Myco

• • Fungi are obligate aerobes, can survive in neutral pH or moist areas or even dry conditions
• General characteristics of fungi:

  • Possess true nucleus
  • Have a nuclear membrane
  • Contain mitochondria
  • Can be observed as yeast (color: tan/yellowish) or mold
  • Characteristics include:
  • Chitin in cell wall
  • Ergosterol in the cell membrane
  • Sexual or asexual reproduction
  • Lack chlorophyll
  • Lack antibiotic susceptibility
  • Saprophytic: living on dead or decayed organic matter in nature
  • Humans are accidental hosts, inhaling spores or through the introduction of fungal elements into tissue by trauma
• General considerations for the identification of yeasts:

  • Colonial morphological features
  • Microscopic morphological features
  • Physiologic studies
  • Rapid commercial yeast identification tests
• Yeast characteristics:

  • Single vegetative cells (unicellular)
  • Moist, creamy, opaque, or pasty colonies on media
  • Reproduce through "budding"
  • Bastoconidium (daughter cell)
  • Enlargement of cells → Parent cell undergoes mitosis → Septum formation → Fission
  • Nucleus is passed into daughter cell
• General considerations for the identification of molds:

  • Growth rate
  • Colonial morphologic features
  • Microscopic morphologic features
• Molds/filamentous fungi characteristics:

  • Appear fluffy, cottony woolly, or powdery colonies
  • Formation of mycelia:
  • Hyphae – Aerial (supports reproductive structures) or Vegetative
  • Septate – frequent cross walls
  • Sparsely septate – irregular intervals
  • Aseptate – absent
  • Colonial topography (Elevation):
  • Verrucose (furrowed or convoluted)
  • Umbonate (slightly raised in the center)
  • Rugose (furrows radiate out from the center)
• Growth rates of molds:

  • Slow growers: 11 - 21 days
  • Intermediate growers: 6 - 10 days
  • Rapid growers: 5 days or less
• Dimorphic and polymorphic fungi:

  • Dimorphic fungi exhibit either a yeast or yeastlike phase and filamentous forms
  • Thermally dimorphic fungi produce a mold form at 25°C to 30°C and a yeast form at 35°C to 37°C
  • Polymorphic fungi have more than one independent form or spore stage in their life cycle
• Reproduction of fungi:

  • Reproduce asexually or sexually
  • Asexual/imperfect fungi produce spores
  • Formation of conidia derived from hyphae
  • Arthroconidia form fragments
  • Microconidia are small, unicellular, round elliptical
  • Macroconidia are large, usually multiseptated, and club- or spindle-shaped
• Sporangiospore:

  • Presence of sporangiospore
  • Asexual spores produced through the structure sporangium
• Sexual/perfect fungi:

  • Joining of two compatible nuclei, followed by meiosis
  • Teleomorph fungi reproduce sexually
  • Synanomorphs are asexual forms of the same fungus
• Fungi phyla:

  • Ascomycota, Basidiomycota, subphylum Mucoromycotina, and Fungi imperfecti (Deuteromycota)
  • Fungi imperfecti have no mode of reproduction
  • 50% of fungi belong to the phylum Ascomycota
• Laboratory diagnosis:

  • Safety issues include using a Class II biological safety cabinet
  • Specimen collection, handling, and transport are crucial for fungal infection diagnosis
  • Proper collection of specimens, rapid transport, and immediate processing in the clinical laboratory are essential for recovery of fungi
  • Viability of fungi decreases over time
• Respiratory tract secretions:

  • Samples include sputum, induced sputum, bronchial washings, bronchoalveolar lavage, and tracheal aspirations
  • Storage at room temp – processed within 2 hrs or refrigerated (4C) for delay
  • Container: sterile, screw top
  • Swab: Dacron swab
  • Mucolytic agent: N-acetyl-L-cysteine
  • Media: Nonselective or with antibacterial agents
• Cerebrospinal fluid collection:

  • Filtered through a 0.45-mm membrane filter attached to a sterile syringe
  • Media used should contain no antibacterial or antifungal agents
  • Processed promptly or kept at room temperature or in a 30°C incubator
  • CSF specimens should never be refrigerated
• Blood cultures:

  • Systems like BACTEC, BacT/ALERT, VersaTREK are adequate for recovery of yeasts
  • Optimal temperature for fungal blood cultures is 30°C, with an incubation time of 21 days
• Eye (corneal scrapings or vitreous humor):

  • Corneal scrapings placed directly onto microscopic slides and inoculated onto non-inhibitory media
  • Vitreous humor aspiration concentrated by centrifugation for smears and culture
  • Samples processed as soon as possible and stored at room temperature
  • Media containing cycloheximide should be avoided
• Hair, skin, and nail scrapings:

  • Wood lamp (UV of >365 nm) used to detect fungi presence
  • Sterile forceps to pull affected hair
  • Samples placed directly in petri dish and inoculated on fungal medium
  • Incubation at 22°C – 30°C
  • Skin and nails cleaned with 70% isopropyl alcohol
• Skin and nails should be cleaned with 70% isopropyl alcohol before sampling
• Only the leading edge of skin lesions should be sampled, as the centers often contain nonviable organisms
• Samples collected from lesions may be obtained by scraping the skin or nails with a scalpel blade or microscope slide
• Deeper scrapings are needed for KOH when sampling nails
• Vaginal samples should be transported to the laboratory within 24 hours of collection using culture transport swabs
• Swabs should be kept moist in sterile tubes
• Both selective and inhibitory agars should be plated for vaginal cultures
• Urine samples should be processed as soon as possible after collection
• All urine samples should be centrifuged, and the sediment cultured using a loop for adequate isolation of colonies
• If processing is delayed, urine specimens should be refrigerated at 4°C
• All tissues should be processed before culturing by mincing, not grinding
• Tissue pieces should be pressed into the appropriate culture media
• Bone marrow may be collected in a heparinized syringe
• Sterile body fluids should be concentrated by centrifugation before culturing
• Direct microscopic examination methods include potassium hydroxide preparation, KOH with Calcofluor white stain, India ink, and tissue stain
• KOH breaks down keratin in samples like skin, hair, nails, and tissue
• India ink is used to examine CSF for encapsulated yeast C. neoformans
• Periodic acid Schiff (PAS) stain is used for the detection of fungi
• Primary media for culturing is Sabouraud dextrose agar
• Cultures should be incubated at 30°C for 21 to 30 days
• Germ tube test is used to differentiate yeast based on their ability to create a germ tube
• Superficial mycoses infect the outermost layer of skin or hair