INTRODUCTION TO MEDICAL MYCOLOGY

Lecture 14

 Mycology
Myco- fungus Logy - study

Definitions
Mycologists--scientists who study fungi Mycology--scientific discipline dealing with fungi Mycoses--diseases caused in animals by fungi Mykos = mycete = fungus

 I. FUNGI

Diverse group of heterotrophs. Many are ecologically important saprophytes (consume dead and decaying matter) Others are parasites. Most are unicellular. multicellular, but yeasts are

Most are aerobes or facultative anaerobes. Cell walls are (polysaccharide). made up of chitin

Over 100,000 fungal species identified. Only about 100 are human or animal pathogens. Most human fungal infections are nosocomial and/or occur in immunocompromised individuals (opportunistic infections). Fungal diseases in plants cause over 1 billion dollars/year in losses.

General knowledge of the fungi

Eukaryotic microorganisms Rigid cell walls: made from chitin, glucans, mannans Plasma membranes: made from ergosterol Lysine synthesis by L- amino adipic acid (AAA) pathway.

General knowledge of the fungi

Both sexual and asexual spore may be produced Store their food as glycogen (plant; starch) Fungi are heterotrophic organisms, lack of chlorophyll (plant; autotrophic)

General knowledge of the fungi

Yeast : unicellular, 370C Mold : multicellular, hyphae, 250C Dimorphic fungi (thermally dimorphic fungi) : mold phase & yeast phase

Fungal Structure
Thallus-body
Molds & fleshy fungi have these structures
Long filaments of cells (hyphae): Septate hyphae (cross wall) :most fungi Aseptate hyphae (coenocytic ) :no cross wall, continous mass with many nuclei .

Yeasts
Facultative Anaerobes Fermentation : ethanol and CO2 Non-filamentous unicellular fungi
Spherical or oval

Reproduction:
a) fission, b). budding

Yeast Reproduction
FISSION even reproduction, nucleus divides forming two identical cells, like bacteria BUDDING uneven reproduction, parent cells nucleus divides and migrates to form a bud and then breaks away

YEAST
Unicellular Microscopy.: Oval to round

(Diameter : 3-15 m)

Macroscopy.: Pasty colonies (resemble bacteria)

Yeast

MOULD
Multicellular Micr.: . Hypha(e) (dia: 2-10 m) . Spores / conidia. Macr.: .Surface texture: Cottony/ powdery/ wooly/velvety/granular/glabrous Pigmentation :obverse & reverse

MOULD
Hypha : septate, aseptate Mycelium: a. Vegetative b. Aerial /fertile/ reproductive

Classification of Hyphae
A. Existence of septa
Septate Nonseptate ( coenocytic )

B. Shape and Morphology

Racquet Spiral Nodular Rhizoid -Root-like Pectinate Favic - chandelier

Dimorphic Fungi
Capable of growing in mould or yeast form under different environmental conditions (temperature, CO2, nutrients)

Fungal Classification
Four groups of true fungi Zygomycetes (common bread moldRhizopus) Basidiomycetes (puffballs & common mushrooms) Ascomycetes (Dutch elm disease/rye smut) Deuteromycetes (fungi imperfection)

Classification ( cont) :
First three groups is based on their method of sexual reproduction 4th group, the Deuteromycetes, have NO sexual reproduction

 Asexual Reproduction
The progeny will be identical to parent: Spores are called
Conidiospores Blastospores, Chlamydospores Sporangiospores

Fragmentation of hypahe -(Arthrospores)

Fungal spores are for reproduction, do not ensure resistance to environmental conditions

Asexual Spores
Conidiospores Chlamydospores Sporangiospores Blastospores Arthrospore

Asexual spores ( cont)

Conidiospore
Multiple (chains) or single spores formed at the end of an aerial hypha Not enclosed within a sac
Aspergillus spp. Penicillium spp Most of the common household molds & mildews are conidial fungi

Aspergillus sp.

Asexual spores ( cont)

Blastospores
A bud coming off the parent cell Candida albicans

 Chlamydospore
Formed within hyphae Thick-walled spore The chlamydospore is a method of producing a substantial resting spore very quickly. Nutrient is shunted from adjacent cells into a preferred cell and it swells up, converts nutrient materials to oil droplets for efficient storage, then rounds off with a thick, often roughened outer wall for protection
Candida albicans

Asexual spores
Sporangiospores
Hundreds formed within a sac (sporangium) at the end of an aerial hypha Rhizopus spp. Mucor spp Absidia spp

Spora-spora aseksual

Sporangiospore
1.Mucor
2.Rhizopus 3.Absidia

Cunninghamella

1.Mucor

2.Rhizopus
3.Absidia

Cunninghamella

(1).arthrospore (2).chlamydospor(3).phialospore

SEXUAL Spores
1. Zygospore 2. Ascospore 3. Basidiospore 4. Oospore

Fungi-Taxonomic classification is based on sexual spores

SEXUAL SPORE CLASS

Zygospore----------Zygomycetes Basidiospore--------Basidiomycetes Ascospore----------Ascomycetes None/Unknown---- Deuteromycetes (Fungi Imperfecti)

Ascomycetes
Asexual phase Conidiospores (Penicillium and Aspergillus) budding yeast

Sexual phase (morels, lichens )

Zygomycetes
Asexual phaseSporangiumbread mold (Rhizopus stolonifer) Sexual phase--- sporgangium ---shotgun fungus (lives on dung) it shoots its sporgangium explosively towards light or fly pathogen (Entomophthora muscae-these types of fungi have been used as agents for biological control of insects

