INSTITUTE OF HEALTH TECHNOLOGY, DHAKADepartment of Laboratory Medicine

BSc in Health Technology (Laboratory)- 1st Year

MYCOLOGY Lecture No. 01(Introduction)

By

Sk. MIZANUR RAHMANLecturer, Mycology

MS in Biotechnology & Genetic Engineering (UODA)MS in Microbiology (SUB)

The Fungi

Learning outcomes- Students should be able to:

• Establish familiarity with the scientific terminology peculiar to mycology

• Describe the dimorphic nature of the pathogenic fungi used in making a clinical diagnosis.   

• Emphasize the eukaryotic nature of the fungi. • To explore the nature of the pathogenesis of fungal

infections. • To gain familiarity with the classification of medically-

important fungi. • To develop an understanding of the nature and mode of

action of anti-fungal agents

Recommended TextbooksTheory • Medical Mycology and Human Mycoses ES Beneke and AL

Rogers Star Publishing Company 1996 Belmont CALaboratory • Identifying Filamentous Fungi: A Clinical Laboratory

Handbook St-Germain G and Summerbell R Rogers Star Publishing Company 1996 Belmont CA

Additional Reading• - Introductory Mycology Alexopoulos CJ Mims CW

Blackwell M Fourth Edition John Wiley &Sons Inc 1996 New York NY

• - Medical Mycology KJ Kwon-Chung and JE Bennett Lea &Febiger 1992 Philadelphia PA

• - Medical Microbiology A Laboratory Manual: Section II WG Wu Third Edition Star Publishing Company 1995 Belmont CA

Medical Mycology Iceberg

What is a Fungus ?

• Eukaryotic – a true nucleus • Do not contain chlorophyll • Have cell walls• Produce filamentous structures• Produce spores

Species of Fungi

• 100,000 – 200,000 species

• About 300 pathogenic for man

Fungal Morphology and Structure

Eukaryotic organisms, distinguished by a rigid cell wall composed of chitin and glucan, and a cell membrane in which ergosterol is substituted for cholesterol as the major sterol component.Fungal taxonomy relies heavily on morphology and mode of spore productionFungi may be unicellular or multicellular. The simplest grouping based on morphology divides fungi into either yeast or mold forms.

Features of Fungi and its value in our life:

The fungi are a ubiquitous and diverse organisms, that degrade organic matter.Fungi have heterotrophic life; they could survive in nature as:

Saprophytic: live on dead or decaying matterSymbiotic: live together and have mutual advantageCommensal: one benefits and other neither benefits nor harmed.Parasitic: live on or within a host, they get benefit and harm the other.

Fungi mainly infect immunocompromised or hospitalized patients with serious underlying diseases. The incidence of specific invasive mycoses continues to increase with time The list of opportunistic fungal pathogens likewise increases each year “It seems there are no non-pathogenic fungi anymore ! “This increase in fungal infections can be attributed to the ever-growing number of immunocompromised patients.

Characteristics of fungi A. eukaryotic, non- vascular organisms

B. reproduce by means of spores (conidia), usually wind-disseminated

C. both sexual (meiotic) and asexual (mitotic) spores may be produced, depending on the species and conditions

D. typically not motile, although a few (e.g. Chytrids) have a motile phase.

E. like plants, may have a stable haploid & diploid states

F. vegetative body may be unicellular (yeasts) or multicellular moulds composed of microscopic threads called hyphae.

G. cell walls composed of mostly of chitin and glucan.

More Characteristics of FungiH. fungi are heterotrophic ( “other feeding,” must feed on

preformed organic material), not autotrophic ( “self feeding,” make their own food by photosynthesis).

- Unlike animals (also heterotrophic), which ingest then digest, fungi digest then ingest. -Fungi produce exoenzymes to accomplish this

I. Most fungi store their food as glycogen (like animals). Plants store food as starch.

K. Fungal cell membranes have a unique sterol, ergosterol, which replaces cholesterol found in mammalian cell membranes

L. Tubule protein—production of a different type in microtubules formed during nuclear division.

Structure•The body of fungi is termed thallus (non- reproductive) •The thalli of yeast are small, globular and are single celled•The thalli of mold are composed of long, branched tubular filaments called hyphae.

Structure• The thallus of a mold is composed of hyphae

intertwined to form a tangled mass called mycelium.

