Diagnosis of Mycotic diseases.pptx

8/21/2019 Diagnosis of Mycotic diseases.pptx

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Seminar

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Introduction

General features of fungi Morphology / structure of the fungi

Types of mycosis

Specimen collection Classification of diagnostic methods:

Microscopy & staining

Culture techniques

Biochemical tests

Serological identification

Special methods

Candida 2


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The fungi are saprophytic (derives nourishment

from dead organic matter) and parasitic eukaryotic

organisms.

Although formerly considered to be plants, theyare now generally assigned their own

kingdom, Mycota.

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They resemble plants because they have rigid cell

walls and are non motile.

Unlike plants, the fungi lack chlorophyll and are

unable to photosynthesize. The fungi also lack the

multicellular complexity and organization of mostanimals.

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Historically, the fungi were regarded as relatively

insignificant causes of infection.

It is now well documented that the fungi are the

common cause of infection, particularly in

immunocompromised patients.

Opportunistic invasive fungal infections remain an

important cause of morbidity and mortality.

The most common fungi that cause disease in transplant

recipients and other immunocompromised patients are

Candida and Aspergillus species 5


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Fungi exhibit two basic structural forms:

YEAST- these are unicellular with spherical orovoid bodies

MOULD - are multicellular organisms.

YEAST MOULD

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YEASTS:Yeasts are unicellular organisms that are round to ovaland range in size from 2 to 60m.

The microscopic morphologic features usually appearsimilar for diff. genera and are not particularly helpful intheir separation.

MOLDS (MOULDS):The mold (or mould) form of growth refers to theproduction of multicellular, filamentous colonies.

These colonies consist basically of branching cylindricaltubules varying in diameter from 2 to 10 mm andtermed hyphae.

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Hyphal growth occurs by apical elongation.

The mass of intertwined hyphae that

accumulates during active growth in the mold

form is called a mycelium.

Germ Tube

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GENERAL FEATURES OF FUNGI:

Fungi seen in the clinical laboratory can generally beseparated into two groups based on the appearance of

colonies formed.

The yeasts produce moist, creamy opaque or pasty

colonies on media,

Whereas the filamentous fungi or molds produce

fluffy, cottony, wooly or powdery colonies.

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Several pathogenic species of fungi that exhibit

either yeast (or yeast like) and filamentous form are

referred to as being dimorphic.

The dimorphism is temperature dependent; the

fungi are designated as thermally dimorphic

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Dimorphic fungi have the ability to grow vegetatively

at 25C as moulds and at 37C as yeasts, spherules or

enlarging endospores.

These fungi include :

Blastomyces dermatitidis, Histoplasma capsulatum,

Paracoccidioides brasiliensis,

Coccidioides immitis, Penicillium marneffei,

Sporothrix schenckii.

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In many species of fungi, individual cells are

separated by cross-wall, or septa, such fungi

are described as septate.

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The septa are incomplete, however, and pores

allow adjacent cytoplasm to mix.

In other fungal species, the cells have no septa, and

the cytoplasm and organelles of neighboring cells

mingle freely. These fungi are said tobe coenocytic.

The common bread mould Rhizopus stolonifer iscoenocytic, while the blue-green mould that

produces penicillin,penicillin notatum, is septate

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The hypha is the morphological unit of fungus

and is visible only with the aid of a microscope.

Hyphae have broad diversity of forms and many

hyphae are highly branched with reproductive

structures.

A thick mass of hyphae is called a mycelium.This

mass is usually large

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The study of fungi is called mycology, and a

person who studies fungi is called a mycologist.

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Based on normal habitat ,classified as

anthropophilic(humans),

zoophilic(animals) geophilic(soil)

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http://en.wikipedia.org/w/index.php?title=Anthropophilic&action=edit&redlink=1http://en.wikipedia.org/wiki/Zoophilichttp://en.wikipedia.org/wiki/Geophilichttp://en.wikipedia.org/wiki/Geophilichttp://en.wikipedia.org/wiki/Zoophilichttp://en.wikipedia.org/w/index.php?title=Anthropophilic&action=edit&redlink=1


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The superficial mycosis:

These are infections limited to the outermost layers ofthe skin, the nails and hair, and the mucous membranes.

The principal infections in this group are thedermatophytoses and superficial forms of candidosis.

These diseases affect millions of individuals worldwide,but are readily diagnosed and usually respond well totreatment.

