Medical Microbiology

Bacteriology
Virology
Immunology
Medical
Microbiology

Mycology Helminthology
Protozoalogy
Parasitology
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 Immunosuppression

•Immunosuppressive drugs
•Anticancer drugs
•Broad-spectrum antimicrobials
•HIV-disease Opportunistic Fungal Infections
 Fungi : General Characteristics
• Are eukaryotic (a true nucleus)
Cell membrane
• Have ergosterol which is specific target for
antifungal agents (cholestrol in mammalian cells)
Cell Wall

Lacks Contains
• Peptidoglycan • Peptidomannan
• Techoic acids • Glycan (target for
• Lipopolysaccharide new antifungal agents)

• Produce filamentous structures

• Produce spores
Fungi : Metabolism
• 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 Groups

On the basis of Morphology

1. Molds (filamentous fungi)
• Most fungi are composed of filamentous
(tubular) structures called hyphae. May be
septated OR Aseptated

Aseptate hyphae Septate hyphae

 Types of 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

Reproductive
Hyphae & conidia

Mycelium
Aerial
(thallus)
hyphae

Vegetative
Surface of hyphae
media
 Fungi Groups
On the basis of morphology
2. Yeasts
• Unicellular (rounded or oval)
• Reproduce by budding
• The only example of pathogenic yeast
is Crptococcus neoformans
 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
 Fungi Groups

On the basis of Morphology

4. 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 : Reproduction
Sexual reproduction
Involves the union of 2 nuclei OR 2 sex
cells or 2 sex organs
+ +
-
- 3. Basidiospore
Gametes at Spores produced
tips of hyphae
on a basidium
unite

Ascus
Male
Female cell
cell

1. Zygospore 2. Ascospore
Two identical cells Fusion of nuclei of two
form the zygote 4. Oospore
cells in an ascus (sac)
Female cell fertilized
by male cell
 Fungi Groups

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 : Reproduction
Asexual Reproduction

Is the main method of reproduction. It includes

1. Fragmentation of hyphae
Each fragment grows into a new hyphae
2. Fission of cells into 2 daughter cells
(like binary fission in bacteria)
3. Budding of cells
Each bud produces a new individual e.g Candida
4. Formation of asexual spores

Note : A single fungus may have both modes of

reproduction.
 Fungi : Reproduction
Asexual Spores
A. Thallospores
1. Blastospores
Produced by budding from thallus

2. Arthrospores Cubical
Formed within lumen of hyphae Rounded
Size less than the size of hyphae
Can be cubical or rounded

3. Chlamydospores
Produced by swelling from thallus
 Fungi : Reproduction
Asexual Spores Spores
disseminate

B. Sporangiospores
Produce sacs filled with spores called sporangium
Hyphae that carry sporangium are called sporangiophores

C. Exogenous Spores (Conidia)

Spores produced on the external part of Conidiophore
a fertile hypha called conidiophore
Conidia

1. Microconidia
Are unicellular
 Fungi : Reproduction
Asexual Spores
C. Exogenous Spores

2. Macroconidia
Are multicellular
Have different shapes

Spindle-shape Cylindrical-shape Rocket-shape

 Fungi : Pathogenesis

• Most fungi are opportunistic

o Cause disease in immunosuppresed patients
o Few are primary pathogens
• Source of infection
 Endogenous
o Normal flora in immunosuppressed patients
o A cause of hospital acquired infection
E.g. Candida albicans (normal flora in mouth, GIT,
GUT in females)
 Exogenous
o 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 : airborne
o GIT : food & water-born
o Blood
o Skin : contact
 Fungi : Pathogenesis
Steps of Infection
1. Adherence by adhesins
• e.g Candida. Molds have no adhesins
2. 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 fungi
4. Tissue injury
• No classical endotoxins or exotoxins of bacteria
• Disease is due to:
• Prolonged presence of fungus & Inflammatory &
immunological response
 Fungi : Immunity to infection
• Innate (Non-specific) Immunity
• Works against all microbes
• Acquired Immunity
A. Cellular :
The main mode of immune response
B. Humoral :
Abs have limited role in some fungal
diseases
E.g. Candida & Cryptococcal infections
 Human Mycosis
Terminologies
A. Anatomical (according to site of infection)
• Dermatomycosis : mycoses of skin
• Pulmonarmycosis : mycoses of lungs
• Cardiovascular mycoses
B. Mycological (according to causative agent)
• Candidiasis
• Aspergillosis
• Cryptococcosis
• Histoplasmosis
 Humans Mycosis : Types
1. Superficial Mycosis
Affects only upper most horny layer of skin,
hairs & nails e.g. Tinea versicolor
2. Cutaneous Mycosis
Ringworm fungi & Candidiasis
3. Subcutaneous OR Implantation
Occurs by implantation of spores into wounds
e.g. thorn-prick mycosis
Mycetoma or Madura foot
4. Systemic Mycosis
Affects deeper tissues : lungs, meninges
Multi-organ disease
 Humans Mycosis : Types
Opportunistic Fungal Infections
• Are due to:
o Fungal flora like Candida
o Fungal saprophytes in environment like
Aspergillus
• Occur in persons:
o Organ transplantation
o Immunosuppressive drugs
o Anticancer drugs
o Broad-spectrum antimicrobials
o HIV-disease
o Drug addicts
 Fungi : Lab Diagnosis
• 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 saprophytic
o Are common lab contaminants
 Fungi : Lab Diagnosis

I. Direct Microscopic Examination

A: Unstained (KOH) Preparation
Digests keratin in tissues but not fungus
A 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
 Fungi : Lab Diagnosis
I. Direct microscopic Examination
B. Stained Preparation
• A rapid, easy & cheap method
1. 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 growth
3. SDA+chloramphenicol+cycloheximide (.5%)
Cycloheximide to inhibit saprophytic fungi
4. 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, creamy
o Molds are fluffy/cottony
b) Microscopic stained preparation
• To see fungal elements
 Candida
Gram

Candida albicans
in Sputum-Gram

Candida albicans-EM

Microsporum
LPCB

Aspergillus
LPCB
Penicillium-EM
 Fungi : Lab Diagnosis
c) Biochemical reactions
• like sugar fermentation
• Commercial kits are available
Germ tube test
For 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 Culture
Is 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. Sporangia
3. Hyphae
o May be brown or colorless
4. Granules
o Are tightly packed hyphae
o 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 only
VI. 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
 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
 Antifungal drugs :
Mechanisms of Action
3. Inhibitors of cell 1. Inhibitors of plasma
wall synthesis membrane synthesis
Echinocandins Azoles
Allylamines

2. Inhibitors of plasma
4. Inhibitors of nucleic membrane function
Fungal Cell Polyenes
acid synthesis
Flucytosine

5. Inhibitors of
Cell division
Grisofulvin
 Antifungal drugs: Polyenes
• 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 B
2. 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
Azoles
• 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
Allylamines
• Naftifine
• Terbinafine
• Inhibit an enzyme in the pathway of
ergosterol synthesis
• Are used locally for dermatophytosis
• Terbinafine can be taken orally
 Antifungal drugs
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
Topical Antifungal Drugs
Polyenes
• Nystatin
Azoles
• Clotrimazole
• Miconazole
• Ketoconazole
Allylamines
• Terbinafine
 Antifungal Susceptibility testing
• Practically there was no need for
antifungal susceptibility testing because:
A) Limited number of antifungal drugs
B) 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