FUNGI

dr. S. Yumna T, MSc, MClinSc(Hons)

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5 KINGDOMS

• Animalia : invertebrates, vertebrates

• Planta : flowering plants

• Fungi : yeasts, molds (moulds), mushrooms

• Protista : protozoans

• Monera : bacteria

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Overview

• >200,000 fungal species, most saprophytic• Ubiquitous: water, soil, air, human, animal,plant• Many : decomposer, food production (cheese),

antibiotic (Penicillium notatum)• <200 fungal species : potential pathogenic for

humans• Few species : most clinically important fungal

infection• Fungi can cause infection (mycoses), poisoning

(mycotoxin, aflatoxin), allergy (asthma).4

Characteristics of major fungal groups

• Structure

• Metabolism

• Reproduction

• Modes of fungal growth

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Structure

• Eukaryotic • Cell wall : chitin

(polymer of N-acetylglucosamine), mannan, glucan

rigidity to cell wall

• Non motile• Fungal

membrane: ergosterol

From: Sherris Medical Microbiology p 632 6

Metabolism

• Non photosynthetic

• Heterotrophic metabolism heterotrophs

- requires exogenous C for growth- chemotrophic : secrete degradative enzymes soluble nutrient passive or active transport

• Most: obligate aerobes, None: obligate anaerobes

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Reproduction

• Asexual

- forms conidia by mitosis

• Sexual

- genetic recombination

- meiosis forms sexual spores in specialized structure

- spores (ascospores, zygospores, basidiospores)

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A. Aspergillus; B. Penicillium; C. Geotrichum; D. Trichophyton; E. Microsporum; F. Epidermophyton and G. Rhizopus.

From Medical Microbiology, 1990, Murray, et al., p. 300, Fig. 28-2. 10

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Types of conidia…cont’d

• Blastoconidia through a budding process, e.g. Cladosporium sp

• Chlamydoconidia produced from terminal or intercalary hyphal cells

e.g. Candida albicans

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Types of conidia…cont’d

• Phialoconidia produced by ‘vaseshaped’ conidiogenous cells (phialide)

e.g. Aspergillus fumigatus

• Macroconidia : large and complex

• Microconidia : small and simple

• Endospore : conidia are enclosed in a sac (sporangium)

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Modes of fungal growth:

Fungi can be divided into two basic morphological forms, yeasts and molds (moulds).

1. Yeasts

- Unicellular

- Reproduce asexually by blastoconidia formation (budding) or fission

- Larger than bacterial cells (5-8 X)

- ex. ‘baker yeast’ : Saccahromyces cerevisiae

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Modes of fungal growth:

• Yeasts

- Yeast can form pseudohyphae.

as a result of a sort of incomplete budding where the cells remain attached after division.

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Modes of fungal growth:

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Modes of fungal growth:

2. Molds

- Multicellular

- Higher form

- Composed of filaments : hyphae mycelium

- Reproduce sexually or asexually

- Most fungi occur in the hyphae form as branching, threadlike tubular filaments.

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Modes of fungal growth:

- Structure of hyphae/filamentous structure:

1. coenocytic/continuous/aseptate

lack of cross wall

e.g. Zygomycetes

2. septate : have cross wall pores

- clamp connection

e.g. Aspergillus sp

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Modes of fungal growth:

A. Yeast cells reproducing by blastoconidia formation; B. Yeast dividing by fission; C. Pseudohyphal development; D. Coenocytic hyphae; E. Septate hyphae; F. Septate hyphae with clamp connections From Medical Microbiology, 1990, Murray, et al., p. 299, Fig. 28-1.

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Clamp connections are formed by the terminal hypha during elongation

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Modes of fungal growth:

3. Fungi dimorphic (dimorphism)

fungus can exhibit either the yeast form or the hyphal form, depending on growth conditions.

-Very few fungi exhibit dimorphism.

-Yeast: enriched medium

-Mold : requires minimal nutrients

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Yeasts and Molds

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Yeast form

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Classification of fungal phyla and class

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Fungi imperfecti : lack of sexual reproduction

rRNA genes analysis : for classification

Medical grouping

• Superficial fungi : skin and its appendages • Subcutaneous : skin subcutaneous or

lymphatic spread• Opportunistic : environmental or ‘normal’

fungi that can cause infection in immunocompromised host

• Systemic fungi : not part of ‘normal microorganism, most virulent,systemic/visceral infection

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Mycoses

• Superficial

• Cutaneous

• Subcutaneous

• Opportunistic

• Systemic

Infection from environment or endogenous

Only dermatophytic infections are communicable

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Pathogenesis

• Host – pathogen interaction

• Pathogenesis :

- adherence, e.g. C. albicans (mannoprotein adhesin) >< fibronectin receptor in host epithelial cells

- invasion, e.g. trauma : Sporothrix schenckii

Coccidioides immitis : airborne 28

Pathogenesis

- invasion : C. albicans (hyphal form) specific enzymes

-tissue injury

- no classic toxin produced in vivo

- host inflammatory immune response

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Laboratory diagnosis

• Specimen collection

• Equipment

• Transport

• Criteria for rejection

• Types of examination

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Specimen collection

Good collection :• Sterile technique• Area most likely to be infected:

