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).

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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
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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. 9
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Types of conidiacontd
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 conidiacontd
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
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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. Dont 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
Sabourauds 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 25-30 C:all fungi will grow
Paired with 30-75 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 C

> 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)
Mycelium
mass 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
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http://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, geophilic
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Cutaneous mycoses
Transmission : infected skin scales
1. Tinea pedis (athletes 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 patient
e.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
Microscopy
Most 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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