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The subcutaneous mycosis:

These are infections involving the dermis, subcutaneoustissues and adjacent bone.

These infections are usually acquired as a result of thetraumatic implantation of organisms that grow assaprobes in the soil and on decomposing vegetation.

These infections are most frequently encounteredamong the rural populations of the tropical andsubtropical regions of the world, where individuals gobarefoot and wear the minimum of clothing.

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The disease may remain localized at the site of

implantation or spread to adjacent tissue.

More widespread dissemination of the infection,

through the blood or lymphatics, is uncommon,

and usually occurs only if the host is in some way

debilitated or immunocompromised.

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The systemic mycoses:

These are infections that usually originate in the

lungs, but may spread to many other organs.

These infections are most commonly acquired as a

result of inhaling spores of organisms that grow as

saprobes in the soil or on decomposing organic

matter, or as pathogens on plants.

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FUNGUS

1. Blastomyces dermatidis

2. Coccidioides immitis

3. Histoplasma capsulatum

4. Pneumocystis carinii

5. Candida albicans

6. Cryptococccus neoformans

7. Dermatophytes

(Trichophyton,

Epidermophyton)

INFECTION

Blastomycosis

San Joaquin valley fever

Histoplasmosis

Pneumocystis pneumonia

Thrush, vaginitis,candidiasis.

Cryptococcosis

Tinea (ringworm, athletes

foot)

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Mycosis Oral manifestation

Blastomycosis(Gilchrist Disease)

Tiny ulcerations on oral mucosa

Histoplasmosis(Darling Disease)

Nodular , ulcerative or vegetative lesions on buccal mucosa,gingiva, tongue, palate & lips.

Cryptococcosis(Torulosis )

Non specific single or multiple ulcers

Coccidiomycosis(San Joaquin valley fever)

Non specific Proliferative granulomatous & ulcerated lesions inoral mucosa.

Candidiasis

(Candidosis)

Oral thrush, Actinic chelitis

Mucormycosis Reddish-black turbinate, with sputum & nasal discharge.Necrosis of paranasal sinuses & orbital floor.

Sporotrichosis Non specific ulceration of oral, nasal & pharyngeal mucosa,with regional lymphadenopathy

RhinosporidiosisWarts or red soft polypoid growth of tumor like nature in oral

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PRIMARY PATHOGENS

1. Histoplasma capsulatum

2. Blastomyces dermatidis

3. Coccidioides immitis

4. Paracoccidioides brasilienses

PATHOGENS WITH INTERMEDIATE VIRULENCE

1. Spoprothrix schenkii

2. Dermatophytes

SECONDARY PATHOGENS

1. Cryptococcus neoformans

2. Candida

3. Aspergillus

4. Mucorales (Rhizopus, mucor, absidia) 23


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COLLECTION AND TRANSPORT OF THE CLINICAL

SPECIMEN:

The diagnosis of fungal infections is dependent

entirely on the selection and collection of anappropriate clinical specimen for culture.

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SPECIMENS INCLUDE

Skin scrapings.

Hair and nails.

Respiratory tract secretions.

Cerebrospinal spinal fluid.

Blood. Smears from Mouth and vagina.

Urine.

Pus. Ocular specimen.

Tissue.

Bone marrow.

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1- Microscopy

2- Culture techniques

3- Biochemical reactions

4- Serological identification:

5- Special Tests

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Gram Stain

Acid fast stain

Potassium Hydroxide (KOH)

Calcofluor white stain

Grocott Methenamine silver stain

PAS stain

Wright stain

India Ink

Methylene blue

Acridine orange stain

Giemsa stain

MICROSCOPY AND STAINS

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Gram stain: Is most commonly performed on most clinical specimens

submitted for bacteriology & will detect most fungi , ifpresent.

Eg: cryptococcus .

These stains define the structure so that morphologic criteria

can be useful for identification.

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Principle: The difference in composition

between gram- positivecell wall, which

contain thick peptidoglycan with numerousteichoic acid cross-linkages & gram negative

cell walls, which, contain thinner amount of

peptidoglycanaccounts for gram staining

difference between two groups .

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The extensive techoic acid cross

links contribute to the ability of

gram-positive organisms to

resist alcohol decolorization.

Gram-negative organism allow

the crystal voilet stain to wash

out with the decolourizing step

& may appear colourless after

this step & appear pink after

takin counterstain.

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Gram stain is usually a poor stain to use when

examining a specimen for a fungus.

