general mycology 2006-07

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Medical Microbiology Bacteriology Virology Mycology Protozoalogy Helminthology Parasitology Immunology Medical Microbiology The study of fungi causing diseases in humans

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Page 1: General Mycology 2006-07

Medical Microbiology

BacteriologyVirology

Mycology

Protozoalogy

Helminthology

Parasitology

Immunology

Medical Microbiology

The study of fungi causing diseases in humans

Page 2: General Mycology 2006-07

Objectives

• To impart sufficient basic science of the medically important fungi to assist you in diagnosing mycotic diseases

• To characterize the following different types of mycoses, defining the tissue they affect

Superficial Cutaneous Subcutaneous Systemic Opportunistic• To differentiate the etiologic agents of these mycosis• To impart sufficient clinical knowledge • To raise your index of suspicion for mycotic diseases• Treatment

Page 3: General Mycology 2006-07

Medical Mycology• Fungi were discovered before bacteria &

viruses• Most fungi cause skin or cosmetic infections

while bacteria & viruses cause fatal diseases• Clinical Mycology has entered “Golden Age”

in modern medicine due to:

•Organ transplantation•Immunosuppressive drugs•Anticancer drugs•Broad-spectrum antimicrobials•HIV-disease

Immunosuppression

Opportunistic Fungal Infections

Page 4: General Mycology 2006-07

• Are eukaryotic (a true nucleus) Cell membrane • Have ergosterol which is specific target for

antifungal agents (cholestrol in mammalian cells)Cell Wall

Fungi : General Characteristics

Contains• Peptidomannan• Glycan (target for new antifungal agents)

Lacks• Peptidoglycan• Techoic acids• Lipopolysaccharide

• Produce filamentous structures• Produce spores

Page 5: General Mycology 2006-07

• For source of carbon & nitrogen need to live on plants, animals or humans

• Are aerobic or facultative anaerobic• Optimum growth temp is 25-30OC

(environmental)• Can tolerate a wide range of pH (2-9)

but generally like acidic pH• Light inhibits fungal growth

Are present on the earth where organic materials exist

Fungi : Metabolism

Page 6: General Mycology 2006-07

On the basis of Morphology1. Molds (filamentous fungi)• Most fungi are composed of filamentous

(tubular) structures called hyphae. May be septated OR Aseptated

Fungi Groups

Aseptate hyphae Septate hyphae

Page 7: General Mycology 2006-07

Types of Hyphae

Reproductive Hyphae & conidia

Mycelium(thallus)

Surface ofmedia

Aerial hyphae

Vegetative hyphae

• Vegetative hyphae: penetrate the media and absorb food

• Aerial hyphae : are directed above the surface of media

• Reproductive hyphae : Aerial hyphae that carry different spores

• Mycelium : A collection of hyphae

Page 8: General Mycology 2006-07

On the basis of morphology2. Yeasts• Unicellular (rounded or oval)• Reproduce by budding• The only example of pathogenic

yeast is Crptococcus neoformans

Fungi Groups

Page 9: General Mycology 2006-07

Fungi Groups

On the basis of morphology

3. Yeast-Like• Unicellular (rounded or oval)• Reproduce by budding but buds fail

to detach and may form short chains of cells called pseudohyphae

• Pseudohyphae are produced during infection and have diagnostic value

• Example: Candida

Page 10: General Mycology 2006-07

On the basis of Morphology4. Dimorphic Fungi• Able to grow in two different forms• As molds at room temperature • As yeasts on incubation at 370C & during

infection in body “Mold in the cold, yeast in the heat”

• Example: Histoplasma capsulatum

Fungi Groups

Page 11: General Mycology 2006-07

Sexual reproduction Involves the union of 2 nuclei OR 2 sex cells or 2 sex organs

+

Gametes at tips of hyphae

unite

-

1. ZygosporeTwo identical cells form the zygote

2. AscosporeFusion of nuclei of two cells in an ascus (sac)

3. BasidiosporeSpores produced on a basidium

Ascus

4. OosporeFemale cell fertilized by male cell

+

-

Fungi : Reproduction

Femalecell

Malecell

Page 12: General Mycology 2006-07

On the Basis of nature of sexual spores fungi can be classified as:

