microbiology, virusology, immunology department medical mycology as. o.b. kuchmak

Microbiology, virusology, immunology department

Medical Mycology

as. O.B. Kuchmak

Medical Mycology

TheseLecture

Students

The Sea of Knowledge

Medical Mycology Outline

1. Introduction, Actinomycetes2. Yeasts, Dermatophytes3. Filamentous Fungi, Dimorphic Fungi4. Dimorphic Fungi5. Opportunistic Fungi

OBJECTIVES

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

–To impart sufficient clinical knowledge

to raise your index of suspicion for mycotic diseases.

What is a Fungus ?

• Eukaryotic – a true nucleus

• Do not contain chlorophyll

• Have cell walls

• Produce filamentous structures

• Produce spores

KINGDOM CHARACTERISTIC EXAMPLE

Monera Prokaryocyte BacteriaActinomyces

Protista Eukaryocyte Protozoa

Fungi Eukaryocyte * Fungi

Plants Eukaryocyte PlantsMoss

Animals Eukaryocyte * ArthropodsMammals

Man

KINGDOM CHARACTERISTIC EXAMPLE

Monera Prokaryocyte BacteriaActinomyces

Protista Eukaryocyte Protozoa

Fungi Eukaryocyte * Fungi

Plants Eukaryocyte PlantsMoss

Animals Eukaryocyte * ArthropodsMammals

Man

ALL EUKARYOTIC CELLS CONTAIN STEROLS

•Mammalian cells – cholesterol

•Fungal cells - ergosterol

Cocci 0.8 u

Bacilli 4-6 u

Spirochetes 8 - 10 u

Viruses 0.08 u

Protozoa 15 u

Nematodes 10 mm

Fungi 10 – 15 u

SIZE COMPARISON OF PATHOGENS

Species of Fungi

• 100,000 – 200,000 species

• About 300 pathogenic for man

The taxonomy of the Kingdom Fungi is evolving and is controversial. Formerly based on gross and light microscopic morphology, studies of ultra structure, biochemistry and molecular biology provide new evidence on which to base taxonomic positions.

Medically important fungi are in four phyla:

1.Ascomycota - Sexual reproduction in a sack called an ascus with the production of ascopspores

2.Basidiomycota -Sexual reproduction in a sack called a basidium with the production of basidiospores

3.Zygomycota - sexual reproduction by gametes and asexual reproduction with the formation of zygospores

4.Mitosporic Fungi (Fungi Imperfecti) - no recognizable form of sexual reproduction. Includes most pathogenic fungi.

Classification

◘ ASCOMYCOTA Trichophyton Microsporum Blastomyces◘ BASIDIOMYCOTA Cryptococcus neoformans◘ DEUTEROMYCOTA Epidermophyton Sporothrix Candida species ◘ ZYGOMYCOTA Rhizopus nigrans

Actinomyces(True Bacteria)

• Tradition• Clinical infection resembles mycoses• Actinomyces grow on mycotic media• Actinomyces grow slowly (24-48 h)• Gross colonies resemble fungi

– (rough,heaped, short aerial filaments)

• Resemble mycelia microscopically, with branched mycelia in tissue and smears.

What is Medical Mycology ?

PATHOGENIC FUNGI

• NORMAL HOST• Systemic pathogens - 25 species• Cutaneous pathogens - 33 species• Subcutaneous pathogens - 10 species

• IMMUNOCOMPROMISED HOSTOpportunistic fungi - 300 species

MYCOTIC DISEASES(Four Types)

1. Hypersensitivity– Allergy

2. Mycotoxicosis– Production of toxin

3. Mycetismus (mushroom poisoning)– Pre-formed toxin

4. Infection

Hypersensitivity

Farmer’s lung Moldy hay

Malt worker’s disease Moldy barley

Cheese washer’s lung Moldy cheese

Wood trimmer’s disease Moldy wood

PARASITIC STATE

1. Increased metabolic state

2. Modified metabolic pathways

3. Modified cell wall structure– Carbohydrate content– Lipid structure– RNA aggregates

PATHOGENICITY OF FUNGI

1. Thermotolerance

2. Ability to survive in tissue environment

3. Ability to withstand host defenses

REVIVED INTEREST IN MYCOLOGY

• Increased frequency of mycotic diseases• Increased awareness by physicians• Better trained laboratory personnel• More invasive procedures used on patients• Increased use of immunosuppressive drugs• Increase in immunosuppressive disease

