Overview of Mycology

Overview of Mycology

• Mycology is the study of fungi, including molds and yeasts
• Molds exhibit filamentous growth, while yeasts have a pasty or mucoid form
• There are over 50,000 valid fungal species, with multiple names possibly due to variances in size, color, host, or geographical location
• Fungi are gram-positive and require oxygen
• Fungi are eukaryotic, containing a nucleus with a membrane, endoplasmic reticulum, and mitochondria
• Fungi are heterotrophic and need organic nutrients for energy, similar to animals and bacteria, whereas plants make their own food
• Fungi rely on enzymes to get energy from organic substrates
• Saprophytes live on dead organic matter
• Parasites live on other living organisms
• Fungi are important in carbon and element recycling

Role of Fungi in the Economy

• Mushrooms (Basidiomycetes) and truffles (Ascomycetes) are industrial uses of fungi
• Fungi serve as a natural food source for animals
• Yeast is a food supplement that provides vitamins
• Penicillium is used to ripen cheese, adding flavor to Roquefort, etc.
• Fungi are utilized to change texture and flavor in the manufacturing of food
• Fermentation is an industrial use of fungi
• Fruit juices go through fermentation to produce ethyl alcohol
• Saccharomyces cerevisiae is used as brewer's and baker's yeast in fermentation.
• Fats, proteins, acids, and industrial alcohol are fermented by fungi

Antibiotics and Plant Pathology

• In 1928, Alexander Fleming first discovered antibiotics, from observing how a fungus suppresses bacteria on a culture plate
• Fungi cause the majority of plant diseases

Medical Importance and Mycology

• 50-100 recognized species are human pathogens
• Many prefer to be free-living saprophytes, and only accidentally become pathogens
• Pathogenic fungi must tolerate the host site temperature and possess an enzymatic system to parasitize animal tissues
• Fungal infections have been on the rise recently

Medical Mycology - History

• From 1941 to 1973, reported deaths in the U.S. from scarlet fever, typhoid, whooping cough, diphtheria, dysentery, and malaria decreased from 10,165 to 107
• Deaths attributed to mycoses increased from 324 to 530 during the same period

Medical Mycology - Modern Man

• Increased mobility/travel to geographical areas facilitates exposure to endemic fungi
• Immunocompromised individuals are more susceptible to fungal infections
• AIDS patients, individuals taking tetracycline (acne) and birth control pills, those with excessive use of antibiotics, and people taking immunosuppressants are immunocompromised populations
• Environmental factors like air and water pollution, processed foods, and fad diets compromise the immune system, leading to fungal infections
• Incidence of fungal infections increase with ageing populations

Mycoses Immunology

• Antibody mediated immunity (B-cell humoral) stimulates antibody production in response to fungal infections
  • Serological tests are used to identify fungal diseases
• Cellular mediated immunity (T-cell) is effective in resisting fungal infections

Fungal Structures: Hyphae and Mycelium

• Hyphae are fundamental, tube-like structural units of fungi
  • Septate hyphae are divided by cross walls
  • Aseptate hyphae lack cross walls
• Mycelium is a mass of hyphae that form the vegetative part of a fungus
  • Aerial mycelium grows in the air
  • Vegetative mycelium absorbs nutrients
  • Fertile mycelium produces conidia or spores for reproduction

Fungal Spores

• Sporulation is the merging of nuclear material or genes to form spores
• Self-fertile fungi are homothallic; those with mating types are heterothallic
• Sexual spores result from the fusion of nuclei

Sexual Spores

• Ascospores form in a sac-like cell called an ascus, where eight spores are often formed (Ascomycetes)
• Basidiospores are formed through the union of two nuclei on a club-shaped structure called a basidium (Basidiomycetes)
• Zygospores are thick-walled and formed during sexual reproduction in Phycomycetes