1. Zygomycetes • Have zygospores & are non-pathogeneic

2. Ascomycetes• Have ascospores & are non-pathogeneic

3. Basidiomycetes• Have basidiospores & are non-pathogeneic

Fungi Groups

Page 13: General Mycology 2006-07

4. Deuteromycetes (Fungi Imperfecti)

• Sexual or perfect state not present or not discovered

• Not placed in any of the above three classes

• Most pathogenic molds, yeasts, yeast-like & dimorphic fungi belong to this group

• Most propagate by asexually

Fungi Groups

Page 14: General Mycology 2006-07

Asexual Reproduction

Is the main method of reproduction. It includes1. Fragmentation of hyphae Each fragment grows into a new hyphae2. Fission of cells into 2 daughter cells (like binary fission in bacteria)3. Budding of cells Each bud produces a new individual e.g

Candida4. Formation of asexual spores

Note : A single fungus may have both modes of reproduction.

Fungi : Reproduction

Page 15: General Mycology 2006-07

Fungi : ReproductionAsexual Spores

A. Thallospores

1. BlastosporesProduced by budding from thallus

2. ArthrosporesFormed within lumen of hyphaeSize less than the size of hyphaeCan be cubical or rounded

3. ChlamydosporesProduced by swelling from thallus

CubicalRounded

Page 16: General Mycology 2006-07

B. SporangiosporesProduce sacs filled with spores called sporangiumHyphae that carry sporangium are called sporangiophores

Fungi : ReproductionAsexual Spores Spores

disseminate

C. Exogenous Spores (Conidia)Spores produced on the external part of a fertile hypha called conidiophore

Conidia

Conidiophore

1. MicroconidiaAre unicellular

Page 17: General Mycology 2006-07

Fungi : ReproductionAsexual Spores

C. Exogenous Spores

2. MacroconidiaAre multicellularHave different shapes

Spindle-shape Cylindrical-shape Rocket-shape

Page 18: General Mycology 2006-07

• Most fungi are opportunistico Cause disease in immunosuppresed patientso Few are primary pathogens

• Source of infection Endogenous

o Normal flora in immunosuppressed patientso A cause of hospital acquired infection

E.g. Candida albicans (normal flora in mouth, GIT, GUT in females)

Exogenouso Main source from environment

Fungi : Pathogenesis

Page 19: General Mycology 2006-07

Mode of transmission• Most fungal diseases are not communicable

between humans or animals• In the past fungal diseases were skin

infections (contact was the mode of transmission)

• In modern medicine due to immunosuppression can be transmitted by o Respiratory tract : airborneo GIT : food & water-borno Bloodo Skin : contact

Fungi : Pathogenesis

Page 20: General Mycology 2006-07

Steps of Infection1. Adherence by adhesins• e.g Candida. Molds have no adhesins2. Invasion• Trauma to skin or MM is essential in fungal

infection because infective element in most fungi is spore which is non-invasive

• Some fungi have invasive power by pseudohyphae like candida

3. Antiphagocytic effect like dimorphic fungi4. Tissue injury• No classical endotoxins or exotoxins of bacteria• Disease is due to:• Prolonged presence of fungus & Inflammatory &

immunological response

Fungi : Pathogenesis

Page 21: General Mycology 2006-07

• Innate (Non-specific) Immunity• Works against all microbes• Acquired Immunity

A. Cellular : The main mode of immune responseB. Humoral : Abs have limited role in some fungal diseasesE.g. Candida & Cryptococcal infections

Fungi : Immunity to infection

Page 22: General Mycology 2006-07

TerminologiesA. Anatomical (according to site of

infection)• Dermatomycosis : mycoses of skin• Pulmonarmycosis : mycoses of lungs• Cardiovascular mycosesB. Mycological (according to causative

agent)• Candidiasis• Aspergillosis• Cryptococcosis• Histoplasmosis

Human Mycosis

Page 23: General Mycology 2006-07

1. Superficial MycosisAffects only upper most horny layer of skin, hairs & nails e.g. Tinea versicolor

2. Cutaneous MycosisRingworm fungi & Candidiasis

3. Subcutaneous OR ImplantationOccurs by implantation of spores into

woundse.g. thorn-prick mycosisMycetoma or Madura foot

4. Systemic MycosisAffects deeper tissues : lungs, meningesMulti-organ disease

Humans Mycosis : Types

Page 24: General Mycology 2006-07

Opportunistic Fungal Infections• Are due to:

o Fungal flora like Candidao Fungal saprophytes in environment like

Aspergillus• Occur in persons:

o Organ transplantationo Immunosuppressive drugso Anticancer drugso Broad-spectrum antimicrobialso HIV-diseaseo Drug addicts