Mycotic Diseases(Four Types)

1. Hypersensitivity– Allergy

2. Mycotoxicosis– Production of toxin

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

4. Infection

The Clinician Must Distinguish Between:

• COLONIZATION

• FUNGEMIA

• INFECTION

EYE

SKIN

UROGENITAL TRACT

ANUS

MOUTHRESPIRATORY

TRACT

Portal of Entry

•SKIN

•HAIR

•NAILS

•RESPIRATORY TRACT

•GASTROINTESTINAL 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..

Classification of fungi

They are classified by several methods:1- Morphological classification2- Systematic classification3- Clinical classification

Fungal Morphology Yeast

Hyphae (threads) making up a mycelium

Mould

Encapsulated yeastCryptococcus neoformans

• Unicellular fungi• Fission yeasts divide symmetrically• Budding yeasts divide asymmetrically

Saccharomyces and Candida

Yeasts

Figure 12.3

Yeast Reproduction• FISSION• “even” reproduction, nucleus divides forming

two identical cells, like bacteria

• BUDDING• “uneven” reproduction, parent cell’s nucleus

divides and migrates to form a bud and then breaks away

Molds• Multicellular, tubular structures (hyphae)• Hyphae can be septate (regular crosswalls) or

nonseptate (coenocytic) depending on the species (grow by apical extension)

– Vegetative hyphae grow on or in media (absorb nutrients); form seen in tissue, few distinguishing features

– Aerial hyphae contain structures for production of spores (asexual propagules); usually only seen in culture

• The fungal thallus consists of hyphae; a mass of hyphae is a mycelium.

Molds

Figure 12.2

Moulds are multicellular organisms consisting of threadlike tubular structures called Hyphae that elongate by apical extension. Hyphae are either:

Coenocytic: hollow and multinucleateSeptate: divided by partitions or cross-walls

Hyphae form together to produce a mat-like structure called a Mycelium.

Vegetative hyphae, grow on or under surface of culture medium, Aerial Hyphae: project above surface of mediumAerial H. produce Conidia (asexual reproductive elements) Conidia can easily airborne and disseminate the fungus. Many medical fungi are termed dimorphic because they exist in yeast and mould forms.

Septate hyphae Non-Septate hyphae Mycelium

Dimorphic Fungi

• Growth as a mold or as a yeast• Most pathogenic fungi are dimorphic fungi• At 37o C yeast-like• At 25o C mold-like• Can also occur with changes in CO2• Fungi grow differently in tissue vs

nature/culture; often dictated by temp

• Some fungi are dimorphic depending on environmental conditions

• These organisms produce both yeast-like and mold-like thalli

• Many are pathogenic

• Candida albicans

Dimorphism

Figure 12.4

DimorphismMany pathogenic fungi are dimorphic, forming moulds at ambient temperatures but yeasts at body temperature.

Clinical Classification of Mycoses

•Cutaneous•Subcutaneous•Systemic•Opportunistic

Cutaneous Mycoses

Skin, hair and nailsRarely 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 FungiUbiquitous 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.

• Systemic mycoses Deep within body• Subcutaneous mycoses Beneath the skin• Cutaneous mycoses Affect hair, skin,

nails• Superficial mycoses Localized, e.g., hair

shafts• Opportunistic mycoses Caused by normal

microbiota or fungi

Fungal Diseases (mycoses)

DIAGNOSIS

Diagnosis

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

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 Serology Antibodies

• Latex Agglutination IgM• Immunodiffusion IgG• EIA IgG & IgM• Complement Fixation IgG

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

ANTIGEN DETECTION PRESENTLY AVAILABLE

Cryptococcosis Histoplasmosis Aspergillosis

Diagnosis

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

DIRECT FLUORESCENT ANTIBODY

CAN BE APPLIED TO

1. HISTOLOGIC SECTIONS2. CULTURE

• Viable organisms• Non-viable organisms

Diagnosis1. Wet Mount2. Skin test3. Serology4. Fluorescent antibody5. Biopsy and histopathology6. Culture7. DNA probe

Inflamatory Reaction

• Normal host–Pyogenic–Granulomatous

• Immunodeficient host–Necrosis

Polymorphic Nuclear Leukocytes

Giant Cell

GMS (Gomori Methenamine Silver Stain)

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

IncubationTemperature

• 370 C - Body temperature

• 250 C - Room temperature

Diagnosis

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

DNA Probes

• Rapid (1-2 Hours)• Species specific• Expensive