Mycology: Fungal Biology

Mycology Terms

• Moulds are multicellular fungi
• Yeasts are single-celled fungi
• Mycosis describes a fungal infection
• Systemic mycosis is a multiorgan infection caused by fungi
• Opportunistic mycosis is a fungal disease primarily in immunocompromised patients
• Dimorphic fungi have both a nonmould (yeast) and mould phase
• Saprobes are organisms living on decaying organic material

Fungal Structure

• Hyphae are branching filaments forming the mycelium
• Septate hyphae have cellular separation or cross-walls, ranging from 3 to 6 μm in diameter
• Sparsely septate (formerly aseptate) hyphae contain few cellular separations, ranging from 5 to 15 μm; also referred to as coenocytic
• Pseudohyphae are chains of budding cells resembling true hyphae but constricted at the septa, forming branches that begin with septation, and have smaller terminal cells
• Vegetative hyphae are for food absorption below the agar surface
• Aerial hyphae support reproductive structures (conidia) and extend above the agar

Conidia

• Conidia are sporelike asexual reproductive structures formed only by imperfect fungi
• Conidia morphology is important for fungal identification and classification
• Microconidia are small, single-celled conidia
• Macroconidia are large, multicellular conidia
• Arthroconidia result from hyphae fragmentation, separated by disjunctor cells in some fungi
• Blastoconidia form through budding
• Chlamydoconidia form from enlarged terminal cells with thick walls, surviving adverse conditions;
• Chlamydospores are similar structures found in hyphae produced by some yeasts
• Poroconidia form by being pushed through a small pore in the parent cell
• Phialoconidia are tube-shaped and branched
• Annelloconidia are vase-shaped; the parent outer cell wall looks saw-toothed as released

Sexual and Asexual Reproduction

• Sexual reproduction involves specialized fungal structures called spores and is undertaken by perfect fungi
• Ascospores are spores in a saclike structure
• Basidiospores are spores in a club-shaped structure
• Oospores result from the fusion of cells from two different hyphae
• Zygospores result from the fusion of two identical hyphae
• Asexual reproduction involves nucleus and cytoplasm division only and is undertaken by imperfect fungi
• Imperfect fungi are the only group that produces conidia

Fungal Media

• Sabouraud dextrose agar (SDA) is a general-purpose, nutritionally poor, and mildly selective medium for fungi, no longer commonly used
• SDA has an acidic pH (5.6) in one formulation to inhibit most bacteria; modified SDA (Emmons) has a neutral pH
• Sabouraud-brain heart infusion agar (SABHI) is a nonselective medium for isolating all fungi and contains dextrose, peptone, and brain heart infusion
• SABHI can be made selective for dimorphic fungi with cyclohexamide, chloramphenicol, and gentamicin
• Brain heart infusion agar with blood (BHIB) is used to grow most fungi, especially from sterile body sites, and contains brain heart infusion and sheep blood
• BHIB can be made selective for dimorphic fungi with cyclohexamide, chloramphenicol, and gentamicin
• Cyclohexamide inhibits saprophytic fungi, chloramphenicol inhibits many gram-positive and gram-negative bacteria, and gentamicin inhibits primarily gram-negative bacteria

Selective and Differential Agars

• Selective agars have antimicrobials to enhance the growth of specific fungi while inhibiting bacteria/undesired growth
• Inhibitory mould agar (IMA) grows most fungal pathogens and specifically recovers cyclohexamide-sensitive Cryptococcus, it contains gentamicin and chloramphenicol
• Dermatophyte test medium (DTM) isolates dermatophytes, it contains cyclohexamide, gentamicin, and phenol red (pH indicator)
• Differential agars enhance pigment development, conidia production, and mould-to-yeast transition
• Potato dextrose agar (PDA) enhances conidia and pigment development of Trichophyton rubrum
• Bird seed (niger seed) and caffeic acid agars are selective and differential media used to grow C. neoformans
• C. neoformans colonies on bird seed/caffeic acid agars form black to brown colonies due to phenol oxidase; chloramphenicol enhances selectivity
• Cornmeal agar with Tween 80 differentiates Candida spp.
• Agars containing rice, casein, and other nutrients differentiate Trichophyton spp.

