Medical Mycology Reference Laboratory

Laboratory of Parasitology and MycologyInstitute of Microbiology and Immunology

Medical Faculty in Belgrade, 2010

MEDICAL MYCOLOGY

1. The morphology and biology of the fungi

Classification of medically important fungi

• Fungal morphology and structure • Antifungals - Mechanisms of action • Fungi - biology and physiology• Fungal infections (FI) - classification

1. The morphology and biology of the fungi

Classification of medically important fungi

• Fungal morphology and structure • Antifungals - Mechanisms of action • Fungi - biology and physiology• Fungal infections (FI) - classification

The goal of teaching: 2. Common fungi which primarily cause skin,

hair and nail FI • Malassezia - morphology, biology

Pathogenesis of pitiriasis versicolor,

Pathogenesis of seborrheic dermatitis • Trichophyton - morphology, biology• Microsporum - morphology, biology• Epidermophyton - morphology, biology

Pathogenesis of dermatophytes

2. Common fungi which primarily cause skin, hair and nail FI

• Malassezia - morphology, biology

Pathogenesis of pitiriasis versicolor,

Pathogenesis of seborrheic dermatitis • Trichophyton - morphology, biology• Microsporum - morphology, biology• Epidermophyton - morphology, biology

Pathogenesis of dermatophytes

3. Common fungi which causes superficial / invasive / oprtunistic FI

• Candida - morphology, biology

Pathogenesis • Cryptococcus - morphology, biology.

Pathogenesis • Aspergillus - morphology, biology.

Pathogenesis • Fusarium - morphology, biology.

Pathogenesis

3. Common fungi which causes superficial / invasive / oprtunistic FI

• Candida - morphology, biology

Pathogenesis • Cryptococcus - morphology, biology.

Pathogenesis • Aspergillus - morphology, biology.

Pathogenesis • Fusarium - morphology, biology.

Pathogenesis

4. Other fungi which cause superficial / invasive / oprtunistic FI

• Penicillium - morphology, biology

Pathogenesis • Zygomycetes (Mucor, Rhisopus, Absidia) -

morphology, biology.

Pathogenesis • Pneumocystis - morphology, biology.

Pathogenesis

4. Other fungi which cause superficial / invasive / oprtunistic FI

• Penicillium - morphology, biology

Pathogenesis • Zygomycetes (Mucor, Rhisopus, Absidia) -

morphology, biology.

Pathogenesis • Pneumocystis - morphology, biology.

Pathogenesis

www.mycology.adelaide.edu.au

www.medicalmycology.org

www.edae.gr/myco2.htm

www.pfdb.net

www.doktorfungus.org/mycoses/index.htm

www.lumen.luc.edu/lumen/DeptWebs/microbio/med/review/mycology.htm

www.mikologija.org.rs

MYCOLOGY WWW

EMPIRE FEATURES EXAMPLE      

Monera Prokaryotes Bacteria,Blue-green algae 

Protista Eukaryotes Protozoa

Fungi Eukaryotes * Fungi

Planta  Eukaryotes Plants 

Animalia Eukaryotes * Arthropods / Mammals

Man

 

Mykos / Fungi Logos/ Science Mycology/ science of fungi Fungus = fungi

Fungi - eukaryotesThere are about 250 000 species of fungi.

About 100 species are important for people

OSNOVNA GRAĐA ĆELIJE GLJIVAFungal cell structure

All fungi possess:

- Cell wall (chitin, glucan, mannan, cellulose)

- Cytoplasmic membrane (ergosterol)

- Nucleus

- Mitochondria

All fungi possess:

- Cell wall (chitin, glucan, mannan, cellulose)

- Cytoplasmic membrane (ergosterol)

- Nucleus

- Mitochondria

Fungal cell structure

MORPFHOLOGICAL DIVISION OF FUNGI:

YEASTS (unicellular fungi) MOULDS (multicellular fungi)

DIMORPHIC (YEASTS/37ºC; MOLDS/25ºC)

MORPFHOLOGICAL DIVISION OF FUNGI:

YEASTS (unicellular fungi) MOULDS (multicellular fungi)

DIMORPHIC (YEASTS/37ºC; MOLDS/25ºC)

Cytoplasmic membrane / CM / Polyenes • Binding to ergosterol CM

(compromise function of CM)

Ergosterol / Azoles • Inhibition of enzymes for the synthesis of

ergosterol

Cell wall / Ehinocandins • Inhibits the synthesis of glucan

Nucleic acid / 5-Fc • Inhibits the synthesis of NA

Antifungals and primary sites of activityMechanisms of action

Fungal morphology and biology

YEASTSYEASTS MOULDS MOULDS

Candida Candida Aspergillus Aspergillus

Fungal morphology and biology

KVASNICE KVASNICE

PLESNI

PLESNI

Dimorphic fungi Dimorphic fungi

Blastomyces (Paracoccidioides) brasiliensisBlastomyces (Paracoccidioides) brasiliensis