Humans Mycosis : Types

Page 25: General Mycology 2006-07

• Diagnosed by demonstration of fungal diagnostic elements (yeasts, hyphae, microconidia, macrocondia) in specimens

Specimens

• Skin scrapings, pieces of nails & hair, sputum, pus etc

• Presence of fungus does not mean infections:o Fungi are saprophytico Are common lab contaminants

Fungi : Lab Diagnosis

Page 26: General Mycology 2006-07

Fungi : Lab Diagnosis

I. Direct Microscopic ExaminationA: Unstained (KOH) PreparationDigests keratin in tissues but not fungusA Rapid method

Method• Place the specimen on glass slide• Add a drop of KOH (20%)• Place a cover slip• Gentle heating for 5-10 min (indirect heat)• Examine under x40 objective• See the fungal elements : hyphae,

microconidia, macrocondia

Page 27: General Mycology 2006-07

I. Direct microscopic ExaminationB. Stained Preparation• A rapid, easy & cheap method1. Lactophenol cotton blue (LPCB) stain Place a drop of alcohol on slide Immerse the specimen in it Add 1-2 drops of LPCB Place a coverslip and see under

microscope• Used to see fungal elements in

dermatophyte cultures

Fungi : Lab Diagnosis

Page 28: General Mycology 2006-07

I. Direct microscopic Examination

B. Stained Preparation 2. Gram-staining• For yeasts : stain gram-positive, are much

larger than bacteria

3. India ink preparation (negative staining)

• Detects thick shining capsule against blue background e.g Cryptococcus neoformans in CSF

Fungi : Lab Diagnosis

Page 29: General Mycology 2006-07

II. Culture• Common media used 1. Sabourauds Dextrose Agar (SDA)• Most commonly used fungal medium with low pH

5• Bacteria may grow on this media which may

mask fungal growth 2. SDA+chloramphenicol (.05%)

Chloramphenicol to inhibit bacterial growth3. SDA+chloramphenicol+cycloheximide (.5%)

Cycloheximide to inhibit saprophytic fungi4. Blood Agar

Yeast & yeast-like fungi grow rapidly like bacteria

Fungi : Lab Diagnosis

Page 30: General Mycology 2006-07

Identification of growth from culture by:

a) Macroscopic (colonial morphology)

• Color from both sides of plate (recto-verso examination)

• Shape & size• Texture of colony:

o Yeasts are typically smooth, creamyo Molds are fluffy/cottony

b) Microscopic stained preparation• To see fungal elements

Fungi : Lab Diagnosis

Page 31: General Mycology 2006-07

CandidaGram

Candida albicansin Sputum-Gram

MicrosporumLPCB

Candida albicans-EM

Penicillium-EM

AspergillusLPCB

Page 32: General Mycology 2006-07

c) Biochemical reactions • like sugar fermentation• Commercial kits are availableGerm tube testFor identification of Candida albicans• Place yeasts in serum and incubate at

37oC for 3 hrs• Most C. albicans will produce tube-like

projections called germ tubes• Seen under microscope

Fungi : Lab Diagnosis

Page 33: General Mycology 2006-07

III. Slide CultureIs used to:• See whole morphological details of

fungus especially yeast• Prevent disturbing the fungal

morphology• Hyphae & spores remain intact

Fungi : Lab Diagnosis

Page 34: General Mycology 2006-07

VI. Serological Tests (Abs Detection)For diagnosis of deep mycoses• Precipitation reaction• Agglutination reaction• Inert particle agglutination• Electrophoretic tests• Complement fixation• Indirect fluorescent antibody• Immunosorbent assays

