Mycology Terms

Moulds: Multicellular Fungi Yeasts: Single-cell Fungi Mycosis: Fungal Infection Systemic Mycosis: Multi-organ infection caused by fungi Opportunistic mycosis: Fungal disease that occur primarily in immunocompromised patients Dimorphic fungi: Fungi that show both a non-mould and mould phase Saprobe: Organism capable of living on decaying organic Material

The Characteristics of Fungi

Body form unicellular filamentous (tube-like strands called hypha (singular) or hyphae (plural) mycelium = aggregate of hyphae sclerotium = hardened mass of mycelium that generally serves as an overwintering stage. multicellular, such as mycelial cords, rhizomorphs, and fruit bodies (mushrooms)

The Characteristics of Fungi

Heterotrophy - 'other food' Saprophytes or saprobes - feed on dead tissues or organic waste (decomposers) Symbionts - mutually beneficial relationship between a fungus and another organism Parasites - feeding on living tissue of a host. Parasites that cause disease are called pathogens.

Fungi as Saprobes and Decomposers

Fungi as Symbionts (Mutualism)

Fungi as Parasites & Pathogens

Heterotrophic by Absorption
Fungi get carbon from organic sources Hyphal tips release enzymes Enzymatic breakdown of substrate Products diffuse back into hyphae

Nucleus hangs back and directs

Product diffuses back into hypha and is used

Fungal Structures
Hyphae long, branching filaments that come together to form the mycelium
Septate hyphae with ceparation or cross walls Aseptate or Sparsely septate Pseudohyphae chain of cells formed by budding

Vegetative Hyphae functions as food absorption and are the portion that extends below the agar surface Aerial Hyphae extends above the agar forming conidia

Conidia

Types of Conidia

Arthroconidia resulting from the fragmentation of hyphae into Conidial morphology individual cells is important in fungal Balstoconidia conidia formed identification as the result of budding Microconidia small Chlamydoconidia terminal cells in the hyphae enlarge and Macroconidia large have thick walls Poroconidia formed by being pushed through a small pore Phialoconidia tube shaped that can be branched Annelloconidia vase shaped conidia

Arthroconidia

Blastoconidia

Chlamydoconidia

Poroconidia

Phialoconidia

Annelloconidia

Figure 31.5-3

Key Haploid (n) Heterokaryotic Diploid (2n) Spore-producing structures Spores Mycelium ASEXUAL REPRODUCTION PLASMOGAMY Heterokaryotic stage

KARYOGAMY

SEXUAL REPRODUCTION Zygote

GERMINATION

GERMINATION

MEIOSIS

Spores

SEXUAL REPRODUCTION
Fungal nuclei are normally haploid, with the exception of transient diploid stages formed during the sexual life cycles Sexual reproduction requires the fusion of hyphae from different mating types Fungi use sexual signaling molecules called pheromones to communicate their mating type Plasmogamy is the union of cytoplasm from two parent mycelia In most fungi, the haploid nuclei from each parent do not fuse right away; they coexist in the mycelium, called a heterokaryon In some fungi, the haploid nuclei pair off two to a cell; such a mycelium is said to be dikaryotic

 Hours, days, or even centuries may pass before the occurrence of karyogamy, nuclear fusion During karyogamy, the haploid nuclei fuse, producing diploid cells The diploid phase is short-lived and undergoes meiosis, producing haploid spores The paired processes of karyogamy and meiosis produce genetic variation

ASEXUAL REPRODUCTION
In addition to sexual reproduction, many fungi can reproduce asexually Molds produce haploid spores by mitosis and form visible mycelia

FUNGAL SPECIES IDENTIFICATION METHODS Mycology: Lab Methods

Stains used: Lactophenol Cotton Blue - very popular for quick evaluation of fungal structures; stains chitin in cell walls of fungi. Periodic Acid - Schiff Stain (PAS) - stains polysaccharide in the cell wall of fungi. Fungi stain pinkred with blue nuclei. Gomori Methenamine Silver Stain - silver nitrate outlines fungi in black due to the silver precipitating on the fungi cell wall.

