general mycology 2006-07

Medical Microbiology

BacteriologyVirology

Mycology

Protozoalogy

Helminthology

Parasitology

Immunology

Medical Microbiology

The study of fungi causing diseases in humans

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

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

• 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

• 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

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

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

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

yeast is Crptococcus neoformans

Fungi Groups

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

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

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

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

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

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

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

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

Fungi : ReproductionAsexual Spores

C. Exogenous Spores

2. MacroconidiaAre multicellularHave different shapes

Spindle-shape Cylindrical-shape Rocket-shape

• 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

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

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

• 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

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

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

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

• 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

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

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

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

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

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

CandidaGram

Candida albicansin Sputum-Gram

MicrosporumLPCB

Candida albicans-EM

Penicillium-EM

AspergillusLPCB

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

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

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

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

albicans

Fungi : Lab Diagnosis

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

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

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

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

• 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

• 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

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

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

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

Antifungal drugs Allylamines

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

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

Polyenes• Nystatin

Azoles• Clotrimazole• Miconazole• Ketoconazole

Allylamines• Terbinafine

Topical Antifungal Drugs

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