General properties of fungi

Dr. Pendru Raghunath ReddyAssistant Professor of MicrobiologyDr. VRK Women’s Medical College

Introduction• Mykes (Greek word) : Mushroom• Fungi are eukaryotic protista; differ from bacteria

and other prokaryotes.1. Cell walls containing chitin (rigidity & support), mannan & other

polysaccharides 2. Cytoplasmic membrane contains ergosterols3. Possess true nuclei with nuclear membrane & paired

chromosomes4. Cytoplasmic contents include mitochondria and endoplasmic

reticulum5. Divide asexually, sexually or by both6. Unicellular or multicellular7. Most fungi are obligate or facultative aerobes

• Cell wall consists of chitin not peptidoglycan like bacteria

• Thus fungi are insensitive to antibiotics as penicillins

• Chitin is a polysaccharide composed of long chain of n-acetylglucosamine.

• Also the fungal cell wall contain other polysaccharide, β-glucan, which is the site of action of some antifungal drugs.

• Cell membrane consist of ergosterol rather than cholesterol like bacterial cell membrane

• Ergosterol is the site of action of antifungal drugs, amphtericin B & azole group

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Diverse group of heterotrophs.

– Many are ecologically important saprophytes (consume dead and decaying matter)

– Others are parasites.

Most are multicellular, but yeasts are unicellular. Most are aerobes or facultative anaerobes. Cell walls are made up of chitin (polysaccharide). 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).

In addition to those species which are generally recognized as pathogenic to man it is firmly established that under unusual circumstances of abnormal susceptibility of patient, or the traumatic implantation of the fungus, other fungi are capable of causing lesions. Those are called (Opportunistic fungi)

Predisposing factors

• Use of Antibiotics,• Use of steroids,• A debilitating condition

of the host, as Diabetes.• A concurrent disease

such as leukaemia• Immunosuppressive

conditions

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Fungal Morphology Molds Yeasts

Many pathogenic fungi are dimorphic, forming hyphae at ambient temperatures but yeasts at body temperature.

04/11/2023 Dr.T.V.Rao MD 9

Understanding the Structure of Fungus

• Simplest fungus :- Unicellular budding yeast• Hypha :- Elongation of apical cell produces

a tubular, thread like structure called hypha• Mycelium :- Tangled mass of hyphae is called

mycelium. Fungi producing mycelia are called molds or filamentous fungi.

• Hyphae may be septate or non-septate (coenocytic hyphae)

Mycelium

• Mass of branching intertwined hyphae– Vegetative– Aerial

Vegetative types

• Favic chandeliers

• Nodular organs

• Racquet hyphae

• Spiral hyphae

Classification of fungi

1.Morphological classification

2.Systematic classification

1. Yeasts

2. Yeast-like fungi

3. Filamentous fungi (molds)

4. Dimorphic fungi

Morphological classification

Yeasts

1. These occur in the form of round or oval bodies which reproduce by the formation of buds known as blastospores

2. Yeasts colonies resemble bacterial colonies in appearance and in consistency

3. The only pathogenic yeast in medical mycology is Cryptococcus neoformans

Yeast colonies

Mucoid colonies

Cryptococcus neoformans

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Yeast-Like1.These are fungi which occur in the form of

budding yeast-like cells and as chains of elongated unbranched filamentous cells which present the appearance of broad septate hyphae. these hyphae intertwine to form a pseudomycelium.

2. The yeast like fungi are grouped together in the genus Candida.

Candida Colonies

Candida albicans

SEM

Filamentous FungiThe basic morphological elements of

filamentous fungi are long branching filaments or hyphae, which intertwine to produce a mass of filaments or mycelium

Colonies are strongly adherent to the medium and unlike most bacterial colonies cannot be emulsified in water

The surface of these colonies may be velvety, powdery, or may show a cottony aerial mycelium.

mycelium: septate mycelium: non septate

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Colony Morphology

Dimorphic FungiThese are fungi which exhibit a yeast form in the

host tissue and in vitro at 370C on enriched media and mycelial form in vitro at 250C

Examples:Histoplasma capsulatumBlastomyces dermatitidisCoccidioides immitisParacoccidoides brasiliesisPenicillium marneffeiSporothrix schenckii

Histoplasma capsulatum - Dimorphism

• Filamentous mold in environment– Thin septate hyphae, microconidia, and

tuberculate macroconidia (8-14 µm)• Budding yeast (2-4 µm) in tissue

– Dimorphic transition is thermally dependent and reversible (25°C 37°C).