Asexual Spores

• Asexual spores are the most common spore type
• Conidia are asexual fungal spores borne externally on a conidiophore, referred to as macro- and microconidia (Ascomycetes & Deuteromycetes)
  • Macroconidia are multicellular; Microconidia are unicellular
• Arthroconidia (Arthrospores) are asexual spores formed by mycelium disarticulation
• Blastoconidia / Blastospores are asexual spores formed from a budding process along the mycelium or another blastospore (class Ascomycetes)
• Chlamydospores are thick-walled, resistant asexual spores formed by direct differentiation, concentrating protoplasm and nutrients in the mycelium
• Sporangiospores are asexual spores contained in a sporangium at the end of a sporangoiphore (Phycomycetes)
• Thallospores are asexual spores produced on a thallus (hypha), and can be found in Deuteromycetes

Miscellaneous Mycology Terms

• Ascus: Sac-like structure in Ascomycetes containing (usually eight) ascospores
• Asexual Reproduction: Spores formed directly from vegetative mycelium/specialized hyphae
• Chromoblastomycosis: Subcutaneous mycosis from traumatic inoculation of dematiaceous fungus
• Coenocytic: Aseptate hypha or cell with numerous nuclei
• Conidiophore: Specialized hypha branch where conidia develop
• Dematiaceous: Pigmented, dark color, usually gray to black
• Dermatophyte: Fungi causing superficial mycoses
• Diphasic (dimorphic): Ability of fungi to grow as yeast or filamentous stages
• Ectothrixic: Fungus grows on the outside of a hair shaft
• Endothrixic: Fungus grows and penetrates into the hair shaft
• Endogenous: Derived from an internal source
• Exogenous: Derived from an external source
• Eukaryotes: Organisms with a true nucleus (like fungi)
• Fungemia: Fungal blood infection
• Fungi Imperfecti: Fungi class with septate hyphae, known for asexual reproduction only
• Germ Tube: Small projections from yeast cells, indicating hyphal formation
• Hyaline: Colorless, clear
• Mold: General term for filamentous fungi
• Mycetoma: Clinical syndrome of localized, tumorous lesions in cutaneous/subcutaneous tissues; often on the foot
• Mycosis: A disease caused by a fungus
• Mycotoxins: Toxins of fungal origin
• Oospore: Zygospore - Sexual spore (Phycomycetes)
• Perfect Fungi: Fungi with sexual and asexual reproductive stages
• Phycomycetes: Fungi class with coenocytic mycelium/stiff sporangiophores
• Pseudohyphae: Chain of elongated budding cells that fail to detach
• Ringworm: Circular/ring-like skin lesions caused by dermatophytes
• Rhizoids: Root-like structures
• Saprobe (Saprophyte): Organism getting nourishment from dead organic matter
• Sexual Reproduction: Zygote/spore formation after fusion of two haploid nuclei
• Sporangiophore: Hypha or stalk bearing a sporangium
• Sporangium: Sac/cell containing asexually produced spores
• Spore: Reproductive body of a fungus; resistant body for adverse environment
• Sterigmata: Structure from a basidium, supporting basidiospores
• Sporotrichosis: Mycosis from Sporothrix schenckii inoculation, lymphocutaneous type
• Telemorph: Sexual form of a fungus
• Thallospore: Spore formed by parts of the thallus changing
• Thallus: Vegetative body of a fungus
• Tinea (Ringworm): Prefix with infected area to indicate cutaneous mycosis
• Yeast: Pasty/mucoid fungus, microscopically shows budding cells
• Zoophilic: Dermatophytes which are parasitic on animals/humans

Fungi Classification by Geography

• Classification is based on geographic distribution, noting that fungal diseases are localized to particular areas
  • Histoplasmosis occurs in the Central Mississippi and Ohio River Valley
  • Coccidioidomycosis occurs in the San Joaquin Valley

Fungi Classification by Epidemiology

• Epidemiologic grouping is based on how fungal diseases spread
• Few are contagious (e.g., ringworm of the scalp)
• Mycoses depends on host susceptibility