7. Better laboratory diagnostic tools

EYE

SKIN

UROGENITAL TRACT

ANUS

MOUTHRESPIRATORY

TRACT

PORTAL OF ENTRY

•SKIN

•HAIR

•NAILS

•RESPIRATORY TRACT

•GASTROINTES-TINAL TRACT

•URINARY TRACT

EYE

SKIN

UROGENITAL TRACT

ANUS

MOUTHRESPIRATORY

TRACT

COLONIZATION

Multiplication of an organism at a given site without harm to the host

EYE

SKIN

UROGENITAL TRACT

ANUS

MOUTHRESPIRATORY

TRACT

INFECTION

Invasion and multiplication of organisms in body tissue resulting in local cellular injury..

GEOGRAPHIC DISTRIBUTION

The present ease and frequency of world-wide travel make it more likely that physicians in the United States will be confronted with a variety of unfamiliar mycoses acquired in distant parts of the country or of the world.

MORPHOLOGY

Fungi exist in three morphologic form:

1. Yeasts 2. Molds. 3. Dimorphs.

• Pathogenic fungi can exist as yeasts or as hyphae (figure 4). A mass of hyphae is called mycelia. Yeasts are unicellular organisms and mycelia are multicellular filamentous structures, constituted by tubular cells with cell walls. The yeasts reproduce by budding. The mycelial forms branch and the pattern of branching is an aid to the morphological identification. If the mycelia do not have SEPTA, they are called coenocytic (nonseptate). The terms "hypha" and "mycelium" are frequently used interchangeably. Some fungi occur in both the yeast and mycelial forms. These are called dimorphic fungi.

Clinical Classification of Mycoses

CutaneousSubcutaneousSystemicOpportunistic

Cutaneous 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

Ubiquitous saprophytes and occasional pathogens that invade the tissues of those patients who have:

•Predisposing diseases: Diabetes, cancer, leukemia, etc.

•Predisposing conditions: Agammaglobulinemia, steroid or

antibiotic therapy.

MOST MYCOTIC AGENTS

ARE SOIL SAPRPHYTES

EPIDEMIOLOGY

Mycotic Diseases Are NOT

Contagious

1. Wet Mount2. Skin test3. Serology4. Fluorescent antibody5. Biopsy and histopathology6. Culture7. DNA probes

DIAGNOSIS

Diagnosis

1. Wet Mount2. Skin test3. Serology4. Fluorescent antibody5. Biopsy and histopathology6. Culture7. DNA probes

DIRECT MICROSCOPIC OBSERVATION

• 10 % KOH

• Gentle Heat

KOH Wet Mount

Diagnosis

1. Wet Mount2. Skin test3. Serology4. Fluorescent antibody5. Biopsy and histopathology6. Culture7. DNA probes

SKIN TESTING(DERMAL HYPERSENSTIVITY)

Use is limited to :

– Determine cellular defense mechanisms– Epidemiologic studies

Diagnosis

1. Wet Mount2. Skin test3. Serology4. Fluorescent antibody5. Biopsy and histopathology6. Culture7. DNA probes

FUNGI ARE POOR ANTIGENS

FUNGAL SEROLOGYANTIBODIES

• Latex Agglutination IgM

• Immunodiffusion IgG

• Complement Fixation IgG

Most serological tests for fungi measure antibody. Newer tests to measure antigen are now being developed

