycology. PF Lehmann tinea capitis Ectothrix species Microsporum canis and M. audouinii October 14, 2008 Usually fluoresce in Woods light Not usual after puberty

Trichophyton tonsurans most common world wide Mycology. PF Lehmann tinea capitis Endothrix species October 14, 2008 Trichophyton tonsurans most common world wide Doesn’t fluoresce in Woods light. Continues after puberty

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 Mycology. PF Lehmann October 14, 2008

Mycotic keratitis Infection 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 periods Anamorph 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

Treatment of sporotrichosis Mycology. PF Lehmann Lymphocutaneous disease Saturated potassium iodide KI works (large amount over several weeks) Azoles (e.g.; itraconazole) Systemic disease amphotericin B, azoles. KI does not work October 14, 2008 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 pathogens Main 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 dermatitidis Similar 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

Coccidioidomycosis Pulmonary 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 Mycelium Arthrospores Growth on Sabouraud agar in vitro at room temp and 37 C Form found in desert soils Converts to parasitic form during infection(

Coccidioidomycosis - Dissemination 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

Treatment and Resistance Amphotericin B Newer azoles CNS infections lifelong if immunosuppressed, e.g. in AIDS DTH skin test positive – Good prognosis Rising 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 dermatitidis Yeast with broad base bud at 37 C and in infection conidiophores at room temp

Pulmonary and systemic blastomycosis 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

Cryptococcosis Etiologic agent: Cryptococcus neoformans var. neoformans (pigeon manure) throughout world var. 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

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

Bird seed agar: phenoloxidase production by Cr. neoformans PF Lehmann Mycology October 16, 2008 Bird seed agar: phenoloxidase production by Cr. neoformans but not by Candida albicans turns colony dark 131. Mixed culture of C. neoformans and C. albicans on bird seed agar (Guizotia seeds) showing the distinctive brown colonies of C. neoformans, due to the selective absorption of pigment from the media, compared to the white colonies of C. albicans.

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

Rapid Diagnosis via Detection of Antigen PF Lehmann Mycology October 16, 2008 Rapid Diagnosis via Detection of Antigen Latex 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 137. Negative cryptococcal antigen latex test.

Cryptococcosis Treatment Amphotericin B + 5-FC combined Azoles (used for long term therapy or following initial treatment)

PF Lehmann Mycology October 16, 2008 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) 136. A positive cryptococcal antigen latex test. It should be noted that the detection of cryptococcal capsular polysaccharide antigen in spinal fluid is now the method of choice for diagnosing patients with cryptococcal meningitis. In AIDS patients, cryptococcal antigen can be detected in the serum in nearly 100% of cases. However, in non-AIDS patients antigen detection in serum is less sensitive with only about 60% of patients with cryptococcosis reported as being positive. Note, serum specimens should be pretreated with pronase to enhance detection of antigen and avoid false negative results.

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

Prophylaxis Instituted when CD4 count <200 ml trimethoprim-sulfamethoxazole**** if not tolerated dapsone aerosolized pentamidine others

Microsporidiosis 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 AIDS Also 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

Opportunists associated with neutrophil deficiencies

Neutrophils (and macrophages) kill spores by phagocytosis

Extracellular killing by neutrophils E.g. invasive candidiasis and invasive aspergillosis Successful attack on large structures

Aspergillosis In USA, most commonly caused by Aspergillus fumigatus Other species occasional including voriconazole-resistant A. lentulus which looks like A. fumigatus

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)

Pulmonary phagocytes fail to kill spores in presence of high dose steroid treatment conidia

PF Lehmann Mycology October 16, 2008 Hyphae branch (usually 45º) as mycelium expands and penetrates blood vessel walls Septate branching hyphae that are angiotropic 034. Grocott’s methenamine silver (GMS) stained tissue section of lung showing dichotomously branched, septate hyphae of Aspergillus fumigatus.

Infarcts follow blood vessel wall penetration

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

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.

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

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

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

Zygomycosis (Mucormycosis) Systemic disseminated zygomycosis Neutropenia is main predisposing factor. Hyphae are angiotropic and usually irregular compared to Aspergillus

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

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)

Skin and Mucosal resistance Predominantly T cells important for resistance Candida AIDS defining in HIV-positive Release of cytokines from Th1 cells stimulates epidermal growth

Severe esophageal candidiasis in AIDS ulcerative erosions and barium leak

In areas where skin remains wet

Vaginitis: satellite lesions and cottage cheese-like discharge

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

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

Disseminated candidiasis in neutropenic patients Often see skin lesions

Dissemination not uncommonly includes eye and vitreous fluid

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

PF Lehmann Mycology October 16, 2008 076. Skin scraping mounted in 10%KOH from superficial candidiasis showing clusters of budding yeast cells and branching pseudohyphae.

Direct smear from urine with pseudohyphae and yeasts PF Lehmann Mycology October 16, 2008 Direct smear from urine with pseudohyphae and yeasts 077. Direct smear of urine from a patient with candidiasis of the kidney showing C. albicans in mycelial or tissue phase with blastoconidia budding from the pseudohyphae.

Chronic mucocutaneous candidiasis Rare Candida on dry skin and nails. Masses of antibodies Susceptibility is multifactorial T cell (anergy may be restricted to Candida) Endocrinopathies Zinc deficiency

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

PF Lehmann Mycology October 16, 2008 Candida species and basic growth form on Sabouraud agar (a high glucose medium) 079. Candida albicans on Sabouraud's dextrose agar showing typical cream coloured, smooth surfaced, waxy colonies. Yeast colonies Yeast cells

Special test for C. albicans

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) Inside fungal cell RNA translation 5-FC 5-FC 5-FU permease deaminase DNA synthesis inhibition

Fungal sterols as a target Fungal sterols are generally C28 sterols; especially ergosterol. (Humans cells have C27 sterols i.e.; cholesterol

(terbinafine = Lamisil®, naftidine) 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

Inhibit lanosterol demethylase Azoles Inhibit lanosterol demethylase Fungistatic Miconazole Ketoconazole Fluconazole Itraconazole Voriconazole Posaconazole 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) Acetyl CoA 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 b(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 b-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 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 acidification HA H+A-