basic diagnostic of fungal and viral infection

8/12/2019 Basic Diagnostic of Fungal and Viral Infection

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DIAGNOSIS AND

THERAPY OF VIRAL AND

FUNGAL INFECTION

Rizalinda SjahrilBasics of Diagnosis and Therapy2009


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Isolation and Identification of

Viruses CELL and ORGAN CULTURE

Cells are derived from tissue source, allowed to grow innutrient media until a confluent one cell layer is obtained.

Organ culture uses a tissue fragment with a specializedfunction (e.g. Fetal trachea with ciliated epithelial cells)

Primary cell culture: cells derived from the initial growth of cells

from a tissue source

Secondary cell culture: redispersed and regrowth of primary cell

culture.

Cell lines: cells that have transformed spontaneously and

become immortal, or cells are obtained from cancerous tissue


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VirusesDetection of Viral growth

Observing CPE; alteration or cytopathic effect in cells

due to viral infection. Alteration may be change inmorphology or death of cells

Observing hemadsorption or interference on cells thatdo not show CPE.

Interference: cells that do not show CPE after infected

with a virus, but the cells may show CPE if infected withanother virus (challenged). But the challenging viruscannot infect the cell culture in the presence of the firstvirus

Expressing infection on lymphocytes (EBV and HIV)


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Viruses Quantitation of Viruses

Hemagglutination Assay:Viruses have attachment proteins (=hemagglutinins) that can be boundon red blood cells, thus causing hemagglutination.

Dilution of virus preparation reacted with a constant amount of redblood cells will show the decreasing amount of hemagglutination.Agglutinated RBCs settle dispersed on the bottom, but unagglutinatedRBCs forms a tight button shaped sedimentation on the bottom of thewell/tube. The titer is the reciprocal of the last dilution that still showsagglutination.

Plaque AssayVirus at several different concentration is reacted into a one layer ofsemisolid culture cells. The growth of any one virus is therefore limitedin its initial place, forming a round cleared area (plaque) due to thedeath of infected cells. The number of plaques is then counted andcalibrated according to the dilution factor. Viral titer is the number ofPlaque forming units per millimeter (Pfu/ml)


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Viruses In Vivo Isolation methods

Embryonated hens eggisolation andpropagation of influenza A virus

Animal inoculation suckling mouse


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Viral Identification Tentative identification based on CPE

characteristics may suggest a virus Further identification are performed by;

Neutralization test

Serology

Cytology Histology

Electron Microscopy


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Immunology for identification

of virus Antigen-antibody reaction :

Precipitation

Agglutination

Neutralization

Complement fixation

Immunofluorescence or radioisotope or enzymelabelling

Antibody detection

Antigen detection


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Nucleic Acid Detection

Analysis of Nucleic Acid:

Agarose gel electrophoresis

Restriction endonuclease Digestion DNA hybridization

Polymerase Chain Reaction

Nucleic acid sequence analysis


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Antiviral Therapy

General considerations Viruses comprises of DNA or RNA, capsid and many

have lipid or lipoprotein envelope Viruses uses the cellular structures for replication

unique for the virus Target of inhibition includes each of the replication

steps: Attachment

Penetration

Uncoating

RNA-directed DNA synthesis

Assembly

Release


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Antiviral Agents

Summary of Antiviral Agents

Mechanism of Action Antiviral agent Viral spectrum

Inhib of viral uncoating Amantadine, rimantadine Flu A

Neuraminidase inhibition Oseltamivir, Zanamivir Flu A, Flu B

Inhib of viral DNApolymerase

e.g. Acyclovir, Famciclovir,Valacyclovir

e.g. ganciclovir

HSV,VZV

CMV, HSV, VZV

Inhib of viral reversetranskriptase

e.g. Zidovudine, dideoxyinosine,dideoxycytidinee.g. Lamivudine

HIV

HIV, HBV

Inhib of viral protease e.g. Saquinavir, Indinavir HIV

Inhib of viral proteinsynthesis

Interferon HBV, HCV, HPV

Inhib of viral RNApolymerase

Ribavirin RSV, HCV, Lassafever

Antisense inhibition of

viral mRNA synthesis

Fomivirsen CMV


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Fungi Cell Structure

Typical eukaryotic features

Nucleus and nucleolus, linear chromosome

Sitoplasm contains organelles; mitochondria, Golgi

apparatus

Rigid cell wall distinguishes fungus from mammaliancells, and a different composition of cell wall thatdistinguishes them from bacteria and plants