 Gridley Stain - Hyphae and yeast stain dark blue or rose. Tissues stain deep blue and background is yellow. Mayer Mucicarmine Stain - will stain capsules of Cryptococcus neoformans deep rose. Fluorescent Antibody Stain - extremely specific method of detecting fungi in tissues or fluids. Papanicolaou Stain - good for initial differentiation of dimorphic fungi. Works well on sputum smears. Gram Stain - generally fungi are gram positive; but Actinomyces and Nocardia are gram variable. Acid-Fast Stain - used to differentiate the acid-fast Nocardia from other aerobic Actinomyces. Giemsa Stain - used for blood and bone marrow specimens. India Ink - demonstrates the capsule of Cryptococcus neoformans in CSF specimens.

Fungal Culturing: Tubed media is used rather than plated media. Less chance for spore release into the environment. Less chance for dehydration. Easier storage. The agar in a tube is inoculated in a straight line. Preliminary identification is based on differential growth patterns on various media. Media: Sabouraud's dextrose agar - classic medium, recommended for most studies. Sabouraud's dextrose agar with chloramphenicol (inhibits bacterial growth). Mycosel agar - a commercially produced agar containing chloramphenicol to inhibit bacterial growth, and cycloheximide to inhibit saprophytic fungi and some yeasts (including C. neoformans). Aspergillus and Scopulariopsis are opportunistic pathogens. Cycloheximide will prevent their growth. Bacteria-like fungi (such as Actinomycetes) are inhibited by chloramphenicol.

 Brain heart infusion slant (BHI) - more enriched than Sab-Dex. For recovery of H. capsulatum. Potato-dextrose agar (PDA) and Corn-meal agar - used in slide cultures to induce spore formation, which aids in identification. Special applications agar: Caffeic Acid Agar - Cryptococcus neoformans will produce melanin resulting in black colonies (must protect media from light). Birdseed Agar - used to isolate Cryptococcus neoformans from contaminated cultures. KT Medium & Kelley Agar - used to convert dimorphic fungus Blastomyces dermatitidis from mycelial to yeast form. Modified Converse Liquid Medium (Levine's) - used to promote spherule production by Coccidioides immitis.

Fungal growth requirements: Temperature - room temperature (25-30 C.) for most fungi. Nocardia sp. and some dimorphic organisms grow best at 37 degrees C. Any fungus capable of growing at 37 C, should be considered potentially pathogenic. Atmosphere - True fungi are aerobic but there are a few anaerobes among the bacteria-like fungi. Time - Some yeasts grow overnight. Saprophytes are fast growers (several days). Generally cultures are held at least 4 weeks. Paracoccidioides brasiliensis may require 4-5 weeks; 10 weeks are recommended if Histoplasma capsulatum is suspected. Routine cultures should be examined every other day.

Colony Morphology (macroscopic features): Surface topography - some fungal colonies may be free growing, covering the entire surface of agar; others may grow in a restricted manner. Surface texture - cottony or wooly (floccose), granular, chalky, velvety, powdery, silky, glabrous (smooth, creamy), or waxy. Pigmentation - Fungi may be colorless or brightly colored. Color may be on fungus itself, on its sporulating apparatus, on the agar, or on the bottom of the colony (reverse pigmentation). Mycelium Vegetative mycelium provides nutrition. Aerial mycelium reproductive.

Microscopic evaluation: Teased Preparation - wet mount Slide Culture - gives undisturbed microscopic morphology. Cellophane Tape Preparation Biochemical studies - used to ID yeast and yeast-like organisms. Carbohydrate fermentation Growth and utilization of a carbohydrate under anaerobic conditions as determined by acid and gas production. Specimen is inoculated beneath the broth. Bromcresol purple is the indicator. Acid production turns purple to yellow. Gas is detected by appearance of bubbles trapped in the fermentation tube. Observe every 48 hours for 14 days.

 Carbohydrate assimilation Determines ability to utilize a carbohydrate as sole source of carbon. Bromcresol purple indicator turns from purple to yellow. Tubes unchanged (as determined by comparing to a blank tube) after 10 days are negative. Nitrogen assimilation Utilizes 3 tubes with differing sources of nitrogen. Bromthymol blue is the indicator (blue to yellow is positive).