Culture Considerations

• Fungal cultures should be incubated at 30°C and grow over several days to weeks and should be maintained in a high-humidity environment
• The tease mount method uses a dissecting needle to pull apart a fungal colony but may damage the structure
• The cellophane tape method transfers aerial hyphae to a microscope slide for examination
• The slide culture method grows fungal colonies on the side of an agar block overlaid with a cover slip, which is removed for examination (prevents damage)

Direct Examination Methods

• A saline wet mount views fungal elements like hyphae, conidia, and budding yeasts and is used for vaginal secretions to diagnose vaginitis
• A lactophenol cotton blue wet mount stains and preserves fungal elements in culture isolates
• Potassium hydroxide (KOH) dissolves nonfungal materials in skin, nail, and hair samples
• Gram stain shows yeasts
• India ink reveals capsules surrounding Cryptococcus neoformans in CSF, however, direct antigen detection assays have generally replaced it
• Calcofluor white stain is a fluorochrome that stains chitin in the cell wall of fungi, viewed under ultraviolet light, white to blue to green depending on wavelength

Body Sites and Fungal Pathogens

• Blood: Candida spp., Blastomyces dermatitidis, Histoplasma capsulatum, and Cryptococcus neoformans
• Cerebrospinal Fluid: Cryptococcus neoformans, Candida spp., Histoplasma capsulatum, and Coccidioides immitis
• Hair: Microsporum and Trichophyton
• Nails: Aspergillus, Epidermophyton, and Trichophyton
• Skin: Candida, Microsporum, Trichophyton, Epidermophyton, and Blastomyces dermatitidis
• Lungs: Candida albicans, Aspergillus, Rhizopus, Penicillium, Histoplasma capsulatum, Blastomyces dermatitidis, and Coccidioides immitis
• Throat: Candida albicans and Geotrichum candidum
• Urine: Candida albicans and Candida glabrata
• Genital Tract: Candida albicans

Yeasts

• Yeasts commonly cause vaginitis and urinary tract infections (UTIs) and newborn infections and meningitis
• Candida albicans is the most common cause of yeast infections

Yeast Identification

• Saline wet mounts and Gram stains show budding yeast
• Yeasts are discovered in routine urinalysis
• India ink preparations are used to show the capsule surrounding Cryptococcus neoformans
• Yeasts grow on SABHI at 22-30°C
• Yeasts form cream-colored, mucoid to smooth colonies within days; yeast colonies on blood agar can resemble Staphylococcus colonies
• Cornmeal agar with Tween 80 differentiates Candida spp. by enhancing hyphae, pseudohyphae, and conidia formation
• C. albicans has chlamydospores with blastoconidia clusters along the hyphae
• C. tropicalis produces long-branched pseudohyphae and blastoconidia singly or in short chains; C. tropicalis does not produce chlamydospores
• Germ tubes are hyphaelike extensions of young yeast cells, nonseptate, and don't constrict at point of origin
• Pseudohyphae look like germ tubes but are septate and constricted at their point of origin
• Germ tube procedure: Yeasts incubated with serum at 37°C for up to 3 hours
• C. albicans is positive for germ tube production, Candida tropicalis is the negative control (although some strains can be + after 3 hours)
• Assimilation tests determine the aerobic use of carbohydrates
• Agar slants with carbs are inoculated with yeast, the medium contains bromcresol purple, tubes incubated at room temperature and read 7/14 days
• Commercial carbohydrate tests are available
• The urease test is used to identify Cryptococcus spp., which are urease positive
• C. albicans is used for the negative control for urease
• A positive urease test is indicated by a pink to purple color
• Chromagars allow identification of several yeast species metabolizing different substrates, producing different colored colonies