Fungi Classification by Taxonomy

• Scientific grouping according to morphologic and cultural characteristics
• KINGDOM: Plantae
  • PHYLUM: Thallophyte - Entire plant is somatic; no roots, or leaves.
    • SUBPHYLUM: Schizomycotina
      • CLASS: Schizomycetes (bacteria)
        • ORDER: Actinomycetales
          • FAMILY: Actinomycetaceae
          • GENUS: Actinomyces
            • SPECIES: A. isarelii, A. bovis, A. naeslundii
          • GENUS: Nocardia
            • SPECIES: N. asteroides, N. brasiliensis, N. carviae
          • FAMILY: Streptomycetaceae
          • GENUS: Streptomyces
            • SPECIES: S. madurae, S. pelletierii, S. paraquavensis, S. somaliensis
  • SUBPHYLUM: Myxomycotina
    • CLASS: Myxomycetes (slime molds)
  • SUBPHYLUM: Eumycotina (true fungi)
    • CLASS: Phycomycetes (algae fungi)
      • GENUS: Rhizopus sp., Mucor sp., Others
    • CLASS: Ascomycetes
      • GENUS: Penicillium sp., Aspergillus sp., Many others
    • CLASS: Basidiomycetes
      • GENUS: Amanita sp. (many poisonous toadstools), Agaricus sp. (edible mushrooms)
    • CLASS: Deuteromycetes (fungi imperfecti)
      • Yeast that reproduce by budding.
      • GENUS & SPECIES:
        • Cryptococcus neoformans, Candida albicans; Diphasic yeast (yeast-like when grown at 37 degrees)
      • GENUS & SPECIES:
        • Blastomyces dermatitidis, Blastomyces brasiliensis, Histoplasma capsulatum, Coccidioides immitis, Sporotrichum schenkii
      • Filamentous fungi - no yeast stage
        • GENUS & SPECIES:
          • Geotrichum sp., Microsporum sp., Trichophyton sp., Epidermophyton sp.

Topographic Grouping of Fungi

• Classification is based on the type of mycoses produced

Superficial Mycoses

• Confined to outermost skin/hair layers with no host cellular/inflammatory response
• Disease is recognized on a cosmetic level

Cutaneous Mycoses

• Fungi have an affinity for keratin in skin, nails, and hair
• Most cutaneous infections from dermatophytes
• They prefer non-living layers so host response is inflammation/eczema (named by body area)
• The three genera of cutaneous mycoses include Microsporum, Epidermophyton, and Trichophyton
• These cause infection of skin, hair and nails

Cutaneous Mycoses by Genera

• Microsporum affects hair and skin rarely, and causes nail infections
  • Can remit spontaneously, and frequently occurs in children
• Trichophyton affects hair, skin and nails in both children and adults
  • Causes chronic infections
• Epidermophyton affects skin and nails, with rare hair infections
  • Commonly occurs in adults

Dermatophytes

• Dermatophytes produce septate, hyaline mycelium
• Each has a preferred host
  • Microsporum affects children, rarely adults
  • Trichophyton affects both children and adults
  • Epidermophyton affects adults, rarely children

Subcutaneous Mycoses

• Subcutaneous mycoses - infections in deeper layers of skin and often muscle tissue
• Man is an accidental host
• Introduction usually from trauma
• Infection tentatively identified by tissue reaction

Systemic Mycoses

• Attack deep tissues and organ systems, create symptoms that resemble diseases
• The two categories of systemic disease:
  • Those caused by truly pathogenic fungi with the ability to cause disease in the normal human host when the inoculum is of sufficient size.
• Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis, Paracoccidioides brasiliensis*
  • Opportunistic fungi, low virulence organisms, and infections will occur when patient's defenses are lowered.
• Aspergillus sp., Candida albicans, Cryptococcus neoformans*

Lab Methods: Specimen Collection

• Collection, handling, and processing of clinical mycology specimens
• Properly collect all specimens
• Physician or nursing staff collections
• Skin is cleaned with 70% alcohol, and surface saprophytes are removed
• Nails are cleaned and clipped, then minced before inoculation to media
• Collect hair from the scalp edge with a Wood's lamp (fluorescence)
  • Obtain hair by plucking, brushing, or using sticky tape
• Collect body fluids via normal sterile procedures

Lab Methods: Specimen Preparation

• Hair & nails are sent in a dry envelope, in a proper container
• Send frozen, or on dry ice
• Package with biohazard regulations, use tube media (no plates)
  • Use aluminum screw-capped mailing tubes with cardboard outer tubes
• Include inside labeling: patient ID, specimen source, suspected organism
• Include Outside labeling: "WARNING: POTENTIAL PATHOGEN"