Hyphae, micro- and macroconidia Yeast within histiocyte

Systematic classification

• Based on sexual spores formation: 4 classes

1. Zygomycetes 2. Ascomycetes reproduce sexually3. Basidiomycetes4. Deuteromycetes (fungi imperfectii)

Zygomycetes • Lower fungi• Broad, nonseptate hyphae• Asexual spores -

Sporangiospores: present within a swollen sac- like structure called Sporangium

• Examples: Rhizopus, Absidia, Mucor

Ascomycetes

• Sexual spores called ascospores are present within a sac like structure called Ascus.

• Each ascus has 4 to 8 ascospores

• Includes both yeasts and filamentous fungi

Ascomycetes• Narrow, septate hyphae• Asexual spores are called conidia borne on

conidiophore• Examples: Penicillium, Aspergillus

BasidiomycetesSexual fusion results in the formation of a club shaped organ called base or basidium which bearspores called basidiospores

Examples: Cryptococcus neoformans, mushrooms

Deuteromycetesor Fungi imperfectii

• Group of fungi whose sexual phases are not identified

• Grow as molds as well as yeasts• Most fungi of medical importance belong

to this class• Examples: Coccidioides immitis,

Paracoccidioides brasiliensis, Candida albicans

Reproduction and sporulation

Types of fungal spores

1.Sexual spores

2.Asexual spores

Sexual spores• Sexual spore is formed by fusion of cells and

meiosis as in all forms of higher life• Ascospores

– Ascus– Ascocarp

• Basidiospores

• Zygospores

Asexual spores

These spores are produced by mitosis

1. Vegetative spores

2. Aerial spores

Vegetative spores

• Blastospores: These are formed by budding from

parent cell, as in yeasts

• Arthrospores – formed by segmentation & condensation of hyphae

• Chlamydospores – thick walled resting spores

e.g. C.albicans

Aerial spores

1. Conidiospores Spores borne externally on sides or tips of hyphae are called conidiospores or simply conidia

2. Microconidia

3. Macroconidia

4. Sporangiospores

• Microconidia - Small, single celled

• Macroconidia – Large and septate and are often multicellular

Immunity to fungal diseases

The defense to fungal infection involves both innate and aquired immunity

The passive protection is provided by intact skin and mucosal surfaces

The fatty acids like sebum also provide protection because of their antifungal activity

The alveolar macrophages are important in engulfing cells in lungs which are removed by ciliary action and coughing

A variety of innate defense factors in saliva such as lysozymes and lactoferrin contribute to mucosal protection

Cellular immunity

For the most of fungi, cellular immunity is mainstay of host defenses

Monocyte-macrophage activity, especially in respiratory tract, appears to be major defence mechanism against most fungi

Renal transplant recipients have peculiar predilection for cryptococcal infection

Humoral immunity

The precise role of humoral immunity in host defense against fungal infections is difficult to determine

Diagnosis of fungal diseases

1. Clinical diagnosis

2. Laboratory diagnosis

Clinical diagnosis

The clinical criteria may give presumptive diagnosis of fungal infections

The superficial and subcutaneous mycoses often produce characteristic lesions that strongly suggest their fungal etiology

In case of systemic mycoses, there is no sign or symptom that specifically suggests a fungal disease

Laboratory diagnosis

Specimens collection

1. Respiratory specimens2. CSF3. Blood4. Tissue, Bone marrow and body fluids5. Skin, hair and nail6. Urine, stool7. Vaginal secretions8. Corneal scrapings

Direct examination

The direct demonstration of fungal elements is essential in establishing diagnosis by detecting presence of causative agent in the clinical material

The following procedures are adopted to directly observe fungi in clinical specimens

1. Wet mounts

2. Histopathology

3. Fluorescent-antibody technique

4. Gram staining

Wet mounts

KOH wet mount

Slide KOH

Most of the specimens can be examined in wet mounts after partial digestion with 10-20% KOH

The clinical specimens like skin, hair and nails should be mounted under cover slip in KOH on slide

This clears material within 5 – 20 minutes, depending on its thickness

A slight warming over a low flame hastens digestion of keratin

KOH can also be supplemented with DMSO to increase clearing of fungi especially in skin scrapings and nail clippings

The KOH can be supplemented with a fluorescent dye, calcofluor white (CFW)

The CFW supplemented KOH especially in corneal scrapings can detect even scanty amount of fungal elements

Tube KOH

The tube KOH is prepared mainly for biopsy specimens, which take longer time for dissolution

The homogenized biopsy tissue is dissolved in 10% KOH and examined after keeping for an overnight in an incubator at 370C

KOH mount

Blastomyces dermatitidisMold (note: septate hyphae)

KOH mount

Ectothrix Endothrix

Other methods

The wet india ink and nigrosin preparations are also very useful in making diagnosis of certain capsulated fungi

Histopathology

The histopathological examination of biopsy specimens is an excellent way to diagnose mycotic infections