Clinically Significant Yeasts

• Candida albicans, the most common yeast isolate, causes candidiasis
• C. albicans is normal flora of the mucous membranes of respiratory, gastrointestinal, and female genital tracts
• Adult C. albicans infections are endogenous; infants acquire infections exogenously from their mothers
• Types of candidiasis: Thrush, vulvovaginitis, onychomycosis, and paronychomycosis
• C. albicans causes systemic infections, including meningitis, UTIs, and heart/lung infections
• Predisposition to C albicans infections include burns, wounds, diabetes mellitus, antimicrobial therapy, pregnancy, leukemia and immune problems
• Culture characteristics: C. albicans grows of fungal media and some bacteria agars
• On cornmeal agar with Tween 80, Candia isolates produce chlamydospores
• A positive germ tube may indicate C. albicans, C. dubliniensis is also positive and will form chlamydospores
• Except for C. krusei, all Candida spp. are urease negative
• Candida spp. are inositol negative
• Other clinically important Candida species: C. glabrata, C. tropicalis, C. krusei, and C. parapsilosis
• Cryptococcus neoformans causes cryptococcosis, which can produce pulmonary infection (mild-moderate) but can lead to systemic infections and meningitis in immunocompromised patients
• Cryptococcosis is also associated with prostate and tissue infections
• Cryptococcus neoformans is acquired via contact with bat/pigeon droppings or contaminated food

Cryptococcus neoformans Identification/Characteristics

• On Gram stain, yeasts appear in spherical form and are not uniform in size
• Hematoxylin and eosin stains are used to show capsules in tissue
• Direct antigen is performed on CSF and serum specimens for cryptococcal antigen
• Culture Characteristics: Brown to black colonies on bird seed or caffeic acid agars
• Only forms blastoconidia
• Biochemical tests include: urease and phenol oxidase positive, inositol utilization, and nitrate reduction is negative
• Formerly named T. beigelii, Trichosporon now includes a number of species associated with human infections (white piedra) and rarely systemic disease (trichosporonosis)
• Trichosporon spp. can be isolated from soil, animals and humans
• Trichosporon spp. form cream-colored, smooth colonies on solid media in about 1 week and produce hyaline hyphae with blastoconidia and arthroconidia
• Trichosporon are urease test positive and can assimilate carbohydrates
• Rhodotorula spp. live in moist areas and has been isolated from soil and dairy products and are generally commensals or contaminants
• Rhodotorula resembles Cryptococcus but is inositol negative and produces a pink pigment
• Geotrichum candidum is a mould is often confused with yeast based on colony morphology and produces true hyphae with rectangular arthroconidia; its clinical significance is questionable

Opportunistic Fungi: Introduction and Characteristics

• Opportunistic fungi only rarely cause disease in healthy individuals but can cause disease in individuals with medical conditions or in immunosuppressed patients
• Most opportunistic fungi form colonies within days (rapid growers)
• Humans generally acquire infections through inhalation of the conidia
• Most opportunistic fungi live on organic matter (saprophytic fungi) in the soil
• Opportunistic fungi are inhibited by antimicrobials media should not contain these substances
• Opportunistic fungi must be repeatedly isolated in patients to be considered significant, identification is based on microscopic morphology, the hyphae are hyaline

Clinically Significant Opportunistic Fungi: Aspergillus spp

• Aspergillus spp. causes aspergillosis, affecting skin, heart, lungs, and the central nervous system; pulmonary aspergillosis affects the bronchi, lungs, or sinuses
• Aspergillus fumigatus is the most common cause of aspergillosis
• A. niger causes otomycosis
• Colony morphology:
• Microscopic appearance: Hyphae are septate; conidiophores terminate in a large, spherical vesicle bearing phialides
• Species identification is based on colony appearance and microscopic characteristics and have septate hyaline hyphae
• Conidiophores arise from a foot cell and support a single vesicle at their tip, flask-shaped phialides, in a single/double row, produce chains of phialoconidia
• A. niger colonies are yellow to black with a yellow reverse
• A. flavus colonies are green to brown with red-brown reverse
• A. terreus colonies are green to yellow with yellow reverse
• A. clavatus colonies are blue to green with white reverse
• A. fumigatus colonies are green to gray with tan reverse
• Zygomycetes include Absidia, Mucor, Rhizomucor, Rhizopus and Syncephalastrum
• Zygomycetes infections are called zygomycoses and mucormycoses and produce allergic reactions in susceptible individuals with rhinocerebral infections being the most common
• Zygomycete infections can rapidly progress to a fatal outcome in immunocompromised patients and diabetics with ketoacidosis
• Spores gain entry to body sites via inhalation etc., causing infection and some produce toxins that cause gastrointestinal disturbances
• Blood Zygomycete infections (fungemia) can lead to CNS disorders
• Colony morphology: Dense growth after several days is dense, the colonies have cotton candy texture, pigmentation ranges from white to gray to brown
• Microscopic appearance: There is sparse hyaline hyphae, they are septate and are ribbonlike and thin walled
• Zygomycetes form rhizoids resembling tree roots for attachment and nutrient absorption
• Absidia have branching sporangiophores between the rhizoid and slight swelling below the columella at the base of the sporangia
• Mucor pp. have a single/branching sporangiophores without rhizoids and rhizoids are absent with no swelling noted below the columella
• Rhizopus spp. produce unbranched sporangiophores that arise opposite rhizoids with no swelling noted below the columella
• Fusarium spp. are opportunistic causing mycetomas, keratitis, and systemic infections
• Fusarium spp produce white, cottony colonies that develop pink/violet centers