Growth on specific agars: Christensen's urea agar - Urea is hydrolyzed by some yeast to form ammonia (pH increases) which turns media from yellow to dark pink. Caffeic acid medium (must protect media from light) Production of melanin by Cryptococcus neoformans resulting in black colonies. Other tests: Germ tube - Candidia albicans & Candidia stellatoidea produce germ tubes when incubated in a protein medium. Demonstration of chlamydospores - Yeast is inoculated by jabbing appropriate agar (Cornmeal with tween 80) and observed every 24 hours for 3 days for chlamydospore production.

Diagnosis of Mycotic Infections

Diagnosis and identification require microscopic examination of stained specimens, culturing in selective and enriched media and specific biochemical and serological tests

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BODY SITES AND POSSIBLE FUNGAL PATHOGENS

Blood Candida sp, Blastomyces dermatitidis, Histoplasma capsulatum, Cryptococcus neoformans CSF Cryptococcus neoformans, Candida sp. Histopasma capsulatum, and Coccidioides immitis Hair Microsporum and Trichophyton Nails Aspergillus, Epidermophyton, and Trichophyton Skin Candida, Microsporum, Trichophyton, Rhizopus, Penicillium, Histoplasma capsulatum, Blatomyces dermatitidis, and Coccidioides immitis Throat Candida albicans, Geotrichium candidum Urine Candida albicans, Candida glabarata GUT Candida albicans

Fungi as Infectious Agents

Molds and yeasts are widely distributed in air, dust, fomites, and normal flora Humans are relatively resistant Fungi are relatively nonpathogenic Of the 100,000 fungal species, only 300 have been linked to disease in animals Fungi are the most common plant pathogens Human mycoses are caused by true fungal pathogens and opportunistic pathogens

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 True or primary fungal pathogen can invade and grow in a healthy, noncompromised host Most striking adaptation to survival and growth in the human host is the ability to switch from hyphal cells to yeast cells Thermal dimorphism grow as molds at 30C and as yeasts at 37C

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THERMAL DIMORPHISM

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Emerging Fungal Pathogens

Opportunistic fungal pathogen has little or no virulence; host defenses must be impaired Vary from superficial and colonization to potentially fatal systemic disease An emerging medical concern; account for 10% of all nosocomial infections Dermatophytes may be undergoing transformation into true pathogens
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Pathogenesis of the Fungi

Portal of entry Primary mycoses respiratory portal; inhaled spores Subcutaneous inoculated skin; trauma Cutaneous and superficial contamination of skin surface Virulence factors thermal dimorphism, toxin-like substances, capsules and adhesion factors, hydrolytic enzymes, inflammatory stimulants

Antifungal defenses are the integrity of the barriers and respiratory cilia Most important defenses are cell-mediated immunity, phagocytosis, and inflammation Long-term immunity can only develop for some
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Control of Mycotic Infections

Immunization is not usually effective Control involves intravenous amphotericin B, flucytosine, azoles, and nystatin In some cases surgical removal of damaged tissues Prevention limited to masks and protective clothing to reduce contact with spores

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Organization of Fungal Disease

Mycoses are presented according to type, level of infection, and degree of pathogenicity
True pathogens: systemic, cutaneous, and superficial mycoses Opportunistic mycoses

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Levels of invasion by fungal pathogens

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Systemic Infections by True Pathogens

Restricted to endemic regions of the world Infection occurs when matter containing conidia is disturbed Spores germinate in the lungs Infection can become systemic Spores may be inoculated into the skin All diseases result in immunity
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Histoplasmosis: Ohio Valley Fever

Histoplasma capsulatum most common true pathogen; causes histoplasmosis Typically dimorphic Distributed worldwide, most prevalent in eastern and central regions of U.S. Grows in moist soil high in nitrogen content Inhaled conidia produce primary pulmonary infection that may progress to systemic involvement of a variety of organs and chronic lung disease Amphotericin B, ketoconazole
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Dimorphic colonies of Histoplasma capsulatum