Gram stain may be used when examining

smears of Candida, Malassezia, and Sporothrix

but should not be relied upon to demonstrate

the yeast of the other dimorphic fungi.

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Acid-fast staining is useful for detecting Nocardia

species and for differentiating them from other aerobicactinomycetes.

Some of the filaments stain red with carbol- fuschin

staining, while others may appear blue because of

counterstaining effect.

The slides are then examined under a bright field

microscope with oil immersion objective

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Fungi lacking cell walls fortified

with mycolic acids cannot resist

decolourization with acidalcohol and are categorized as

being non-acid fast,

On H&E, all fungi show pink

cytoplasm, blue nuclei and nocolouration of the wall.

Eg:, Blastomyces and Histoplas

ma can be appear red and be

acid-fast staining.

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PAS Staining:

Periodic acid

Schiff(PAS) is a staining method used todetect polysaccharides such as glycogen, and

mucosubstances such as glycoproteins, glycolipids and

mucinsin tissues.

Periodic Acid-Schiff Stain is used to detect yeast cells

and fungal hyphae in tissues.

Periodic acid (5%) hydrolyzes the cell wall aldehydes,

which then combine with the modified Schiff reagent.

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The cell walls of fungi stain magenta.

This only works on living fungi;

Procedure is complex, most laboratories have replaced it with

the calcofluor white stain

Result: Cell wall------pink magenta

Background-------green

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GROCOTT METHENAMINE SILVER

It is primarily used for the detection of fungal elements intissues.

Grocott's methenamine silver stain (GMS) will stain both living

and dead fungal organisms.

result : Fungi --------------black

Background--------pale green.charcoal gray--------innerparts of hyphae.

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PRINCIPLE:

The mucopolysaccharide components of thefungal cell wall are oxidized to release aldehyde

groups.

The aldehyde groups then react with the silver

nitrate, reducing it to a metallic silver, rendering

them visible.

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Binds to cellulose and chitin in the cell walls of fungi,

including yeast cells, hyphae, pseudohyphae, and

spherules.

Rapid and specific.

The dye can be mixed with 10% KOH so that

mammalian cells can be dissolved, thus facilitatingvisualization of fungal elements.

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Fungi, Pneumocystisspp., andAcanthamoebaspp.

appear green or white against a dark background

when the stained slide is examined under UVillumination.

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Recommended for mounting and staining yeast and

molds.

Formulated with lactophenol, which serves as a

mounting fluid, and cotton blue.

Organisms suspended in the stain are killed due to the

presence of phenol.

The high concentration of the phenol deactivates lytic

cellular enzymes thus the cells do not lyse.

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Cotton blue is an acid dye that stains the chitin

present in the cell walls of fungi .

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Differential staining of basophilic and acidophilic material.

Polychromatic

Mixture of

*methylene blue*azure B (from the oxidation of methylene blue)

*and eosin Y

*in methanol.

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Like Giemsa Stain, it is primarily used for the

differentiation of intracellular and extracellularcirculating blood parasites.

Eg. Histoplasma. Pneumocystis

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Methylene blue is another contrasting dye

commonly used in the laboratory, primarily fordetection of bacteria and fungi.

It can be mixed with potassium hydroxide and used

to examine skin scrapings for fungal elements.

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INDIA INK

India ink is useful for indicating the presence orabsence of extra cellular polysaccharide capsules of

fungal cells.

The technique is particularly helpful for detecting

Cryptococcus neoformans in CSF.

Because India ink serves as a negative stain, theencapsulated yeast cells can readily be detected

against the dark back ground.

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The ink should be free from artifacts or granular

carbon particles to ensure a good preparation.

Appearance of a distinctive halo surrounding the

encapsulated fungi.

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KOH SOLUTION


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KOH SOLUTION

The KOH Solution is useful in detecting fungal elements in thickmucoid material.

In specimens containing keratinous material, such as skin scales,nails, or hair.

A 10 to 15% solution used to dissolve cellular and organic debris

and facilitate detection of fungal elements.

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Action

Proteinacious host cell components are partiallydigested by alkali while the fungal cell wall remains

intact (although fungal elements will dissolve after

exposure for a few days).

Ink (e.g., permanent blue-black Parker Super Quick Ink)

can be added as a contrasting agent to aid in thedetection of fungi.

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ACRIDINE ORANGE STAIN


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ACRIDINE ORANGE STAIN

Acridine orange is a fluorescent stain.

