Intro Mycol

• Fungi are eukaryotes with a true nucleus and many chromosomes
• Fungi do not have peptidoglycan in their cell walls
• Fungi absorb food directly through their cell walls and reproduce sexually and asexually
• Fungi are absorptive heterotrophs that decompose dead organisms and waste, including cellulose, lignin, and keratin
• Fungi can be saprobic, parasitic, or mutualistic, with digestive enzymes secreted outside of the cell to break down large molecules
• Medical Mycology is the study of fungi, including yeasts and molds, which are considered lower plants
• Fungal cell walls are made of chitin and contain 5-10% protein and 50-60% carbohydrate polymer
• Fungi require a carbon source for growth and are responsible for alkali resistance and resisting osmotic pressure
• Fungi have a cell membrane containing ergosterol or zymosterol, and they provide strength to the cell
• Yeast morphology includes individual oval to round cells that bud to form daughter cells
• Mold morphology consists of multicellular hyphae with or without crosswalls, forming mycelium
• Dimorphic fungi can switch between yeast and mold morphologies based on temperature or CO2 concentration
• Fungal spores are single-celled reproductive structures that can be produced sexually or asexually
• Asexual reproduction in fungi is generally faster but results in less genetic diversity
• During sexual reproduction in fungi, haploid cells fuse to form a zygote, leading to genetic diversity
• Fungal infections can be transmitted through inhalation, traumatic inoculation, medical devices, or person-to-person contact
• Types of fungal diseases include intoxication, mycotoxicosis, mycetismus, hypersensitivity diseases, colonization, and infections
• Fungal infections can be superficial & cutaneous, subcutaneous, systemic, or opportunistic, affecting different body areas
• The clinical microbiology laboratory offers different levels of service for fungal specimen examination and identification
• Fungal specimens for testing include blood, bone marrow, sterile body fluids, tissue, respiratory tract samples, urine, hair/skin/nails, oropharyngeal samples, and vaginal secretions
• Fungal Specimens Transport & Storage:

  • Process and plate within 2 hrs of collection
  • Avoid excessive heat or cold
  • Leave at room temperature
  • Exceptions:
  • CNS samples store at 30°C if there is a delay in processing
  • Store at 4°C if the sample is contaminated with bacteria
  • Urine and respiratory specimens: keep biopsy specimens moist
  • Skin, hair, and nails: store in a clean dry envelope
• Direct Microscopic Examinations for Fungal Culture:

  • Saline or wet preparation to detect fungal elements like hyphae, pseudohyphae, and blastoconidia
  • Lactophenol cotton blue (LPCB) preserves fungal structures and stains chitin in fungal cell walls
  • 10-40% KOH dissolves keratin in tissue cells, fungal cells survive because chitin resists alkali
  • Yeasts stain purple in Gram stain, but are not considered Gram-positive
  • Hyphae often stain in a granular pattern
• Identification of Isolates from Fungal Culture:

  • Request fungal cultures if suspecting a fungal infection, not routinely plated
  • Specimens where fungi are clinically important:
  • Sterile body fluids like CSF, blood, pleural fluid, synovial fluid
  • Immunocompromised patients at any specimen site
  • Sputum growing 4+ yeast or filamentous fungus
  • Fungal isolate repeatedly growing from the same clinical source
  • Sputum growing as much Aspergillus sp. as normal flora
• Identification of Fungal Isolates:

  • Growth conditions:
  • Media: Sabouraud dextrose agar (SAB), SAB with cyclohexamide and chloramphenicol (mycosel agar) pH 7.0, Brain-heart infusion (BHI), Bird seed agar for Cryptococcus neoformans, Cornmeal with Tween 80 agar
  • Temperature: 22-30°C and sometimes 35-37°C
  • Incubation: 1 to 4 weeks until considered NO GROWTH=Negative, systemic fungi may take up to 12 weeks
  • Macroscopic:
  • Growth rate: rapid, intermediate, slow growers
  • Microscopic:
  • Reproductive structures like chlamydospores, hyphae, pseudohyphae, arthroconidia
• Antifungal Therapies:

  • Less antifungal agents available compared to antibacterial options
  • Best options target unique fungal characteristics like fungal cell wall and membrane
  • Fungistatic stops organism growth but does not kill isolate, fungicidal kills organism
  • Delivery of antifungal: topical, oral, intravenous
  • Drugs include Amphotericin B, Nystatin, Flucytosine, Azoles, Echinocandins, Terbinafine
• Drug: Polyene antifungal class - Amphotericin B:

  • Mode of action: Binding to ergosterol in cell membrane resulting in pore formation causing leakage of potassium and other cell components
  • Target organisms: broad usage including Candida sp., Cryptococcus, Aspergillus
  • Problems: renal toxicity in 80% of patients within 2 weeks of therapy
• Drug: Polyene antifungal class - Nystatin:

  • Mode of action: Irreversible interaction with sterols, including ergosterol in plasma membrane resulting in pore formation
  • Target organisms: broad usage especially useful for systemic Candida sp. infections, Cryptococcus, Aspergillus
  • Problems: renal toxicity
• Drug: New class - anti β(1,3) D-glucan synthase - Echinocandins:

  • Mode of action: inhibitor of β(1,3) D-glucan synthase, needed for cell wall β(1,3) D-glucan polysaccharide synthesis
  • Target organisms: broad spectrum against Candida sp. and Aspergillus sp.
  • Problems: rare problems with fever, rash, GI upset
• Drug: Allylamine class - Terbinafine:

  • Mode of action: disruption of ergosterol synthesis by inhibiting squalene epoxidase
  • Target organisms: Candida sp., dermatophytes Trichophyton sp.
  • Problems: rare reports of hepatotoxicity, exacerbation of lupus erythematosus, GI upset
• Drug: Flucytosine (5-fluorocytosine, 5-FC):

  • Mode of action: inhibits protein synthesis and DNA synthesis
  • Target organisms: Cryptococcus sp., utilized for non-invasive Candida sp.
  • Problems: rapid resistance, hepatic toxicity rarely reported
• Drug: Azole class - Azoles:

  • Mode of action: Inhibition of fungal cytochrome P-450 enzyme needed for the synthesis of ergosterol in the cell membrane
  • Target organisms: Candida sp. and 2nd generation drugs can be utilized for the treatment of molds
  • Problems: rash, rare GI disruption, rare report of hepatic disease
• Saprobes absorb nutrients from dead organic matter.
• Parasites absorb nutrients from living hosts.
• Mutualists derive nutrients from other organisms in a way thatbenefits both partners
• Bud to form daughter cells = blastoconidia or blastospores
• Hyphae – long strand of cells• With crosswalls = septate• Without crosswalls = aseptate/nonseptate or coenocytic• Mass/group of hyphae = mycelium
• Pseudohyphae (false hyphae) – Elongated blastoconidia, constricted at their point of attachment, true hyphae are not constricted
• Sporangiospores form inside a sac called asporangium, which is often borne on a spore-bearing stalk, called sporangiophore, fromeither the tips or sides of hyphae.
• Chlamydospores form with a thickened cellwall inside hyphae
• Conidiospores (also called conidia) areproduced at the tips or sides of hyphae butnot within a sac. There are many types ofconidia, some of which develop in chains onstalks called conidiophores.