Lecture 1 (M&P)

Saprobes

Absorbs nutrients from dead organic matter

Parasites

Absorbs nutrients from living hosts

Mutualists

Benefits both partners

Fungi

• Aerobic

• Eukaryotic with membrane-bound nucleus

• Cell wall made up of Chitin (alkali, osmotic, strength)

• Cell membrane made up of ergosterol or zymosterol

• Require carbon source, no photosynthesis

• Fungi is a thallus (does not have roots, stems, and leaves)

Mold (morphology)

• Multicellular

• Hyphase long - strand of cells

• Without wall - septate

• With wall - coenocytic or aseptate

• Mass group of hyphae = mycelium

• Pseudohyphae (false hyphase) = Elongated blastoconidia, constricted at their point of attachment, true hyphae are not constricted

Yeast (morphology)

• Unicellular

• Individual oval/round cells

• Buds to form daughter cells (blastoconidia or blastospores)

• After mitosis, daughter nucleus is \n isolated from the parent \n cell by the formation of a new wall around it

Dimorphic Fungi

• Two Shaped Fungi

• When there is changes in temperature or CO2 some fungi can change into Molds or Yeasts

• Yeast: 35-37 C

• Mold: 25 - 30 C

Spores

• Single celled reproductive structure

• Sexual or Asexual reproduction

• Sexual: Fusion of two haploids to form a zygote

• Asexual: Nuclear and cytoplasmic division or mitosis to produce two or more identical cells

• Extremely small and can easily spread

Asexual Reproduction (Spores)

• Most common and faster

• Less genetic diversity

• Produced by one parent only (through mitosis)

• Genetically identical to that parent

• Released from the parent hypha

• Reproduce asexually by fragmentation, budding, or producing spores

Sporangiospores (Asexual Reproduction)

Spores that develop within a sac (sporangium) at a hyphal tip or side

Chlamydospores (Asexual Reproduction)

Forms with a thickened cell wall inside hyphae.

Conidiospores (Asexual Reproduction)

Spores that are not produced in a sac, but do develop at a hyphal tip or side

Sexual Reproduction

• Greater genetic diversity

• Occurs in response to adverse environmental conditions

• Fungal mating types “+” and “-” not male and female as hyphae are morphologically indistinguishable

• Both mating types present in the same mycelium (homothallic or self-fertile)

Four Stages for Sexual Reproduction

• Plasmogamy: brings together the two haploid nuclei (not fused)

• Karyogamy: fusion of these haploid nuclei and the formation of a diploid nucleus (zygote)

• Meiosis: takes place in the gametangia organs and restores the haploid state

• Gametes of different mating types are generated. At this stage, spores are let into the environment

Zygospore Fungi (zygomycota)

Contains haploid nuclei that are incorporated into spores

Sac Fungi (ascomycota)

• Ascus

• Eukaryotic and haploid hyphae

Club Fungi (basidiamycota)

• Basidia

• Eukaryotic hyphae

• Plasmogamy phase can last a long time

Modes of Transmission

1. Inhalation of conidia (must work with molds in type 2 BSC & wear gloves)

2. Traumatic Inoculation (contact with soil)

3. Medical Devices (catheter)

4. Person to person (yeasts)

Intoxication

Accidental of ingestion of fungal metabolites: alkaloids, psychotropic chemicals, aflatoxin, other toxic substances

Mycotoxicosis

Ingestion of toxins produced from fungus

Mycetismus

Mushroom poisoning resulting from eating toxins found within mushrooms

Hypersensitivity Disease

• Allergic Disease

• Type I hypersensitivity - fungal spore exposure

• Inhalation – asthma

• Skin – eczema

• Eye/sinus(nasal mucous membrane) – rhinitis, hay fever

Colonization

Yeast isolates are commonly identified as normal microbiota of skin and mucous membranes

Infection

• Mild and self-limiting or severe life threatening

• Opportunistic or true-pathogenic isolates regardless of immune status

Superficial & Cutaneous

• Affecting superficial layers of skin, hair, and nails

• Cause little or no inflammation

Subcutaneous Mycoses

• Localized infections of subcutaneous tissue following the traumatic implantation of the aetiologic agent

• Results in significant inflammation

Systemic Mycosis

Deep tissue and organ infection resulting from dissemination of from other area (lung, skin, traumatic injury)

Opportunistic

Immunocompromised

Level I

Direct examination of clinical samples for fungal elements

Level II

• Identification of yeasts using commercially available systems

• Latex agglutination for detection of cryptococcal antigen

• Performance of commercial fungal immunodiffusion tests

Level III

Level II plus identification of filamentous fungi

Level IV

• Level III plus identification of all fungi

• Performance of all fungal serological tests

• Performance of fungal susceptibility tests

Blood (best specimen type)