For the detection of bacteria and fungi in clinicalspecimens.

Acridine orange stains nucleic acid, changing dyesoptical characteristics so that it will fluoresce brightorange under UV-light.

The dye intercalates into nucleic acid in both the nativeand denatured states.

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Neutral pH: bacteria, fungi and cellular material (e.g.,

leukocytes, squamous epithelial cells) stain red-orange.

Acid pH(pH 4.0), bacteria and fungi remain red-orange

but background material stains green-yellow..

Rapid stain, particularly useful in thick or purulent

specimens.

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GIEMSA STAIN


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GIEMSA STAIN Giemsa Stain, combines methylene blue and eosin.

Typically used by hematology laboratories for demonstrating

the differences of nuclei and cytoplasmic features of the bloodcell components.

Also used for detection of blood parasites.

Eg. Histoplasma, pneumocystis.

Intracellular yeasts and inclusions typically stain blue(basophilic)

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Special stains called brightners/whiteners bind to carbohydrate

on the fungal surface and

flouresce.

(Tinopal for Candida)

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Culture media

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Culture media used for fungal growth:

The most commonly used cultures media used for

fungal growth are as follows:

Sabouraud agar

Hay infusion agar

Potato dextrose agar

Potato Dextrose Broth

Yeast Agar

Yeast Broth

Mycological Agar

Malt extract agar

Malt Extract Broth

Mycological Agar

Soy Peptone Yeast Extract

Agar

Water Agar (WA)

Antibiotic Agar (AA)

Acidified Cornmeal Agar(ACMA)

Cornmeal Agar (CMA)

Potato Carrot Agar (PCA)55

Common media for primary fungal isolation include


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Common media for primary fungal isolation include

Sabouraud dextrose agar and brain-heart infusion

agar, either in petri dishes or screwtop tubes.

Sabouraud dextrose agar

brain-heart infusion

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The media may be enriched with 5% to 10% sheep

blood to support the growth of certain fungi.

Media generally contain a source of carbon,

nitrogen and vitamins. Glucose (dextrose) is themost widely utilizable carbon source, and hence is

the most commonly used in growth media.

Fructose and mannose are the next most commonly

utilized sugars by fungi and are found in media from

natural sources

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Mostly natural media based on materials such

as cornmeal, carrots, hay, potatoes, oatmeal,

soil, etc. Are used.

Semi-synthetic media, containing both natural

ingredients and defined components include

Malt Extract Agar, Malt Agar.

Synthetic or defined media which containprecise amounts of a carbon source, vitamins

and minerals is less commonly used.

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Water Agar (WA)--use for isolating fungi from surface-sterilized


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Water Agar (WA) use for isolating fungi from surface sterilized

substrates.

Antibiotic Agar (AA)--use for isolating fungi from substrates notreadily surface-sterilized, or to clean up a culture contaminated

with bacteria.

Acidified Cornmeal Agar (ACMA)--use for isolating fungi fromsubstrates that are likely to be contaminated with bacteria. as the

acidity inhibits bacteria and the medium supports the growth of a

wide range of fungi.

Cornmeal Agar (CMA)--use for growing a wide range of fungi,

particularly members of the Fungi imperfecti; provides a good

balance of mycelial growth and sporulation.

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Potato Carrot Agar (PCA)--considered a relatively

weak medium somewhat comparable to CMA, good

for some Fungi imperfecti.

Malt Agar (MA)--lacks peptone, and is useful for

culturing many Ascomycota; sporulation in some

species is inhibited by peptone.

Malt Extract Agar (MEA)--a good growth medium for

soil fungi, fungi isolated from wood, basidiomycetes,etc. An all-purpose type of medium.

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Potato Dextrose Agar (PDA)--a relatively rich

medium for growing a wide range of fungi.

Potato Dextrose-Yeast Extract Agar (PDYA)---

good for growing cultures derived from

mushrooms.

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CARBOHYDRATE FERMENTATION

Yeasts are able to metabolize some foods, but not

others. In order for an organism to make use of a

potential source of food, it must be capable of

transporting the food into its cells.

It have the proper enzymes capable of breaking the

foods chemical bonds in a useful way.

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Sugars are vital to all living organisms.

Yeast is capable of using some, but not all sugarsas a food source.

2% of suger is added to basal media.

Yeast ferments suger into CO2 & ethanol.