Basidiomycetes
Basidiospore Examples: boletes, puffballs,smuts, stinkhorns and tooth fungi

MYCOSES
Superficial ( skin, hair, cornea) Cutaneous ( Dermatophytosis ) Subcutaneous True systemic (endemic) Opportunistic

Laboratory to diagnosis of fungal infection

Specimen collection and transport Specimen processing Direct examination Selection and inoculation of culture media Identification

Specimen collection and transport

must be material from the actual infection site must be carefully the contamination must be established for the best chance of recovery of causative microorganisms (optimal times)

Specimen collection and transport ( cont)

must be obtained to perform the culture or other techniques request (sufficient quantity) must be used to ensure optimal recovery of microorganisms obtain cultures before the treatment the culture container must be properly labeled

Sampling
In superficial mycoses the scales or infected hairs

may be stored into small sterile glass Petri dishes. Infected nail or skin scrapings are taken from the deeper parts with a blunt scalpel. Specimens from the mucous membranes or from orifices should be collected with dry swabs or preferably swabs soaked in Sabourauds broth.

For the diagnosis of bronchopulmonary infection morning sputum should be collected in a sterile container. For systemic mycosis, pus swab from an ulcer or aspiration from unruptured abscess, or biopsy during surgical operation are collected by strict aseptic technique.

For urinary tract infection, mid-stream urine samples are collected into a wide mouth sterile container.

 For cerebrospinal infections, a lumbar puncture should de performed to collect CSF into sterile test tubes. For Pleural and Peritoneal Effusions, a sample is collected by needle aspiration into sterile container.

Specimen processing
specimen should be examined as soon as possible direct examination :
KOH mount Calcofluor white India ink

culture media

Direct Examination

Selection and inoculation of culture media

Culture media for recovery of fungi from clinical specimens usually used is PDA, SDA or Corn Meal Agar. The recovery rate may be somewhat enhanced by using a variety of isolation media, storage, incubator time and temp.

 If the specimens are contaminated e.g. sputum, incorporate antibiotics such as chloramphenicol (0.5 g/l) in the media used for isolation.
Cycloheximide can also be incorporated for the isolation of dermatophytes in order to get rid of saprophytic fungi The pathological material is spread upon the surface of agar slopes. The fragments of skin, hair or nail are planted with a firm straight pointed wire. Incubate slopes at temperature up to 30oC

Initial observations in the study of fungus isolates

1.Appearance of the growth 2. Rate of growth 3. Colony pigmentationn (Recto & Verso) 4. Growth on media containing antifungal agents 5.Dimorphic fungi

Initial observations in the study of fungus isolates

1.Appearance of the growth - surface and reverse surface of colony were observed - delicate or hairlike hyphae 2. Rate of growth - saprophytes : 3-5 days - dimorphic fungi : 10 days or more - dermatophytes : 14 days or more

Initial observations in the study of fungus isolates

3. Colony pigmentation

4. Growth on media containing antifungal agents

- most strains of the dimorphic fungi can grow - most strains of the rapidly growing saprophytes are inhibited 5. Dimorphic growth - mold form (the environmental and infective form) ; ambient or room temperature (22-25 OC) - yeast form (invasive form) ; near body temperature (30-35 OC)

Recto

Verso

Microscopic Examination:
A Needle Mount is made as soon as spore formation is sufficiently advanced at the center of the colony. Place a drop of lactophenol cotton blue on the glass surface. With a nickel-chrome needle, pick-up a bit of the mycelium and put it directly to the drop of stain. Cover it with a cover-slip and examine under the microscope.

Histopathological Diagnosis
To establish the diagnosis of a fungus disease, identification should be made by a combination of mycologic and histopathologic study.

Because of size, characteristic morphology and histochemical composition, fungi can be studied satisfacorily in tissues.

 Although many fungi can be seen with haematoxylin and eosin stain (H&E), some are not stained by this method. Special stains for fungi are of great help in reaching the etiologic agent. These stains are mainly:
Periodic acid-Schiff stain (PAS). Gomori methenamine Silver stain (GMS). Mayers mucicarmine stain. Gridley fungal stain.

Serological Diagnosis
Serological diagnosis of mycoses usually lacks complete specificity because some of the pathogenic fungi have common antigens. Fractional separation of the active antigenic components of a fungus has not been achieved with complete success. However, serologic techniques are useful in reaching a presumptive diagnosis.

 These methods may include:

Double Diffusion method. Counter-immuno electrophoresis. Latex agglutination. Indirect Immunofluorescence. Indirect haemagglutination. ELISA.

Experimental Animal infection

Experimental animal inoculation is sometimes useful for the isolation of the causative fungus especially in deep mycoses.
White mouse - Histoplasmosis , Cryptococcosis. Hamster for Blastomycosis Albino Rabbit for Candidosis. Guinea-pig for experimantal infections with zoophilic Dermatophytes.

TYPES OF FUNGAL INFECTION

Superficial Fungal Infections

DERMATOPHYTES. YEASTS

aka Tinea

-Pityrosporum.
-Candida sp.

TINEA Infections
T.Corporis- ringworm of body T.Cruris- groin T.Pedis- foot T.Unguium- nail T.Capitis - scalp

Yeasts
Pityrosporum & Candida. Ordinarily commensals. Can become pathogens under favourable conditions.

Candidal Intertrigo

Pityriasis Versicolor
hypopigmented

T.Pedis