Single cell (round / oval / elongated 2-7μm)

• Asexual reproduction:

blastoconydia / blastospore

• Sexual reproduction:

ascospores or basidiospores

Single cell (round / oval / elongated 2-7μm)

• Asexual reproduction:

blastoconydia / blastospore

• Sexual reproduction:

ascospores or basidiospores

YEASTS - morphology and biology

pseudohyphae and chlamydospores!!!

Multicellular / elongated hyphae (5-50 µm)

and conidia / spores:• conidia (asexual reproduction):

arthroconidia (hyphae segmentation) macroconidia microconidia ...

• spores (sexual reproduction): ascospores or zygospores

Multicellular / elongated hyphae (5-50 µm)

and conidia / spores:• conidia (asexual reproduction):

arthroconidia (hyphae segmentation) macroconidia microconidia ...

• spores (sexual reproduction): ascospores or zygospores

MOULDS - the morphology and biology

Hyphae containing compartments (septaes):• complete (septated hyphae)• incomplete (aseptated hyphae)

Mycelium - branched, tubular filaments (hyphae)• basal / vegetative (in medium)• aerial / reproductive (above medium)

Hyphae containing compartments (septaes):• complete (septated hyphae)• incomplete (aseptated hyphae)

Mycelium - branched, tubular filaments (hyphae)• basal / vegetative (in medium)• aerial / reproductive (above medium)

Dykariotes(2 haploid nucleus)

Mitosis

Most fungi are haploid /

heterokaryotic

REPRODUCTION OF FUNGI

YEASTS:

budding occur blastoconydia / blastospore

Unicellular structures (2-7 µm)

MOULDS:

Eg. fragmentation of hyphae - artroconidia conidiogenesis- conidia:

multicellular - macroconidia (15-150 µm)

unicellular - microconidia (2-6 µm)

YEASTS:

budding occur blastoconydia / blastospore

Unicellular structures (2-7 µm)

MOULDS:

Eg. fragmentation of hyphae - artroconidia conidiogenesis- conidia:

multicellular - macroconidia (15-150 µm)

unicellular - microconidia (2-6 µm)

ASEXUAL REPRODUCTION

• Microscopic appearance of hyphae and conidia important to identify nondermatophytes moulds • Microscopic appearance of macroconidia and hyphae essential for identification of dermatophytes moulds

Stages: • merge + (n) - (n) cells → plasmogamy

• resulting zygote (2n) of chromosomes • meiosis → 4 nucleus formed by (n) number of chromosomes

spores

Stages: • merge + (n) - (n) cells → plasmogamy

• resulting zygote (2n) of chromosomes • meiosis → 4 nucleus formed by (n) number of chromosomes

spores

SEXUAL REPRODUCTION Zygomycetes, Basidiomycetes, Ascomycetes

Sexual characteristics are key for taxonomy and systematization of fungi

zygospores ascospores bazidiospores

macroelements

microelements

oxygen aerobic microaerophilic facultative anaerobic

Water

CH

pH 2-8 optimum 5.4 - 5.6

macroelements

microelements

oxygen aerobic microaerophilic facultative anaerobic

Water

CH

pH 2-8 optimum 5.4 - 5.6

Fungal growth

Yeast growth curve:• no well-defined logarithmic

and exponential phase • linear growth (7-14 days)

• counts in colony forming

units / CFU

Yeast growth curve:• no well-defined logarithmic

and exponential phase • linear growth (7-14 days)

• counts in colony forming

units / CFU

Mould growth curve:

• Can not be expressed by counting the living cells (7-14 days)

• measure the weight of the talus

Mould growth curve:

• Can not be expressed by counting the living cells (7-14 days)

• measure the weight of the talus

psychrophiles/0-20˚C mesophiles/20-45˚C termphfiles/45-90˚C

psychrophiles/0-20˚C mesophiles/20-45˚C termphfiles/45-90˚C

YEASTS37oC

YEASTS37oC

MOULDS25oC

MOULDS25oC

Dimorphic

25°C rise to a MOULD37°C in the form of YEASTS

Dimorphic

25°C rise to a MOULD37°C in the form of YEASTS

For isolation of fungi from the patient material it is necessary to use both 25°C and 37°C

Colony growth:

3-7 days (yeasts, non-dermatophytes moulds) 14-21 days (moulds - dermatophytes) 4-6 weeks (dimorphic fungi)

TEMPERATURES FOR FUNGAL GROWTH

ADHERENCE

- attachment to receptors on cells

ADHERENCE

- attachment to receptors on cells

FUNGAL VIRULENCE FACTORS

ENZYMES- proteinase,

phospholipase,elastase, keratinase,

catalase, urease,coagulase, esterase,hemolysin, lipase ...