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CO2 is collected in durhamstube, & ethanol as a

byproduct of the fermentation. Shows positive

result.

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This test is used to detect presence of ureaseenzyme produced by different Candida species.

Christensens urea agar slants are used.

Conversion of the yellow slope to pink or red isconsidered positive.

A negative test is reported when there is no colourchange observed

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U d d b h i li i


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Urease produced by the organisms split urea into

ammonia and CO2.

Urease positivepink colour

Urease negativeyellowcolour

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Fungi has ability to break down milk protein

called casein into small peptides & amino acids.

The hydrolytic reaction creates a clear zone

around the cell as casein protein is converted to

soluble & transparent end products.

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There is no reagent or indicator in the agar. A

zone of clearing around the growth area

identifies the presence of the enzyme caseinase

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Available tests


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Immunodiffusion

Antibodies

AntigensRadioimmunoassay (RIA)

Exoantigen test

Complement fixation

Enzyme-linked immunosorbent assay (ELISA)

Antibodiesandantigens

Radioallergosorbent Test (RAST)

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Immunodiffusionis a diagnostic test which

involves diffusion through a substance such as agar.

Immunodiffusion

o Radial Immunodiffusion (Mancini method).

oOuchterlony Double Diffusion

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Radial Immunodiffusion

- Asingle diffusion techniquewhere Ab is put into gel

and Ag is measured by the size of a precipitin ring

formed when it diffused out in all directions from awell cut into the gel.

Ouchterlony Double Diffusion

- Both Ab and Ag diffuse from wells into a gel medium.

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Ag is added to an

antibody rich media.

The two continue to

react until the zone of

equivalence is

reached.

The area of ring is a

measure of the Ag

concentration.

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Method


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Interpretation

Diameter of ring isproportional to the

concentration

MethodAb in gel

Ag in a well

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Both antigen and

antibody can diffuse

independently.

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Wells are cut in the gel

Reactants are added in the well

Incubate (12-48 hrs) in a moist chamber

Precipitin lines will form

(where the moving front of the antigen meets antibody)

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Fusion of lines at their

junction to form an arc

- Serologic identity /

presence of common epitope

Crossed lines- Demonstrates 2 separate

reactions

- Compared antigens shared no

common epitopes Fusion of 2 lines with spur

- Partial identity

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IgE is the antibody associated with Type I allergic response.

The RAST is a radioimmunoassay test to detect

specific IgE antibodies to suspected or known allergens for the

purpose of guiding a diagnosis about allergy.

The suspected allergen is bound to an insoluble material and the

patient's serum is added.

If the serum contains antibodies to the allergen, those antibodies

will bind to the allergen.


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Radiolabelled anti-human IgE antibody is

added where it binds to those IgE antibodiesalready bound to the insoluble material.

The unbound anti-human IgE antibodies are

washed away. The amount of radioactivity is

proportional to the serum IgE for the allergen.

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RAST rating IgE level (IU/ml) comment

0 < 0.35ABSENT OR UNDETECTABLE

ALLERGEN SPECIFIC IgE

1 0.350.69LOW LEVEL OF ALLERGEN

SPECIFIC IgE

2 0.703.49MODERATE LEVEL OF


3 3.5017.49HIGH LEVEL OF ALLERGEN

SPECIFIC IgE

4 17.5049.99VERY HIGH LEVEL OF


5 50.00100.00ULTRA HIGH LEVEL OF


6 > 100.00EXTREMELY HIGH LEVEL OF


The RAST is scored on a scale from 0 to 6

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A t h i d t th t ti f h D

Radio immuno assay


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A technique used to measure the concentration of hormones, Drugs, enzymes,

viruses, bacterial antigens and other organic substances of biological interest

found in Blood, Tissues and other biological fluids

Principle: Uses an immune reaction[AntigenAntibody reaction] to estimate aligand

Ag + Ag* + Ab AgAb + Ag*Ab + Ag + Ab*

Unbound Ag* and Ag washed outRadioactivity of bound residue measuredLigand conc is inversely related toradioactivity of radiolabelled ligand.

[Ag : ligand to be measured ; Ag*radiolabelled ligand]

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Advantages

Highly specific: Immune reactions are specific

High sensitivity : Immune reactions are sensitive

Disadvantages

Radiation hazards: Uses radio labelled reagents

Requires specially trained persons

Labs require special license to handle radioactive material

Requires special arrangements for

Requisition, storage of radioactive material

radioactive waste disposal.