Lab Methods: Processing Specimens and Body Fluids

• Skin, nails, & hair undergo direct exam via KOH preparation to recover fungus
• Appropriate body fluid processing:
  • CSF is centrifuged; examine sediment microscopically and inoculate media
  • Pleural fluid, sputum, and bronchial aspirations must be fresh; saprophytes would overgrow pathogens such as H. capsulatum, only refrigerate for 2 hours
  • Gastric washings are processed like pleural fluids
  • Genito-urinary specimens use the first morning specimen; centrifuge
  • Blood/bone marrow is inoculated directly to BHI broth/slant, inoculate to other fungal media as well
  • Wound abscess or drainage should be cultured anaerobically for suspected actinomycosis
  • Tissue specimens are examined for pus, caseous material, or granules; mince aseptically and inoculate using sterile saline

Lab Methods: Direct Examination

• Direct examination required on any biological material sent to the lab for fungus culture
  • Look for spores, hyphae, mycelial elements, budding yeast, and mycotic granules
• Wet mount prep (good for yeast examination) done in a natural medium minimizes structure loss
• KOH prep with potassium hydroxide is done on skin scraping, hail, nails, sputum, and vaginal specimens
  • The KOH digests and clears tissue cells, mucus, etc. so fungal elements are visible

Lab Methods: Stains Used

• Lactophenol Cotton Blue (LPCB) quickly evaluates fungal structures and stains chitin
• Periodic Acid - Schiff stain (PAS) stains polysaccharide in cell walls of fungi, fungal stain pink-red with blue nuclei
• Gomori Methenamine Silver stain outlines fungi in black because silver nitrate precipitates on the cell wall
  • The internal parts of hyphae are deep rose to black and the background is light green
• Gridley stains Hyphae and yeast dark blue or rose with a deep blue tissue background
• Mayer Mucicarmine stains capsules of Cryptococcus neoformans deep rose
• Fluorescent antibody stain is simple, specific and sensitive for detecting fungi and other organisms
• Papanicolaou stain is good for initial differentiation via dimorphic fungi, works well on sputum smears
• Gram stains are generally gram positive for fungi, variable for Actinomyces and Nocardia
• Modified Acid-Fast stain is used to differentiate acid-fast Nocardia from other aerobic Actinomyces
• Giemsa stains blood and bone marrow. Histoplasma capsulatum is an intracellular organism, which appears as small oval to pear-shaped yeast-like cells

Lab Methods: Yeast Stains and Fungal Culturing

• India Ink demonstrates the capsule of Cryptococcus neoformans in CSF specimens
• Tube media is generally used for fungal culturing to prevent spore release
  • Tube media also has less dehydration and is easy to store
• Inoculate agar tubes in a straight line.
  • Preliminary identification is based on differential growth patterns on various media

Culture Media

• Carefully select appropriate culture media
• Use more than one media type
• Sabouraud's dextrose agar (Sab-Dex) is a classic medium, is recommended for most studies
• Sabouraud's dextrose agar with chloramphenicol inhibits bacterial growth
• Mycosel agar inhibits bacterial growth and saprophytic fungi
  • Cycloheximide inhibits saprophytes like Aspergillus and Scopulariopsis
  • Cycloheximide also inhibits Cryptococcus neoformans
  • It inhibits bacteria-like fungi (Actinomycetes)

Culture Recovery, and Special Agar

• Brain Heart Infusion slant is more enriched than Sab-Dex, and is used in H. capsulatum recovery
• Potato-dextrose agar (PDA) and Corn-meal agar induce spore formation, aiding identification
• Caffeic Acid Agar allows Cryptococcus neoformans to produce melanin
• Birdseed Agar isolates Cryptococcus neoformans from contaminated cultures
• KT Medium & Kelley Agar converts dimorphic fungus, Blastomycetes dermatitidis, from mycelial to yeast form
• Modified Converse Liquid Medium (Levine's) promotes spherule production by Coccidioides immitis

Growth Requirements

• Grow fungi at room temperature (25-30 C)
  • Nocardia sp. and some dimorphic organisms grow best at 37°C
  • Any fungus capable of growing at 37°C should be considered a potential pathogen
• True fungi are aerobic and there are a few anaerobes
• Some yeasts grow overnight, saprophytes are fast growers (several days), and cultures are held for 4 weeks
  • Paracoccidioides brasiliensis may require 4-5 weeks, and Histoplasma capsulatum require 10 weeks