- edge ringworm- broken hair- sputum ; not saliva, etc

• Adequate• Deliver promptly and process quickly• Avoid overgrowth of bacteria• Label : name, DOB, site, etc

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Specimen collection

A. Adequate collection sufficient for microscopy and culture

B. Inadequate collection

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Equipment

• Bone curette • Blunt scalpel• Spoon excavator• Scissors• Forceps• Nail clippers • Sterile cotton

swab All equipment must be cleaned and sterilized

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Equipment

• Collection container

sealed with parafilm. Don’t use sticky tape

• Black collection cards

• Media

• Inoculating needles

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Mycology specimen collection-1

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Mycology specimen collection-2

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Skin scraping

• Require epidermal scales

• Scrape margin or edge• Active border – red

scaly raised• Several lesions –

separate samples• Blister wall (dinding

vesikel)

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Nails

• Larger sample• Discoloured,

dystrophic, brittle (rapuh)

• Scrape under nail plate

• Collect all debris• Separate samples

from different nails (do not pool)

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Hair

• Hair roots NOT cut hair

• Fluoroscence with woods lamp

• Crusts or scales use scalpel

• Swab exudate if kerion

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Transport

• Skin, Hair, Nail

- room temperature NOT refrigerated

• Sputum, urine, swabs, tissues

- refrigerate if delay in reaching laboratory

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Criteria for rejection

• No ID/label

• Sputum with >25 epi/lpf

• Dry swab

• Insufficient specimen

• 24 hours urine/sputum

• Improper container (leaking/unsterile)

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Direct microscopic examination

• Direct smears, using several types of reagents or stains

- 10 % KOH (in glycerol) : clear away tissue cells and debris, dissolves keratin.

spec: nails, hair, skin scrapings, fluids or exudates

- Glycerol stops drying

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Direct microscopic examination

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Direct microscopic examination

-Calcofluor white

• binds to cellulose and chitin fungal wall fluoresce blue-white or apple green depend on filter

• Dye fluoresces on exposure UV light

•Mix in equal parts with 10% KOH glycerol

•Fluorescent microscope – correct filter

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Types of examination

• Direct microscopic examination

- from clinical specimen (sputum, spinal fluid, skin scraping, etc)

• Culture

• Fungal identification

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Direct microscopic examination

India ink

Detects capsule around yeast cells

Cryptococcus sp in CSF

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Direct microscopic examination

• LP light brown or LPCB (lactophenol catton blue) blue

• Wright, Giemsa : intracellular yeast form of Histoplasma capsulatum

• Gram stain : positive (yeasts)• ZN : negative, except Nocardia sp

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• PAS (periodic acid shift) : red with yellow/light green background

• etc• Histologic examination : biopsy

HE, methenamine silver

Methenamine silver stain of sinus biopsy

(100). All of the black material represents

the invading fungus, Aspergillus flavus.

Two of the characteristic sporebearing

structures can be seen on the nasal cavity side

(arrows).

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Culture

• Slow, days - weeks

• Sabouraud’s dextrose agar (SDA)/SA

- glucose and peptones as nutrients.

- pH is 5.6

• Selective media : plus antibiotic, e.g. chloramphenicol, gentamicin

• Antifungal cycloheximide in SDA: inhibit contaminating molds from environment and some pathogens

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Culture

• Incubation 25-30º C:all fungi will grow

• Paired with 30-75 º C: pathogenic fungi will grow

• Aerobic incubation

• Sterile site, e.g CSF: no need selective agents

• Blood sheep : dimorphic fungi

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Chromogenic agar

Fungal identification

• For yeast

- germ tube test : for Candida albicans

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Yeast colony inoculated into 1 ml horse erum incubation for 2 hours, 35 ºC

> 2 hours : can be false (+)

- biochemical tests

Fungal identification

• Molds

depends on growth rate, colonial appearance, metabolic properties.

- macroscopic : examine both side, including reverse (bottom)

surface structure: velvety,cottony,etc

topography: flat, raised, wrinkled,etc

pigmentation in surface and reverse: white, brown, etc

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Trichophyton rubrum

surface

bottom/reverse appearance

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Fungal identification

• - microscopic :

conidia/spores: shape, size, structure arrangement

hyphae : size, septate, clamp connection

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Mold form • Hypha (e)

Elongated cells or filaments

• Septate and nonseptate hyphae (continuous)

• Myceliummass of hyphae

• Vegetative mycelium : acts as root : nutrients/moisture collector

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…continued• Aerial mycelium

bears conidia or spores : fluffy character (seperti kapas)

• Conidiophore

a hyphae bearing conidia which bears at its tip or sides,

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Superficial mycoses

• Skin (str.corneum) and hair shaft

• Host cellular responses : minimal

• Harmless except cosmetically

Pityriasis versicolor : Malassezia furfur

Tinea nigra : Hortaea werneckii

Piedra (hair shaft): Piedraia hortai

• Dx. Skin scraping microscopic exam.