ANTIGEN DETECTION PRESENTLY AVAILABLE

Cryptococcosis Aspergillosis Histoplasmosis

Diagnosis

1. Wet Mount2. Skin test3. Serology4. Fluorescent antibody5. Biopsy and histopathology6. Culture7. DNA probes

DIRECT FLUORESCENT ANTIBODY

CAN BE APPLIED TO

1. HISTOLOGIC SECTIONS

2. CULTURE

• Viable organisms• Non-viable organisms

Diagnosis

1. Wet Mount2. Skin test3. Serology4. Fluorescent antibody5. Biopsy and histopathology6. Culture7. DNA probe

INFLAMMATORY REACTION

• Normal host–Pyogenic

–Granulomatous

• Immunodeficient host–Necrosis

Polymorphic Nuclear Leukocytes

GMS

Diagnosis

1. Wet Mount2. Skin test3. Serology4. Fluorescent antibody5. Biopsy and histopathology6. Culture7. DNA probes

ISOLATION MEDIA

SABOURAUD DEXTROSE AGAR

(pH ~ 5.6)

•Plain

•With antibiotics

•With cycloheximide

INCUBATION TEMPERATURE

• 37 C - Body temperature

• 25 C - Room temperature

Diagnosis

1. Wet Mount2. Skin test3. Serology4. Fluorescent antibody5. Biopsy and histopathology6. Culture7. DNA probes

DNA Probes

• Rapid (1 Hour)

• Species specific

• Expensive

TREATMENT

THERAPY

Because they are eukaryotic, fungi are biochemically similar to the human host. Therefore it is difficult to develop chemotherapeutic agents that will destroy the invading fungus without harming the patient.

A BASIC TENET OF PATHOLGY IS:

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 cell walls without damaging the host cell.

Cryptococcosis

A sub-acute or chronic infection which may affect the lungs or skin but most commonly manifests as a meningitis

Cryptococcus neoformans

• Spherical

• 5-10 microns

• Narrow- base

• Surrounded by polysaccharide capsule

Cryptococcus neoformans

Virulence factors

1. Anti-phagocytic polysaccharide capsule

2. Antioxidant melanin

3. Ability to grow at 37 C

Ecological Niche

Cryptococcus neoformans

• Pigeon droppings

• Chicken droppings

Cryptococcusportal of entry

• Inhalation

• inoculation

Typical clinical course• Inhalation

• Mild pneumonia

• Vision, lethargy, headache

• Delirium, nuchal rigidity, coma

• Over a period of months

• Death if not treated

Clinical Specimens

Cryptococcosis

•CSF•Sputum•Pus•Biopsy material•urine

Specimen of choice

Cerebrospinal fluid

Serological Tests

Cryptococcosis

• Indirect fluorescent antibody• Tube Agglutination• Latex Agglutination (measures antigen)

Drugs of Choice

Cryptococcosis

•Amphotericin B plus 5-Flucytosine

OR

•Amphotericin B plus Fluconazole

Yeasts

Unicellular forms that are

spherical or ovoid in shape.

Round to oval, single cells

which reproduce by budding

8-12 MICRONS

Pseudomycelium

Clinical SpecimensCandida

• Sputum

• Scrapings from lesions

• Blood smears

• Vaginal discharge

• Urine

• Feces

• Nail clippings

SEROLOGY• There have been several antibody

detection test, but none are significant.

• Beta-Glucan measurement is now showing promise

Therapy

• Nystatin– Vaginitis– Cutaneous

• Fluconazole *

• Itraconazole

Cryptococcosis

A sub-acute or chronic infection which may affect the lungs or skin but most commonly manifests as a meningitis

Yeasts

Unicellular forms that are spherical or ovoid in shape

• Hyphae (filamentous fungi, mycelium)– Septate– Coenocytic (non-septate)

• Dimorphic– Yeast– Mycelium

Yeasts

Unicellular forms that are spherical or ovoid in shape

• Hyphae (filamentous fungi, mycelium)– Septate– Coenocytic (non-septate)

• Dimorphic– Yeast– Mycelium

Dimorphic Fungi

• Yeast Form• Parasitic form• Tissue form• Cultured at 37 C

• Mycelial Form• Saprophytic form• Cultured at 25 C

SPORES

• SEXUAL

• ASEXUAL– Arthrospore– Blastospore– Chamydospore– Conidia

• Microconidia• Macroconidia