ID Techniques for Fungi

• Inoculation involves plating and slanting the specimen
• Plates are inoculated with a large "S" shape to allow rapid growing fungi to be removed
• Slants are inoculated with a straight line
• Incubation is aerobic, but can be anaerobic if Actinomycetes are suspected
• Expect dimorphic fungi, and set room temp roughly 37E
• Consider the type of media and growth rate
• Routine cultures are examined every other day for 4 weeks
• Systemic pathogens take 10 days to 2 week growth, but saprophytic fungi grow within 1 week

Colony Morphology and Mycelium

• Surface topography describes fungal colonies that are either free-growing, or grown in restricted manner
• Colonies can be cottony, granular, velvety, powdery, silky, smooth, creamy, or waxy in texture
• Pigmentation can be colorless or brightly colored on the fungus itself, its sporulating apparatus, on the agar, or on the bottom of the colony (reverse pigmentation)
  • Pigment color is due to color the sporulating apparatus
  • The top pigment and agar discoloration are crucial in determining fungi
• Vegetative mycelium provides nutrition and aerial mycelium is reproductive

Microscopic Evaluation and Hyphae

• Teased Preparation is a method of Microscopic evaluation
• Slide Culture Techniques are great because they allow for undisturbed microscopic morphology
• Transparent Tape Preparation is a method of Microscopic evaluation
• Hyphae structure has plural hyphae and single hyphae
  • Hyphae is either septate or aseptate and is either dematiaceous and hyaline

Spore Structures and Tests

• Spores are used in reproduction and terms that address reproduction exist
• Biochemical studies can be performed to identify yeast and yeast-like organisms
• Grow and utilize carbohydrates under anaerobic-ly observing acid and gas production
• Inoculate under broth that is completely covered, as acid production will turn Bromcresol purple yellow
  • Observe gas production in bubbles every 48 hours for 14 days
• Utilize carbohydrates as a carbon source in carbohydrate assimilation as seen when Bromcresol purple turns yellow
  • Tubes can be negative if unchanged
• Utilize 3 tubes that differ in nitrogen sources in nitrogen assimilation, and bromthymol blue will become blue to yellow if the test is positive
• Use Christensen's urea agar to see if the urea hydrolyzed from yeast forms ammonia that turns media yellow to dark pink
• Conduct a germ tube test of Candida albicans or stellatoidea
• Inoculate with appropriate agar and observe every 24 hours to test for chlamydospores

Hypersensitivity and Saprophytes

• Hypersensitivity (seromycology; skin tests) and serological tests can determine T-cell and B-cell immunity
• Serological tests require drawing sera in pairs during the acute convalescent phases
• Test for complement fixation, agglutination, precipitin, immunofluorescence, immunodiffusion, and counterimmunoelectrophoresis
• The goals of testing antifungal susceptibility, and the serum concentration of antifungal agents are to examine any unusual fungal isolates
• Some progress in attempt to standardize susceptibility testing as a means in evaluating unusual fungal isolates, determining the cause(s) of patient relapse and treatment failure
• Determining serum concentration of antifungal agents is important when absorption factors in kidney or liver
  • HPLC is highly accurate in drug analysis
• Saprophytes can be contaminants or opportunistic pathogens
• Most are inhibited by cycloheximide and are grouped by mycelia
• Mycelia have septate or aseptate hyaline versus dematiaceous properties

Hyaline Members

• Hyaline members can vary in growth rate. surface appearance, and color appearance
• Aspergillus mycelium is septate and hyaline with unbranched condiophores
  • A. fumigatus from a pulmonary source grows well at 37C
• Paecilomyces sp.: is a rapid grower with Brush-like conidiophores and Long, tapered sterigmata
• Penicillium sp. is commonly and rapidly white that becomes bluish-green. Conidiophores characteristically form a brush-shaped structure and Sterigmata is flask shaped
• Scopulariopsis sp. are a moderately slow growing that becomes white turning brown with age. Branched or unbranched conidiophores; sterigmata are coarsely roughened
• Trichoderma sp. Shows a moderately rapid growth with Flask-shaped conidiophores and clustered conidia