Cutaneous and Superficial Fungi: Introduction

• Superficial mycoses infect the outer epithelial layers of skin and top layers of hair/nails
• Cutaneous mycoses infect the deeper layers of skin and more tissue
• Dermatophyte groups various fungi causing infections of the skin, hair and nails
• Dermatophytes are keratinophilic and contain 3 genera: Trichophyton, Epidermophyton, and Microsporum
• In cutaneous/superficial infections rare areas of the body
• Dermatophyte skin infection is termed tinea
• Tinea pedis (athlete's foot) infections of the spaces between the toes is caused by Trichophyton spp. or Epidermophyton spp., characterized by itching and scaling
• Tinea corporis (ringworm) an infection on smooth skinMicrosporum spp. and Trichophyton characterized by circular patches of scaly skin
• Tinea unguium (onychomycosis) and infection of the nails caused by Epidermophyton spp. or Trichophyton spp., causes discoloration, thickening, progressive destruction of the nails
• Tinea capitis (scalp infection) caused by Microsporum spp. or Trichophyton spp., characterized by circular bald patches on the scalp
• Tinea barbae (barber's itch) is hair infection of beard hair hairs caused by Microsporum spp. or Trichophyton spp., and characterized characterized by skin lesions
• Tinea cruris (jock itch) in the groin is infection caused by Trichophyton spp. and Epidermophyton spp., and characterized by itching and scaling of the groin area
• Identification is based on colony morphology and microscopic appearance, hair perforation test may be necessary

Characteristics of Dermatophytes

• Trichophyton colony characteristics: Two colony types seen between 7 and 10 days on SABHI
• Characteristic colonies:
  • Buff granular colonies, rose to tan colored, with yellow, brown, or red reverse
  • White fluffy colonies with a colorless to yellow reverse
• Microscopic characteristics: -Macroconidia that are smooth/thin walled, pencil shaped, contain 3–7 cells, and are few in number -Microconidia that are round to club shaped grapelike clusters and are few to numerous
• Species Identification:
  • T. mentagrophytes produces grapelike clusters of microconidia, some strains produce numerous macroconidia (+hair perforation)
  • T. rubrum produces few/numerous macroconidia, numerous club-shaped microconidia borne singularly on the hyphae (+hair perforation), deep red reverse on PDA
  • T. verrucosum produces only chlamydoconidia on SDA/PDA (no hair perforation), elongated rat-tail macroconidia on thiamine
• Epidermophyton culture characteristics: Yellow colonies with a tan reverse on SABHI and micro characteristics:
  • Macroconidia that are smooth/thin walled and club shaped numbers and micro present
  • E. floccosum invades nails and arthroconidia observed on KOH preparation
  • Khaki-yellow colonies with tan reverse at room temperature

Other Dermatophytes

• Microsporum -Colony culture at SABHI with a salmon-colored reverse, slow growers
  • 4-15 cells in thin to thick -Macroconidia that are rough/thin to thick walled numbers. Micro shaped numbers. Micro
  • species identification: M. audouinii forms with with pointed pointedends, grows poorly humans
  • thickmacroconidia species animals -Numerous elliptical macroconidia 4-6 cellssoil

Superficial Mycoses

• Tinea versicolor in caused stratum corneum, upper skin and with with
• Diagnosis with is made by using and. Lesions will underlamp
• Tinea characterised and hands and feet are caused by infections. Suggest infections on
• in 10% revealoval characteristics Trichosporon.

Subcutaneous Fungi

• Fungi gain entry skin infection (feet the most and that is infection
• Fungi the
• Mycetoma infection and the from is and includes
• is infection is and and with sclerotic bodies