Fungi : Lab Diagnosis

Page 35: General Mycology 2006-07

V. Serological Tests (Ags Detection)• Cryptococcal antigen in CSF• Galactomannan in invasive Aspergillosis• Beta-glucan & D-arabinitol in Candia

albicans

Fungi : Lab Diagnosis

Page 36: General Mycology 2006-07

IV. Histopathology• Fungi occur in tissues as one of the following:1. Yeast Cells

o Intracellular small yeasts like Histoplasma capsulatum

o May have a large distinguishing capsule as in Cryptococcus

2. Sporangia3. Hyphae

o May be brown or colorless4. Granules

o Are tightly packed hyphaeo Combination of yeast & hyphae as in candida

Fungi : Lab Diagnosis

Page 37: General Mycology 2006-07

V. Examination under Wood’s light• Long-wave UV rays • When come in contact with mycotic area

of skin or hair produce fluorescent colours

• Occurs in some mycotic infections onlyVI. Fungal Skin Tests• Has no diagnostic value• Mainly used for epidemiological studies• Does not differentiate between active

and past infection• Observed by induration and swelling• e.g. Histolasmin test & Trichophytin test

Fungi : Lab Diagnosis

Page 38: General Mycology 2006-07

Antifungal drugs

• Fungi closely resemble human cells• Use the same mechanisms to synthesize proteins & nucleic acid as higher animals• Difficulty in selective toxicity• So relatively few antifungal drugs for systemic use• The available systemic drugs are quite toxic

Page 39: General Mycology 2006-07

5. Inhibitors of Cell division Grisofulvin

Fungal Cell

3. Inhibitors of cell wall synthesis Echinocandins

1. Inhibitors of plasma membrane synthesis Azoles Allylamines

2. Inhibitors of plasma membrane function Polyenes

4. Inhibitors of nucleic acid synthesis Flucytosine

Antifungal drugs :Mechanisms of Action

Page 40: General Mycology 2006-07

• Bind to ergosterols, disrupt the cell membrane, leakage of the cytoplasm leading to cell death.

1. Nystatin• Is fungistatic• Not absorbed from GIT so is used locally only• Used in treatment of oral & vaginal candidiasis• Cannot be used in systemic fungal infections• Has cross-resistance to amphotericin B2. Amphotericin B• Is fungicidal• The most effective for serious fungal infections• Is used systemically (orally & IV)• Has toxic effects on nephritic tissues• Liposomal preparations are less toxic but very

expensive

Antifungal drugs: Polyenes

Page 41: General Mycology 2006-07

• Interfere with ergosterol synthesis, leading to defective cell memebrane

A. Imidazoles • Ketoconazole • Clotrimazole• Miconazole• Mostly used locally to treat yeast, molds &

dermatophytes• Systemic use is restricted due to

hepatotoxic & antispermatogenesis effects

Antifungal drugs Azoles

Page 42: General Mycology 2006-07

B. Triazoles New Triazoles• Fluconazole Voriconazole• Itraconazole Genoconazole• Have same mode of action like imidazoles• Are less toxic than imidazoles• Are used to treat systemic infections• Fluconazole crosses blood brain barrier

and is used in treatment of cryptococcal meningitis

Antifungal drugs Azoles

Page 43: General Mycology 2006-07

• Naftifine• Terbinafine• Inhibit an enzyme in the pathway of

ergosterol synthesis• Are used locally for dermatophytosis• Terbinafine can be taken orally

Antifungal drugs Allylamines

Page 44: General Mycology 2006-07

Griseofulvin• Inhibits fungal cell division• Is fungistatic• Concentrates in dead keratinized layers

of skin• Active against dermatophytes only• Used in skin & nail infections• Taken orally for months• Has side effects on stomach

Antifungal drugs

Page 45: General Mycology 2006-07

Flucytosine• Inhibits DNA synthesis• Effective against yeasts • Not effective against most molds• Used in treatment of systemic

yeast infections

Echinocandins• Caspofungin• Acts on fungal cell wall• Used against yeasts & molds

Antifungal drugs

Page 46: General Mycology 2006-07

Polyenes• Nystatin

Azoles• Clotrimazole• Miconazole• Ketoconazole

Allylamines• Terbinafine

Topical Antifungal Drugs

Page 47: General Mycology 2006-07

Antifungal Susceptibility testing

• Practically there was no need for antifungal susceptibility testing because:

A) Limited number of antifungal drugsB) Problems associated with antifungal

susceptibility testing

Due to wide use of these few drugs resistance strains are appearing so there is increasing need for it