Dematiaceous Members

• Fusarium sp. has a Rapid colonial growth and be come a brightly colored. Short conidiophores are often branched, have both macro- and micro-conidia, which are oval to sickle-shaped
  • Has been reported in eye infections
• Alternaria sp. exhibits growth; colonies become very dark with age, can be overgrown
  • conidiophores bear single chains of conidia
• Curvalaria sp. Show rapid growth as velvety colonies that vary in color from grayish-brown to black that form Spirally arranged brown conidia
• Cladosporium sp. has rapid growth and reverse black mycelium
  • The dematiaceous mycelium is septate.

The Phycomycetes

• The Phycomycetes are asexual. and are always susceptible to cycloheximide.
• Some Phyomycetes are rapidly growing growers that have root-like rhizoids
• Spore (sporangia) bearing structures are called sporangiophores.
• Mucor fills a growth tube one day, lacks rhizoids, and has sporangiophores
• Rhizopus features rhizoids, and sporangiophores nodal are originally from the culture
• Syncephalastrum shows rapid growth as a white/dark colonies that grows high
  • The high, septate mycelium is short, exhibits branched sporangiophores

Yeasts: Candida

• Candida albicans often comes with oral thrush, skin infections
• Testing, production of chlamydospore on agar can identify infection
• Torulopsis glabrata is always with the candida infection and always shows during carb fermentation
  • Can be observed with elderly during diabetes
• Cryptococcus neoformans from wild pigeons that grown in melanin/warm temp
  • Identified by India ink staining
• Geotrichum will show with respiration infections, and show on vagina
  • Disease shows when immune is low

Bacteria-like Fungi

• Sulfar granulation can be observed by the Actinomycetes israeli
• To grow, require media, special staining and conditions
• They will always anaerobically grow

Biphasic Fungi and Mycoses: Sporothrix schenkii

• Sporothrix schenckii grows and can cause rose fever with gardeners are affected

Coccidioides immitis

• Coccidioides immitis Is The San Joaquin valley fever
• Exhibits as mycelila/tissue parasites

Histoplasma capsulatum

• Histoplasma capsulatum is Mississippi Valley fever (found in darling)

Deep Tissue Agents

• Blastomyces dermatitidis and North American blastomycosis / Gilcrest disease agent
• Paracoccidioides braziliensis agent of South American Blastomycosis

Types of Mycoses

• Types are based on body sites

Superficial Mycoses

• Affect skin/hair tissues, inflammatory and or destructive, but are the causation
• Malassezia furfur that cause/pityriasis
• Phaennellcomyes werneckii causes Exophiala
• Trichosporon
• Piedraia hortae

Treatment and Cutaneous Mycoses

• Cutaneous Mycoses include C. albicans & three genera are always from skin/hair tissue -*Microsporum, Epidermophyton
  • Septate and hairlike is common

Microscopic Types

• Skin and hair as Microsporum
  • Tryhoohyton always shows in all children -Trychophyton infection
• Epidermohpyton appears in a beaver-wall infection

Subcutaneous Mycoses and Infections

• Causes from soil bacteria
• Chromoblastomycosis
• Chromoblastomycosis (fungal)

Chromoblastomysos Types

• A papule caused spreading/warty or tumor like Pedrosoi is common/wears shoes, wear hygiene/world, tropical

Etiological Agents

• Fungal agents
• Ameduromycotic agents and are clinical pictures with Amoxicilitic and death is a risk
• Is slow and progressive
• Body walled Lymph node are affected/body tries and immune
• systemic is common

Agents and Blastomycosis

• Dissemination of agents from yeast, affect the entire system
• Types of Agents -Dermatitides ,Paracoccidiodomycosis

Capsulation

• Capsulation and bronchio pneumonia shows and occurs with bird/ bat stool
• Coccidiodides is asymptomatic and airborne
• Histoplasmosis produces arthroconidia
• Brazilian Para and Brazilian blaster

Treatment

• Effectiveness caries
• Nystatin is work
• Pimarcin
• B and D flucytosin
• Mica oil