ENZYMES- proteinase,

phospholipase,elastase, keratinase,

catalase, urease,coagulase, esterase,hemolysin, lipase ...

MORPHOGENESIS

- transition from micelial to yeasts

growth phase, and the blastospore in

germ tube

MORPHOGENESIS

- transition from micelial to yeasts

growth phase, and the blastospore in

germ tube

Mycosis (FI)

Mycoallergosis

Mycotoxicosis

Mycosis (FI)

Mycoallergosis

Mycotoxicosis

FUNGAL DISEASES - classification

Mycetism

mushroom poisoning

Mycetism

mushroom poisoning

MEDICAL MYCOLOGY MEDICAL

TOXICOLOGY

“fruiting bodies”

FUNGAL PATHOGENICITY1. Apathogens (saprophytes)

2. Pathogens: opportunistic

pathogenic

FUNGAL PATHOGENICITY1. Apathogens (saprophytes)

2. Pathogens: opportunistic

pathogenic

CLASIFFICATION OF FUNGI AND FUNGAL

INFECTIONS (FI) CLASIFFICATION OF PATHOGENIC FUNGI

In regard to location and the immune status of patients

• Opportunistic / IFI ~ 300 species

• Skin FI ~ 33 species• Subcutaneous FI ~ 10 species• Endemic FI (dimorphic) ~ 25 species

CLASIFFICATION OF PATHOGENIC FUNGIIn regard to location and the immune status of patients

• Opportunistic / IFI ~ 300 species

• Skin FI ~ 33 species• Subcutaneous FI ~ 10 species• Endemic FI (dimorphic) ~ 25 species

Pathogenic fungi:- Dermatophytes Trichophyton Microsporum Epidermophyton- Dimorphic fungi

Pathogenic fungi:- Dermatophytes Trichophyton Microsporum Epidermophyton- Dimorphic fungi

Opportunistic fungi :- Candida- Aspergillus- Fusarium- Zigomicetes- Cryptococcus- Malassezia ...

Opportunistic fungi :- Candida- Aspergillus- Fusarium- Zigomicetes- Cryptococcus- Malassezia ...

immunocompromised persons / risk groups

immunocompetent persons

According to the localization:

1. superficial fungal infection (SFI) - superficial - cutaneous - mucocutaneous

2. deep / systemic / invasive (IFI) - localized - disseminated

*subcutaneously (subcutaneous tissue ...)???

According to the localization:

1. superficial fungal infection (SFI) - superficial - cutaneous - mucocutaneous

2. deep / systemic / invasive (IFI) - localized - disseminated

*subcutaneously (subcutaneous tissue ...)???

MYCOSES - FUNGAL INFECTIONS (FI)

presence and reproduction of fungi on / in the human body

CLINICAL SIGNS - CHARACTERISTIC CLINICAL SIGNS - CHARACTERISTIC

CLINICAL SIGNS - NO CHARACTERISTIC (risk group of patients!)

CLINICAL SIGNS - NO CHARACTERISTIC (risk group of patients!)

onychomycosisonychomycosis

Pulmonary

aspergillosis Pulmonary

aspergillosis

STANDARD MYCOLOGYCAL METHODS

(conventional methods):

1. Microscopic examination of the patient samples-direct microscopic examination (DME)-histopathology (PH)

2. isolation of fungi from patient samples - mycological cultures (MC)

STANDARD MYCOLOGYCAL METHODS

(conventional methods):

1. Microscopic examination of the patient samples-direct microscopic examination (DME)-histopathology (PH)

2. isolation of fungi from patient samples - mycological cultures (MC)

BASIC PRINCIPLES FOT THE DIAGNOSIS OF FI

IMMUNOLOGICAL METHODS

Antibodies (Ab) detection

Antigen (Ag) detection

Other biomarkers detection

IMMUNOLOGICAL METHODS

Antibodies (Ab) detection

Antigen (Ag) detection

Other biomarkers detection

MOLECULAR METHODS

- DNA detection (PCR)

MOLECULAR METHODS

- DNA detection (PCR)

PROVEN IFIPROVEN IFI PROBABLE / POSSIBLE IFI PROBABLE / POSSIBLE IFI

“golden s t a n d a r d”“golden s t a n d a r d”