FUNGI

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

5 KINGDOMS
Animalia : invertebrates, vertebrates Planta : flowering plants Fungi : yeasts, molds (moulds), mushrooms Protista : protozoans Monera : bacteria

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

Structure
Eukaryotic Cell wall : chitin
(polymer of Nacetylglucosamine) , mannan, glucan rigidity to cell wall

Non motile Fungal membrane: ergosterol

From: Sherris Medical Microbiology p 632

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.
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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

Fungi imperfecti : lack of sexual reproduction rRNA genes analysis : for classification
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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 bluewhite 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
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Fungal identification
For yeast - germ tube test : for Candida albicans
Yeast colony inoculated into 1 ml horse erum incubation for 2 hours, 35 C > 2 hours : can be false (+)

- biochemical tests
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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

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

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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 2celled 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.

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

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 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
Most occur on non hairy areas of the trunk

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Periphery of ring : active fungal growth

"Tinea barbae" caused by T. rubrum .

Cutaneous mycoses
3. Tinea capitis (scalp ringworm) Trichophyton sp Microsporum sp

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

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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
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.

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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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