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Case 1

Case•A 25-year-old Caucasian male from Darwin, presented with non-inflammatory, brown pigmented, non-scaling lesions on the palmar aspects of his hands. Direct microscopy showed the presence of fungal elements and the fungus shown below was isolated

60http://www.mycology.adelaide.edu.au/virtual/2007/ID2-Feb07.html#top

Case 1

• Clinical Presentation:

Brown to black, non-scaling macules on the palmar aspect of the hands. Note there is no inflammatory reaction.

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Case 1

• Direct Microscopy :Skin scrapings mounted

in 10% KOH : pigmented brown to dark olivaceous (dematiaceous) septate hyphal elements and 2-celled yeast cells producing annelloconidia

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Case 1

• Culture:Colonies are slow growing,

initially mucoid, yeast-like and shiny black. With age they develop abundant aerial mycelia and become dark olivaceous in color.

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Conidia of Hortaea werneckii

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• What is the diagnosis ?

Tinea nigra

• Causative fungi?

Hortaea werneckii.

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Cutaneous mycoses

• = dermatophytoses

• Infect superficial keratinized tissue (skin, nail, hair). Keratin nutrients

• Causative fungi: dermatophytes

Trichophyton : skin, nails, hair

Epidermophyton : skin, nails. NOT hair

Microsporum: skin, hair. NOT nails

• Habitat : anthropophilic, zoophilic, geophilic66

Cutaneous mycoses

• Transmission : infected skin scales

1.Tinea pedis (athlete’s foot)

Trichophyton rubrum

Trichophyton mentagrophytes

Epidermophyton floccosum

Tinea pedis caused by T. rubrum.

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"Moccasin-type" tinea pedis caused by E. floccosum (left)

• toe web spaces are the major reservoir on the human body for these fungi

• individuals with chronic or subclinical toe web infections are carriers and represent a public health risk to the general population, in that they are constantly shedding infectious skin scales.

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Cutaneous mycoses

2. Tinea corporis (ringworm)

Epidermophyton floccosum

Trichophyton sp

Microsporum sp

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Most occur on non hairy areas of the trunk

Periphery of ring : active fungal growth "Tinea barbae" caused by T. rubrum .

Cutaneous mycoses

3. Tinea capitis (scalp ringworm)

Trichophyton sp

Microsporum sp

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Predominant species depends on the geographic location of the patiente.g. in the USA : T. tonsurans

3 types of hair invasion in tinea capitis:•Ectothrix invasion is characterised by the development of arthroconidia on the outside of the hair shaft. •M. canis, M. gypseum, T. equinum and T. verrucosum

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• Endothrix hair invasion is characterised by the development of arthroconidia within the hair shaft only.

• T. tonsurans and T. violaceum.

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• Favus usually caused by T. schoenleinii, produces favus-like crusts

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Cutaneous mycoses

4. Tinea cruris (jock itch)

Epidermophyton floccosum

Trichophyton rubrum

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Similar to ringworm but most occur in the moist groin area

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Tinea of the buttocks caused by T. rubrum granular strain

Tinea of the buttocks caused by T. rubrum downy strain.

Cutaneous mycoses

5. Tinea unguium (Onychomycosis)

Trichophyton rubrum

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Case 2

• Case

A 59 year old male presented with suspected onychomycosis of the toe nail.

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Case 2

• Direct microscopy of nail scrapings (KOH mount) revealed the presence of septate fungal hyphae breaking up into arthroconidia.

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Case 2

• Culture:

Colonies (SDA) are flat to slightly raised, white to cream, suede-like to downy, with a yellow-brown to wine-red reverse.

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Case 2

• MicroscopyMost cultures show scanty

to moderate numbers of slender clavate to pyriform microconidia. Macroconidia are usually absent

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Case 2

• What is the pathogen causes the disease?

Trichophyton rubrum

Trichophyton rubrum is an anthropophilic dermatophyte.

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Etiology

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Management of dermatophytoses

• Is dependant on clinical setting

• Topical or systemic agent?

• Mycological confirmation

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Other mycoses

Subcutaneous mycoses

•Chronic, localised (skin, connective tissue, bone, muscle)

•Deep tissue involvement rare

•Traumatic implantation

•Sporotrchosis, chromomycosis (chromoblastomycosis), mycetoma

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Systemic mycoses

•Pathogenic fungi

•Deep tissue and organs

•Dimorphic fungi

•Geographically restricted

•Airborne : inhalation of conidia

•Coccidioidomycosis, blastomycosis, histoplasmosis

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Other mycoses

Opportunistic mycoses

•Opportunistic fungi

•Candidiasis (candidosis), cryptococcosis, aspergillosis

•Now increasing:

immunosuppressive

corticosteroid, etc

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Anti fungal

• Amphotericin B and nystatin : bind to ergosterol, form pores that disrupt membrane function cell death

• Imidazole (clotrimazole, ketoconazole, miconazole) dan triazole (fluconazole and itraconazole) : interact with C-14 -demethylase to block demethylation of lanosterol to ergosterol

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