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Events in Histoplasma infection and histoplasmosis

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Coccidioidomycosis: Valley Fever

Coccidioides immitis causative agent Distinctive morphology blocklike arthroconidia in the free-living stage and spherules containing endospores in the lungs Lives in alkaline soils in semiarid, hot climates and is endemic to southwestern U.S. Arthrospores inhaled from dust, creates spherules, and can form nodules in the lungs Amphotericin B treatment
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Events in Coccidioides infection

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Disseminated coccidioidomycosis

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Blastomyces Dermatitidis: North American Blastomycosis

Blastomyces dermatitidis causes blastomycosis Dimorphic Free-living species distributed in soil of a large section of the midwestern and southeastern U.S. Inhaled 10-100 conidia convert to yeasts and multiply in lungs Symptoms include cough and fever Chronic cutaneous, bone, and nervous system complications Amphotericin B
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The dimorphic nature of Blastomyces dermatitidis

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Cutaneous blastomycosis in the hand and wrist

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Paracoccidioidomycosis
Paracoccidioides brasiliensis Distributed in Central and South America Lung infection occurs through inhalation or inoculation of spores Systemic disease is not common Ketoconazole, amphotericin B, sulfa drugs

The morphology of Paracoccidioides

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Subcutaneous Mycoses
Subcutaneous mycoses: when fungi are transferred directly into traumatized skin, they can invade Most species in this group are greatly inhibited by higher temperatures of the blood and viscera Diseases are progressive

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Sporothrix Schenckii
Sporotrichosis (rose-gardeners disease) Very common saprobic fungus that decomposes plant matter in soil Infects appendages and lungs Lymphocutaneous variety occurs when contaminated plant matter penetrates the skin and the pathogen forms a nodule, then spreads to nearby lymph nodes

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The microscopic morphology of Sporothrix schenckii

Clinical appearance of lymphocutaneous sporotrichosis

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Chromoblastomycosis and Phaeohyphomycosis

Chromoblastomycosis: A progressive subcutaneous mycosis characterized by highly visible verrucous lesions
Etiologic agents are soil saprobes with dark-pigmented mycelia and spores Fonsecaea pedrosoi, Phialophora verrucosa, Cladosporium carrionii Produce very large, thick, yeast-like bodies, sclerotic cells

Phaeohyphomycosis differs in the causative species and the appearance of the infectious agent

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Mycetoma
When soil microbes are accidentally implanted into the skin Progressive, tumor-like disease of the hand or foot due to chronic fungal infection; may lead to loss of body part Caused by Pseudallescheria or Madurella

Mycetoma caused by Madurella

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Cutaneous Mycoses
Infections strictly confined to keratinized epidermis (skin, hair, nails) are called dermatophytoses ringworm and tinea 39 species in the genera Trichophyton, Microsporum, Epidermophyton Closely related and morphologically similar Causative agent of ring worm varies case to case

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 Natural reservoirs humans, animals, and soil Hardiness of the dermatophyte spores, presence of abraded skin, and intimate contact promote infection Long infection period followed by localized inflammation and allergic reactions to fungal proteins

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 Ringworm of scalp (tinea capitis) affects scalp and hair-bearing regions of head; hair may be lost Ringworm of beard (tinea barbae) affects the chin and beard of adult males; contracted mainly from animals Ringworm of body (tinea corporis) occurs as inflamed, red ring lesions anywhere on smooth skin Ringworm of groin (tinea cruris) jock itch affects groin and scrotal regions

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 Ringworm of foot and hand (tinea pedis and tinea manuum) is spread by exposure to public surfaces; occurs between digits and on soles Ringworm of nails (tinea unguium) is a persistent colonization of the nails of the hands and feet that distorts the nail bed Treatment of dermatophytes includes topical antifungal agents tolnaftate, miconazole applied for several weeks Lamisil or griseofulvin 1-2 years
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Ringworm lesions Ringworm of the extremities

