Introduction To Mycology

Microbiology (Dr. De Castro) Introduction to Mycology 13 December 07 •

INTRODUCTION TO MYCOLOGY Mycology • The study of fungi and their multiple functions in nature. FUNGUS

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Eukaryotic – a true nucleus

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Do not contain chlorophyll  achlorphyllous Have cell walls Produce filamentous structures Produce spores Grow as saprophytes and decompose dead matters 100,000-200,000 species 300 species are pathogenic in man

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FUNGAL CELL WALL COMPOSITION − Structural Components o Chitin microfibrils [β (1-4)-linked polymer of Nacetylglucosamine] o Chitosan in Zygomycota [β (1-4)-linked polymer of glucosamine] o Β-linked glucans − Gel-Like Components o Mannoproteins (form matrix throughout wall)

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Antigenic glycoproteins, agglutinans, adhesions − on cell wall surface Melanins − dark brown to black pigments − confer resistance to enzyme lysis, confer mechanical strength and protect cell from UV light, solar radiation and dessication Sporopollenin − Aromatic polymer found in spore walls of some fungi, confers properties similar to melanin Plasma membrane − Semi-permeable

Characteristic Prokaryocyte

Protista Fungi Plants

Eukaryocyte Eukaryocyte Eukaryocyte

Animals

Eukaryocyte

Examples Bacteria Actinomyces Protozoa Fungi Plants Moss Anthropods Mammals Man

MEDICAL MYCOLOGY There are four types of mycotic diseases: 1. Hypersensitivity − An allergic reaction to molds and spores − Indoor pollution 2. Mycotoxicoses

Virns, leu, brim

Mycetismus • The ingestion of pre-formed toxin mushroom poisoning Infection

B. MORPHOLOGY OF FUNGI • Yeast • Yeast-like • Mold (hyphae) • Dimorphic Fungi YEASTS 1. Unicellular organisms 2. Round or oval fungi that bud or pinch off 3. Growth resembles bacteria 4. May be dry, pasty or slimy 5. May be gram-positive 6. Much larger than bacteria • Bacteria are 0.1-0.2 microns • Yeast are 3-20 microns (the size of PMNs, RBCs, etc Ex. C. neoformans YEAST-LIKE (PSEUDOHYPHAE) – Candida

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Budding yeast with septum. The septum has formed between the daughter bud and the mother cell but separation of the two has not occurred.

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Candida albicans mother and daughter cells. Cells were grown under conditions that induced hypha formation for 30 mins. The daughter cell is on the right; the mother cell is on the left. The daughter cell has not reached a threshold volume and therefore has not yet formed a hypha.

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C. albicans cell at 3h. 3 hours after the appearance of the germ tube, the hypha has septa.

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C. albicans hyphal cells at 5h. After 5h in hypha-inducing medium, many hyphae are evident. Clumping of the hyphae is also apparent and hyphae are beginning to form hyphae blastospores, which are now budding cells.

A. CLASSIFICATION TAXONOMY Kingdom Monera

Poisoning of man and animals by feeds and food products contaminated by fungi which produce toxins from the grain substrate

HYPHAE (MYCELIUM) • Multi-cellular filamentous structures • Cylindrical, branching filaments composed of tubular cell wall filled with cytoplasm and organelles • Most fungal hyphae are 2-10 µm diameter

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A mass of hypahe is called Mycelia Pattern of branching of mycelia aids diagnosis “hypahe”, “mycelium”, “molds” – interchangeable

Molds (Hyphal Fungi) 1. Seen microscopically as filaments which are variable in length but usually constant in diameter 2. On plates look fuzzy or “carpet-like” Hyphae 1. Septate – filaments have crosswalls or septate Ex. Aspegillus 2. Aseptate – filaments lack crosswalls; also called coenocytic Ex. Mucor

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Microbiology – Intro to Mycology by Dra De Castro 3. 4.