Lysis centrifugation Wampole Isolator System

• Must use for H. capsulatum

• Plate on to media absent of cycloheximide, CAP

Special media BACTEC MYCO/F Lytic Bottle

• 20-30ml blood volume

Continuous monitoring systems

Bone marrow (best specimen type)

Heparinized marrow inoculated into Isolator tubes

• 0.5ml sample minimum

Sterile body fluids (best specimen type)

Tissue (best specimen type)

• Ground and homogenize

• Direct inoculation on to media

• If suspect Zygomycete sp. mince/slice sample

Respiratory tract (best specimen type)

• 1st morning specimen

• Plate flecks of blood, pus

• Decontaminate/liquify as for Mycobacterium

Urine (best specimen type)

10-50ml centrifuge, plate sediment with and without cycloheximide

Hair (best specimen type)

Removes 10-12 affected hairs with forcep

Skin (best specimen type)

Scrape surface of skin at active margin

Nails (best specimen type)

• collect debris from under the nail, or

• scrape outer surface of nail and discard.

• Nails cut into small pieces, pushed into agar have best recovery

Oropharyngeal samples (best specimen type)

Dx thrush by microscopic examination of material

Vaginal secretions (best specimen type)

Dx by clinical symptoms and microscopic examination

External Eye (best specimen type)

Surgically obtained corneal scraping for mycotic keratitis plated bedside directly onto non-inhibitory media

Exudates, Pus and Drainage (best specimen type)

• Examine for granules, plate directly onto media

• Crush granules, examine for hyphae then plate

Transport & Storage

1. Process and plate within 2 hrs of collection

2. Avoid excessive heat or cold

3. Leave at RT (ONLY EXCEPTIONS - CNS store at 30° if delay in processing

4. Keep biopsy specimens moist

5. Skin, Hair and Nails - clean dry envelope

Processing Fungal Specimens

• Direct smears

• Cultures for ID

Saline or wet preparation (Direct Microscopic Examinations)

Detect fungal elements: hyphae, pseudohyphae, blastoconidia

Lactophenol cotton blue (LPCB) (Direct Microscopic Examinations)

Cotton blue stains chitin in fungal cell walls which helps visualize fungal elements

10-40% KOH (Direct Microscopic Examinations)

• Dissolves keratin in tissue cells

• Fungal cells survive because chitin resists alkali

Gram Stain (Direct Microscopic Examinations)

• Yeasts stain purple BUT ARE NOT CONSIDERED GRAM+

Acid Fast Smear (Direct Microscopic Examinations)

• Distinguish different types of lipids in the outer membrane of our organism

• yeast ascospores stain red

• blastoconidia stain blue

India Ink (Direct Microscopic Examinations)

• Fungal organisms that have capsules

Periodic acid Schiff (PAS) (Direct Microscopic Examinations)

• Pink with a blue background

Grocott-Gomori’s methenamine silver stain (GMS) (Direct Microscopic Examinations)

• Black / brown on a green background

Calcofluor white (Direct Microscopic Examinations)

• fluorochrome dye concentrates in fungal cell walls

• organisms will be blue-white or green using UV microscope

Fungal Temperature Growth

• 22-30 C and sometimes 35-37 C

Fungal Incubation Growth

• 1 to 4 weeks until considered NO GROWTH=Negative

• Systemic fungi may take up to 12 weeks

• Yeasts (24 hours) and opportunistic organisms (72 hours) grow fastest

Macroscopic Growth rate

• rapid, <24 hr to 5 days

• Intermediate (1-5 days) - hyaline and dematiaceous molds “contaminants”

• Slow growers (1-4 weeks) - systemic pathogens

Macroscopic Colony Morphology

• Texture - due to height of aerial hyphae

Macroscopic Reproductive Structures (aerial mycelia)

• Lactophenol cotton blue (LPCB)

Antifungal Therapies

• Very few available

• Toxicity issues as similar to human cells; eukaryotic organisms

Fungistatic

Stops organism to grow but does not kill

Fungicidal

Kills organism

Amphotericin B

• WAS 1st line treatment

• Shock can occur to patient

• Binding to ergosterol/cholesterol in cell membrane resulting in pore formation causing leakage of potassium

Nystatin

• Irreversible interaction with sterols, including ergosterol in cell membrane resulting in pore formation causing leakage of potassium

• A topical agent to treat yeast infections

Echinocandins

• Targets β(1,3) D-glucan synthase

Terbinafine

• Skin infections with yeast

• Disruption of ergosterol synthesis

Flucytosine

Taken up by fungal cells by using a fungal-specific cytosine permease it then inhibits protein synthesis and DNA synthesis

Griseofulvin

Arrests metaphase by disrupting mitotic structures, stopping cell division

Azoles

Inhibition of fungal cytochrome P-450 enzyme needed for the synthesis of ergosterol in the cell membrane