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
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