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Superficial Mycoses
Tinea versicolor caused by Malassezia furfur; elicits mild, chronic scaling, mottling of skin; also implicated in folliculitis, psoriasis, and seborrheic dermatitis White piedra caused by Trichosporon beigelii; whitish or colored masses develop scalp, pubic, or axillary hair Black piedra caused by Piedraia hortae; dark-brown to black gritty nodules, mainly on scalp hairs
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Opportunistic Mycoses
All have predisposing factors
Candida dominant opportunistic pathogen Aspergillus accounts for most lung infections Cryptococcus Alternaria Paecilomyces Fusarium Rhizopus Torulopsis

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 Candida albicans Widespread yeast Infections can be short-lived, superficial skin irritations to overwhelming, fatal systemic diseases Budding cells of varying size that may form both elongate pseudohyphae and true hyphae Forms off-white, pasty colony with a yeasty odor

Infections by Candida: Candidiasis

Candida Albicans
Normal flora of oral cavity, genitalia, large intestine or skin of 20% of humans Account for 70% of nosocomial fungal infections Thrush occurs as a thick, white, adherent growth on the mucous membranes of mouth and throat Vulvovaginal yeast infection painful inflammatory condition of the female genital region that causes ulceration and discharge Cutaneous candidiasis occurs in chronically moist areas of skin and in burn patients
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Infections by Candida albicans

Detection of Candida albicans

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Diagnosis and Treatment

Presumptive diagnosis made if budding yeast cells and pseudohyphae are found; germ tube Growth on selective, differential media differentiates Candida species Topical antifungals for superficial infections, amphotericin B and fluconazole for systemics

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Cryptococcosis and Cryptococcus Neoformans

Cryptococcus neoformans causes cryptococcosis A widespread encapsulated yeast that inhabits soil around pigeon roosts Common infection of AIDS, cancer, or diabetes patients Infection of lungs leads to cough, fever, and lung nodules Dissemination to meninges and brain can cause severe neurological disturbance and death
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Diagnosis and Treatment

Negative stain demonstrating encapsulated budding yeast Biochemical tests, serological testing Systemic infection requires amphotericin B and fluconazole

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Pneumocystis (Carinii) Jiroveci and Pneumocystis Pneumonia A small, unicellular fungus that causes pneumonia (PCP), the most prominent opportunistic infection in AIDS patients This pneumonia forms secretions in the lungs that block breathing and can be rapidly fatal if not controlled with medication Pentamidine and cotrimoxazole
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Aspergillosis: Diseases of the Genus Aspergillus

Very common airborne soil fungus 600 species, 8 involved in human disease; A. fumigatus most commonly Serious opportunistic threat to AIDS, leukemia, and transplant patients Infection usually occurs in lungs spores germinate in lungs and form fungal balls; can colonize sinuses, ear canals, eyelids, and conjunctiva Invasive aspergillosis can produce necrotic pneumonia, and infection of brain, heart, and other organs Amphotericin B and nystatin
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Clinical aspects of Aspergillosis

Microscopic appearance of Aspergillus

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Zygomycosis
Zygomycota are extremely abundant saprobic fungi found in soil, water, organic debris, and food Genera most often involved are Rhizopus, Absidia, and Mucor Usually harmless air contaminants invade the membranes of the nose, eyes, heart, and brain of people with diabetes and malnutrition, with severe consequences

Absidia corymbifera 70

Miscellaneous Opportunists
Any fungus can be implicated in infections when immune defenses are severely compromised Geotrichum candidum geotrichosis; mold found in soil, dairy products; primarily involved in secondary lung infections Fusarium species soil; occasionally infects eyes, toenails, burned skin

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Common fungi that can cause uncommon infections

Fungal spores are common sources of atopic allergies Seasonal allergies and asthma Farmers lung, teapickers lung, bark strippers disease Fungal toxins lead to mycotoxicoses usually caused by ingesting or inhaling fungal toxins Aflatoxin toxic and carcinogenic; grains, corn, peanuts; lethal to poultry and livestock Stachybotrys chartarum sick building syndrome; severe hematologic and neurological damage
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Fungal Allergies and Intoxications