The H&E stain is routine procedure in histopathology laboratory and stains most of fungi

H&E stain

Endospores and sporangia of Rhinosporidium seeberi

Cryptococcus neoformans

Blastomyces dermatitidis Rhizopus species

H&E stain

The specific fungal stains, such as periodic acid-Schiff (PAS), Grocott-Gomori’s Methenamine silver (GMS) and Gridely stains are widely used for demonstrating fungi in tissues

Mayer’s mucicarmine stain can also be used specifically to show capsular material of Cryptococcus, endospores and sporangia of Rhinosporidium seeberi

PAS stain GMS stain

Fluorescent-antibody staining

May used to detect fungal antigen in clinical material such as pus, blood,CSF, tissue impression smears and in paraffin sections of formalin fixed tissue

It is less satisfacory for sputum specimens

Detects fungus when there are only few organisms present, as seen in pus from sporotrichosis

Its use is limited by restricted availability of specific antisera

Gram staining

All fungi are gram positive

Gram stained smear of Candida species

Fungal culture

The solid media are employed for fungal culture

The medium commonly employed is emmon’s modification of Sabouraud Dextrose Agar (SDA) [pH – 5.4]

The media may supplemented with antibiotics, such as gentamicin and chloramphenicol to minimize bacterial contamination and cycloheximide to inhibit saprophytic fungi

The special media may be used for isolation and to help rapid identification, when identity of a particular pathogen is strongly suspected Eg: Bird seed agar for Cryptococcus neoformans

Potato dextrose agar, Brain heart infusion (BHI) agar and Czapek Dox agar may also be used for isolation of fungi

Incubation

Cultures are routinely incubated in parallel at room temperature (for weeks) and at 370C for days

Many fungi develop relatively slowly and cultures should be retained for atleast 2-3 weeks (in some cases upto 6 weeks) before being discarded

Yeasts usually grow within 1-5 days

Interpretation of fungal cultures

The significance of fungal isolate depends on its source and identity

The isolation of an established pathogen such as H. capsulatum or Coccidioides species from any specimen is generally regarded as an evidence of infection

In case of commensal or opportunistic fungi following points should be considered

1. Isolation of same strain in all culture tubes2. Repeated isolation of same strain in multiple specimens3. Immune status of patients and4. Serological evidence to confirm significance of isolate

Identification of fungi

Gross or macroscopic examination of cultures

Microscopic examination

1. Tease mount

2. Slide culture

Tease mount

For microscopic examination, fragment of fungal culture is teased out using two teasing needles and placed on a glass slide in drop of LCB stain

To study the morphology of hyphae, spores and other structures, tease mounts are prepared in lactophenol cotton blue (LCB) which contains lactic acid, phenol, glycerol and cotton blue

Microscopic characteristics that should be observed are the following

1. Sepatate versus sparsely septate hyphae

2. Spores or conidia

Slide culture

Is used to study undisturbed morphological details of fungi, particularly relationship between reproductive structures like conidia, conidiophore and hyphae

It is indicated when teased mount of LCB is inconclusive in particular fungal isolate

Procedure

Place sterile microscopic slide on bent glass rod at the bottom of petri dish

A piece of one square centimeter block of cornmeal agar or potato dextrose agar is put up on the slide

Inoculate the fungal strain under identification at four sides of agar block

Cover the inoculated block with sterile coverslip and incubate at 250C in BOD incubator

Slide culture

Add a little sterile distilled water on filter paper to avoid drying of agar

When growth appears, place drop of LCB on slide and coverslip from block

Cellophane tape preparation

A piece of tape is gently laid over portion of fungal colony and slowly lifted and place over a glass slide containing LCB solution

This preparation has come into greater use to overcome obstacles of time consumption and requirement of extra equipment to prepare slide culture

Aspergillus Penicillium

Miscellaneous tests for the identification of molds

1. Hair perforation test

2. Urease test

3. Thiamine requirement

Miscellaneous tests for identification of yeasts

Germ tube production, carbohydrate assimilation, chromogenic substrates, potassium nitrate assimilation, temperature studies and urease

Serologic tests

Detection of antibodies

1. Immunodiffusion2. Countercurrent immunoelectrophoresis (CIE)3. Whole cell agglutination4. Complement fixation5. ELISA

Detection of antigens

6. Latex particle agglutination7. ELISA

Molecular techniques

1. PCR

2. DNA probes

Mycoses

Infection caused by fungus is known as mycosis (Plural mycoses)

Classification of mycoses

Classification of fungal disease according to primary sites of infections

1. Superficial mycoses2. Cutaneous mycoses3. Subcutaneous mycoses4. Systemic mycoses5. Opportunistic mycoses