Vegetative – growth within the surface of the medium (agar) Aerial – filaments that extend above the surface (stick up into the air)

Septa

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Regular cross-walls formed in hyphae. Hyphae with septa are septate, those lacking septa except to delimit reproductive structures and aging hyphae are called aseptate or coenocytic. o Primary septa are formed as a process of hyphal extension and generally have a septal pore, which allows for cytoplasmicand organelle movement. o Secondary or adventitious septa are imperforate, formed to wall off ageing parts of the mycelium. Septate hyphae- Aspergillus ; Aseptate hyphae - Mucor DIMORPHIC FUNGI (FUNGAL DIMORPHISM) Yeast (Parasitic or Pathogenic Form) • Seen in human tissue, exudates, or if cultured in an incubator at 37 °C. Mycelium (Saprophytic or Mold Form) • Form observed in nature or if incubated at 25 °C. • Conversion to the yeast form essential for pathogenicity C. REPRODUCTION OF FUNGI SEXUAL REPRODUCTION ● Used for scientific classification ● Rarely seen in tissue ● Not usually studied in the laboratory ● Probably not infectious ASEXUAL REPRODUCTION ● These are the forms we study in the laboratory ● Asexual reproductive forms are CONIDIA ● Sometimes called SPORES ● Usually round but may be rectangular ● Similar in size to yeasts TYPES OF CONIDIA 1. Arthroconidia o Hyphae break or fragment at separation points forming small pieces that give rise to a new hyphae o E.g. Coccidioides, Geotrichum 2. Blastoconidia o Cells reproduce by budding o Daughter cells pinch off from the mother cell o E.g. Canidia, Cryptococcus o Note: If blastoconidia continue to elongate and fail to pinch off, they are called PSEUDOHYPHAE (yeast-like) 3. Chlamydoconidia o Round and thick-walled conidia that form directly within or at the ends of hyphae; they can then fragment or brek off to form new hyphae o E.g. Candida albicans, dermatophytes OTHER PROPERTIES OF FUNGI ● They resemble mammalian cells ● Cell wall has chitin glucosamine, hexose, hexosamine, protein, etc. ● Special stains may be required to see them WHY ARE WE SEEING MORE MYCOSES TODAY? ● More compromised patients ● Transplant patients ● Anti-cancer drugs ● Invasive catheters (bladder, CNS, vascular) ● Use of broad-spectrum antibiotics ● The host has changed, not the fungi

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HOW DO PEOPLE GET MYCOSES? ● Overgrowth of normal flora (usually candida) after catheters, chemotherapy or broad-spectrum antibiotics ● Inhalation of conidia (Histoplasmosis) ● Trauma / Implantation (Sporothrix) ● Contact with plants (Sporothrix) and animals (dermatophytes) ESTABLISHMENT OF INFECTION WITH MYCOTIC AGENT DEPENDS ON: ● Inoculum size ● Resistance of the host SEVERITY OF DISEASE  In mycotic infections depends more on the host immune system than on the virulence of the fungus  Clinician must distinguish bet : ● Colonization ● Transient fungemia ● True infection PORTAL OF ENTRY ● Skin ● Hair ● Nails ● Respiratory tract ● Gastrointestinal tract ● Urinary tract COLONIZATION ● Multiplication of an organism at a given site without harm to the host INFECTION ● Invasion and multiplication of organisms in body tissue resulting in local cellular injury D. DIAGNOSIS SPECIAL STAINS FOR FUNGI ● Giemsa stain o Nucleus blue with clear or pink cytoplasm ● Gomori’s Methenamine Silver (GMS) / Silver stain o Yeasts and hyphae are brown-black, the counterstain is usually green, stains the cell wall only ● Periodic Acid Schiff (PAS) o Stains cell walls pink o Counterstain is blue or green I. Wet Mount II. Skin Test III. Serology IV. Fluorescent antibody V. Biopsy and Histopathology VI. Culture DIRECT MICROSCOPIC OBSERVATION ● 10% KOH ● Skin crappings suspected to contain dermatophyets or pus from a lesion can be mounted in KOH on a slide and examined directly under the microscope ● Gentle heat SKIN TESTING (DERMAL HYPERSENSITIVITY) ● May interfere with serologic test, causing false positive result ● Use is limited to: o Determine cellular defense mechanisms o Epidemiologic studies SEROLOGY ● May be helpful when it is applied to a specific fungal disease

Microbiology – Intro to Mycology by Dra De Castro ● ●

There are no screening antigens for fungi in general –fungi are poor antigens The efficacy of serology varies with different fungal infections