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Exoantigen tests for the immunoidentification of

fungal pathogens are playing a new and significant rolein the diagnostic laboratory.

Properly performed and controlled exoantigen testslead to rapid, accurate identification of cultures ofmany fungal pathogens.

The tests are particularly valuable in identifyingdimorphic pathogens that are difficult to convert orwith atypical cultures.

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Specific antibodies developed against particular

mycelial antigens will react in a gelimmunodiffusion precipitin test.

The mold form of dimorphic fungi can beidentified definatly by an antigenantibody

reaction.

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Exoantigen immunodiffusion plate showing positiveidentification of Histoplasma capsulatum. Note H and M bandsof identification; EX = culture filtrate; H = Histoplasmaantibodyand antigen, C = Coccidioides antibody and antigen; B= Blastomycesantibody and antigen.

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The ability of antigen-antibody complexesto fix

complementis the basis of CFT.

To detect the fixation of complement, Sheep RBC

coated with amboceptor is used as the indicatorsystem

Very sensitive method to detect Ags & Abs.

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Antigen+Test serum

(Contains Ab)

+ Complement

+ Hemolytic system

Antigen+Test serum

(Contains noAb)

+ Complement

+ Hemolytic system

- Complement fixed

- No hemolysis, +veCFT

- Complement notfixed

- Hemolysis, -ve CFT

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The enzyme-linked immunosorbent

assay (ELISA) has become one of the

most widely used serological tests forantibody or antigen detection.

This test involves the linking of

various label enzymes to eitherantigens or antibodies.

Enzymes used in ELISA include Alkaline Phosphatase, Peroxidase

and Galactosidase.


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For Ab detection,

Ag coated wells used

If Ab present, it binds to Ag

Add goat anti-humanIg antibody conjugated with

enzyme

Add a substrate. Enzyme

acts & colour produced

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For Ag detection, wells

coated with Sp. Ab. Ag in specimen binds to

coated Ab.

Add enzyme conjugated

antiserum.

Add substrate.

Colour produced

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Ag coated wells

Two specific Abs,

(enzyme conj.& test Ab)

added simultaneously

More specific test Abs

attach to Ag

Substrate added

No colour since enzyme

not available

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A number of dermatophytes have the ability topenetrate hair in vitro. The test is particularlyuseful in distinguishing atypical isolates ofTrichophyton mentagrophytes from T. rubrum.

Procedure

1.Place several sterile hairs in a sterile glass Petri

dish. Hair from a blond child is preferred. 2.Add 25 ml of sterile water and 0.1 ml of sterile

yeast extract to the Petri dish.

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c. Transfer a small amount of the fungus colony to the hairs in the

P t i di h


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Petri dish.

d. Incubate at 25 C and examine weekly for up to 4 weeks.

e. Examine hairs by placing a few hairs in a drop of lactophenol on a

microscope slide. Place a cover glass over the preparation and

examine microscopically.

It will be necessary to examine several hairs before concluding anegative result.

3. Results

a. Positive - Presence of perforations (conical or wedge-like holes) inthe hair.

b. Negative - Absence of perforations in the hair.

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Thermotolerance is a useful characteristic that can beused as an aid in the identification of several medically

important moulds. Thermotolerance of some medicallyimportant fungi include:

Fungus Upper Growth Limits C

Aspergillus fumigatus 48-50 C C. carrionii 35-36 C

Fonsecaea pedrosoi 38 C

Rhizomucor pusillus 45-55 C

Trichophyton mentagrophytes 37 C Wangiella dermatitidis 40 C

Xylohypha bantiana 42-43 C

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Nucleic acid probes can identify microorganismsmore rapidly than traditional culture.

Culture identification of fungi is based upon DNAprobes that are complimentary to speciesspecific ribosomal RNA.

The fungal cells are lysed by sonication, heatkilled & exposed to DNA that has been labelledwith a chemiluminescent tag.

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The labelled DNA combines with the organisms

ribosomal RNA to form a stable DNA/RNAhybrid.

Complex is measured in luminometer.

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Cannot be used on fresh clinical specimens.

It is to be used for culture identification only.

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Strain of several bacteria produces germ tubesfrom their yeast cells when placed in a liquid

nutrient environment & incubated at 35C for 3

hrs. Eg: Candida albicans.

By this method several fungi can be

differentiated from fungi, not forming germ

tube.

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Determining the resistance of isolates to

cycloheximide (0.5 mg/ml) is useful when screeningcultures for Blastomyces dermatitidis, Coccidioidesimmitis, Histoplasma capsulatum, Microsporumspp., Paracoccidioides brasiliensis, Sporothrix

schenckii, and Trichophyton spp. Etc.

All of these fungi will grow in the presence of

cycloheximide at 30 C or less, while fungi such asAbsidia, Aspergillus, Mucor, Rhizopus,Scedosporium, and many more are inhibited bycycloheximide.

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Candida : Candidais a genus of yeasts and is the

most common cause of fungal infections

worldwide.

Many species of candida are

harmless commensals or endosymbionts of

hosts including humans; however, when mucosal

barriers are disrupted or the immune system iscompromised they can invade and cause disease.

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Species:

Candida albicans

Candida tropicalis

Candida parapsilosis

Candida guilliermondii

Candida kefyr

Candida krusei Candida lusitaniae

Candida grabrata (no pseudohyphae)

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Holmstrup and Bessermann ( 1983)

Primary candidiasis Acute infections

Acute pseudomembranous candidiasis

Acute erythematous candidiasis

Chronic infections

Chronic pseudomembranous candidiasis

Chronic erythematous candidiasis

Chronic plaque like candidiasis

Chronic nodular candidiasis

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Secondary candidiasis


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y

Chronic Mucocutanous Candidiasis with

endocrinopathy Familial CMC with endocrinopathy

Familial CMC without endocrinopathy

Idiopathic CMC with juvenile onset (20yr)

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Candidiasis is a fungal disease

Other names- Candidosis, Moniliasis , Thrush

Caused by Candida albicans

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Exists in 3 forms

Pseudohyphae

Yeast

Chlamydospore

Reproduction-Asexual budding

Temperature-25-370C

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Common inhabitant- oral cavity, GIT & Vagina


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Most oppotunistic infection in world

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Acute & Chronic diseases- tuberculosis, diabetes

mellitus & anemia

Myxedema, hypoparathyroidism, Addisons

disease

Immunodefeciency AIDS

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Nutritional defeciency like Fe, Vitamin A, Vitamin


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B6

Prolonged hopitalization for chronic illness and

debilitating diseases

Prolonged use of antibiotics, corticosteroids and

cytotoxic drugs

Radiation therapy

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Use of intravenous tubes catheters heart valves


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Use of intravenous tubes, catheters, heart valves

and poorly maintained dentures, heavy smoking

Old age, infancy and pregnancy

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Candida species highly developed mechanisms

to adapt readily to changes in host environment

Shifts b/w different phenotypes in a reversible

and random fashion

Phenotypic switching involves co-ordinated

regulation of phase specific genes and a way toadapt

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It produces genetically altered variants at a high

rate


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rate

These differ in colony morphology, cell shape,

antigenecity and virulence

Produces a number of adhesins that mediate

adherance to host cells, candidal morphogenesis

or signaling

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Morphogenesis


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hyphal form is invasive and pathogenic, while

the yeast is the commensal nonpathogenic form

hyphae are capable of contact-sensing or

thigmotropism- facilitate the penetration

Secreted Hydrolytic Enzymes

Secrete: phospholipase , lipase ,

phosphomonoesterase , hexosaminidase , and at

least three separate aspartic proteinases

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Aspartic proteinases (Sap proteins) fulfill


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p p p p

number of specialized functionsduring the

infective process:

digesting molecules for nutrient acquisition

digesting ordistorting host cell membranes

facilitate adhesion and tissueinvasion

digesting cells and molecules of the host

immunesystem

avoid or resist antimicrobial attack by the host.

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Variety of clinical manifestations

Range from mild superficial mucosal

involvement to severe, fatal disseminated formseen in immunocompromised individuals

Classified into 2 categories -Mucocutaneous

-Systemic

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Mucocutaneous

oral & oropharyngeal ( thrush)


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oral & oropharyngeal ( thrush)

candidal oesophagitis

candidal vulvovaginitis & balanitis

intertrigo or intertriginous candidiasis

Systemic

Eyes, kidney and skin through hematogenous

spread and other visceral organisms

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Acute candidiasis

Chronic cndidiasis

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Gross Appearance

Soft, white, slightly

elevated plaquesresembling

milk curds

Site

Buccal mucosa, tongue,palate, gingiva, floor of

mouth

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Histopathology

Tangled mass of fungal hyphae


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Tangled mass of fungal hyphae