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Squalene Allylamines Squalene-2,3-Epoxide

FUNGAL SEROLOGY LATEX AGGLUTINATION IMMUNO-DIFFUSION COMPLEMENT FIXATION

ANTIBODIES IgM IgG IgG

Most serology tests for fungi measure antibody. Newer tests to measure antigen are now being developed. ANTIGEN DETECTION PRESENTLY AVAILABLE ● Cryptococcosis ● Histoplasmosis DIRECT FLUORESCENT ANTIBODY ● Can be applied to: o Tissue o Culture BIOPSY AND HISTOPATHOLOGY ● Normal host o Pyogenic o Granulomatous ● Immunodeficient host o Necrosis CULTURE ● A definitive diagnosis requires a culture and identification ● Pathogenic fungi are usually grown on SABOURAUD DEXTROSE AGAR ● Isolation media o Sabouraud Dextrose Agar (pH is approximately 5.6) o Plain o With antibiotics o With cycloheximide ● Incubation temperature o 37°C: body temperature

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25°C: room temperature Two cultures are inoculated and incubated separately at 25°C and 37°C to reveal DIMORPHISM The culture are examined macroscopically and microscopically They are not considered negative for growth until 4 weeks of incubation

E. TREATMENT THERAPY Since they are eukaryotic, fungi are biochemically similar to the human host. Thus, it is difficult to develop chemotherapeutic agents that will destroy the pathogenic fungus and not harm the patient A BASIC TENET OF PATHOLOGY A cause of irreversible cell injury is cell membrane damage IN FUNGAL THERAPY We attempt to induce cell injury by causing the cell membrane of the fungus to become permeable PROBLEM Finding an agent that will selectively injure fungal cells without damaging host cells ALL EUKARYOTIC CELLS CONTAIN Mammalian Cells  Cholesterol Fungal Cells  Ergosterol Ergosterol Synthesis

Acetyl-CoA Acetoacetyl-CoA HMG-CoA Mevalonic Acid

Lanosterol Azoles Morpholines Polyenes Ergosterol

PRIMARY ANTIFUNGALS 1. Polyene derivatives  Amphotericin B MOA:  Binds to sterols  Ergosterol is a constituent of the cell walls of the fungi (eukaryotic)  There is a greater avidity for ergosterol than for cholesterol in the cell walls of animal and human cell  The binding to the cell wall alters the permeability of the cell wall and results in the leakage of intracellular contents of the fungus  Nystatin 2. Azoles (Ketoconazole, Flucanozole, Itraconazole, Voriconazole)  There are few rare serious side effects from each itraconazole and flucanozole 3. Griseofulvin  A slow acting drug used for skin and nail infections. It accumulates in the stratum corneum and prevent hyphal penetration through these layers 4. Allylamines  Terbinafine (Lamisil) 5. 5- fluorcytosine  Interferes with RNA synthesis 6. Echinocandins  Caspofungin MOA Polyenes Azoles Griseofluvin 5-FC

Ergosterol in cell membrane Interfere with ergosterol synthesis Forms a barrier to fungal growth Inhibits RNA syntheis

F. CLINICAL CLASSIFICATION  Cutaneous  Subcutaneous  Systemic  Opportunistic Superficial Mycoses  Skin, hair and nails  Rarely invade deeper tissue  Dermatophytes Subcutaneous Mycoses  Confined to subcutaneous tissue and rarely spread systemically. The causative agents are soil organisms introduced into the extremities by trauma Systemic Mycoses  Involve skin and deep viscera  May become widely disseminated  Predilection for specific organs Opportunistic Fungi

Microbiology – Intro to Mycology by Dra De Castro 

Ubiquitous saprophytes and occasional pathogens that invade the tissues of those which have: o Predisposing diseases: diabetes, cancer, leukemia, etc o Predisposing conditions: agammaglobinemia, steroid and antibiotic therapy

Yung 1st week trans bout myco.. antay antayin nio lang ha.. di pa nman kelngan. Or kelangan nio pa ba? O well, bsta mgpapass kami.. late late konti. Kasi kakabigay lng ng mga copies.. peace!.. doble doble na trans na ggwins.. whaaa!.. No trans days are so over.. hay.. kakaiyak.malas namin lately..

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