Intermingled with desquamated epithelium,

keratin, fibrin, necrotic debris,leukocytes and

bacteria

White plaque can be wiped away leaving a

normal area or an erythematous area

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Antibiotic sore mouth under category oferythematous candidiasis

Sequale to course of broad spectrum antibiotics

Gross :Lesions appear red or erythematous

Site- Any

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Central Papillary Atrophy of


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Central Papillary Atrophy ofTongue

Asymptomatic, symmetriclesions of tongue

Dorsal aspect in posteriorregion

Erythematous appearance-loss of filliform papillae

Strong relationship b/wlesion and chronic smoking

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Leukoplakia type candidiasis

Gross:Firm white, persistent plaques

Site:Lips, tongue , cheeks

Persist for years

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Roed Peterson Incidence of candida is seen in


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Roed-Peterson- Incidence of candida is seen in

lesions of leukoplakia.

Occurrence of cytological atypia in lesions of

leukoplakia.

Cawson & Binnie definite relationship b/w

chronic candidiasis and oral epidermoid

carcinoma

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Specimens

swab, scrapings, blood, CSF, biopsies, urine,exudates, material from removed iv. Catheters

1. Microscopic examination

plaque smear---Gram stain---pseudohyphae,

budding cells.

20% KOH/ Calcoflour white: skin & nail scrapings.

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2. H & E

Both yeast and hyphae


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Both yeast and hyphae

superficially + deep

3. Special stains

PAS, Gomori Methanamine Silver

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Culture


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Culture

Blood/ cornmeal/

sabourauds agar

oval, budding yeast

cells, 3-6m.

Soft cream colored

colonies with a

yeasty odor.

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Sabauruodsagar slant

for culture of candidiasis

Morphologic tests

Germ Tube Test


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Filamentous extension from a yeast cell that is

about one half the width and 3-4 times the

length of the cell

1. Small portion of an isolated colony suspendedintest tube containing 0.5ml sheep serum.

2. Tube inoculated at 35o for no longer than 2 hrs

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Serology:


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Limited specificity and sensitivity (Ab detection)

EIA/latex agglutination(ag detection)

cell wall mannan.

beta 1,3,D glucan

No clear criteria for establishing diagnosis.

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l d d


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Nucleic acid detection:

Hybridisationamplification

analysis

Detection of fungal metabolites

D-arabinitol (Bernard et al)

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Seen microscopically in exfoliative and biopsyspecimens

PAScandidal hyphae and yeasts

PAS method demonstrates stains carbohydrates

contained in abundance by fungal cells walls;organism identified as bright magenta

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Hyphae are approx 2 microns in diameter and vary

in length and may show branching.


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in length and may show branching.

Hyphae accompanied by variable no. of yeasts,

squamous epithelial cells & inflammatory cells.

10% to 20% KOH may be used to evaluate

specimens for presence of fungal organisms.

KOH lyses the background of epthelial cells allowing

more resistant yeasts and hyphae.

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Characteristic pattern of

parakeratosis, neutrophilic

microabscess, a thickned

spinous layer and chronic

inflammation of connective

tissue

Tubular hyphae embedded in

the parakeratin layer

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THANK YOU...

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Bibliography


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Bailey & Scotts Diagnostic microbiology. Twelfthedition.

R.Ananthanarayan, CKJ paniker text book of

microbiology 6th edition.

Laboratory diagnosis on fungal infections- a review.Dr.

Shruti nayak, dr. Amarnath shenoy, dr. U. S. Krishna

nayak

Text book of microbiology by prof. C.P.Baveja. Microbiology & microbial infections: Mycology. Topley

& Wilson. 10thedition.

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Murray, P. R., E. J. Baron, M. A. Pfaller, F. C. Tenover,


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Murray, P. R., E. J. Baron, M. A. Pfaller, F. C. Tenover,

and R. H. Yolken (eds.). Manual of clinical microbiology,

6thedition.

Medical Microbiology: A guide to microbial infections,

pathogenesis, laboratory diagnosis & control. David

Greenwood. Sixteenth edition. Oral Candida: Clearance, Colonization, or Candidiasis?

JDR 74(5); 1995.

Candida albicansSecreted Aspartyl Proteinases in

Virulence and Pathogenesis .Microbiology and

Molecular Biology Reviews 400 428 67(3);400

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