Identification of medically important molds are

based on the morphology and development of

reproductive elements (conidia and spores)


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Fungal terms:

Conidia:

Spore:

Conidiophore:

Macroconidia:

Microconidia:

Chlamydoconidia=arthroconidia: chlamydia thatdevelops within the hyphae

Ascospore: a sexual spore

asexual form of reproductive elements

sexually produced elements

a stalk structure where conidia buds outLarge sized conidia

Small sized conidia


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Diagnosis of fungal infection

1. Direct Microscopic Examination: wet mount

2. Antigen Detection: Fluorescent antibody

3. Culture & Isolation

4. DNA Detection

5. Skin test

6. Serology

7. Histopathology (biopsy)


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Specimens : Skin scraping, sputum, Pus

Methods:

1. Potassium hydroxide test: Skin scraping + KOH10% and place under a coverslip* strong alkali digests the tissue elements such as epithelial cells,

leukocytes, debris

2. Gram sputum or pus usually gram positive

3. Direct Immunofluorescence : to identify fungi infixed tissue section; using calcifluor that binds topolysaccharides in cellulose and chitin, and thefluorescence is viewed under ultra violet light.

4. Gomori Methenamine Silver (GMS) stain fungalcell black in tissue section.


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Specimen: liqour

Method :latex agglutination test

Examples:

Cryptococcus

Histoplasma


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Specimens : Skin scraping, sputum, pus, liquor, blood

Medium :Sabouraudsdextrose agar (contains only glucose and peptones, pH 5.6).

Most bacteria fail to grow/grow poorly in this medium.

Temp. 25o-30oC, paired culture at 35oC and 25oCfor dimorphic fungi

May be added with cycloheximide to inhibit the growth of somesaprophytic fungi /contaminants from the environment

Selective medium: Blood agar (or other enriched media) containingchloramphenicol and gentamicin to obtain pure culture.

**But note that adding cycloheximide in Saboraud agar may inhibitthe growth of Cryptococcus neoformans, and chloramphenicol mayinhibit the yeast form of some dimorphic fungi.

Identification: to determine YEAST or MOLD

1. Colony: mycelium, septation, branching, pigmentation, spore orconidia production.

2. Microscopic: morphology of conidia and conidiophore

3. Biochemical reactions.

4. DNA Probe


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Skin test (dermal hypersensitivity testing): nowused most often to evaluate a patientsimmunity

Serology tests :Fungi is a poor antigen

1. Latex agglutination test : IgM

2. Immunodiffusion test: Ig G

3. Complement fixation test : IgG

Frequently false positive due to cross reactions.Antibody detection: must be done 2-3 months

after the onset of disease


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Antifungal Chemotherapy

Most fungal infections are self-limiting andrequire no chemotherapy

For superficial mycoses topical therapy isgiven

For deep mycoses that are uncontrolled bythe immune system require prolonged use of

relatively toxic antifungals.


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Antifungal affecting the

membrane sterols

Polyenes: Nystatin andamphotericin B bind tosterols in the cytoplasmic membrane of theeukaryotic cells forming membrane channelscausing leak of small molecules from thecytoplasm. ACTIVE against most fungi

Azoles: imidazole, ketokonazole, triazoles,fluconazole, itraconazole inhibits the enzyme

which is crucial for ergosterol synthesis Allylamines: inhibits squalene epoxidase during

the ergosterol synthesis leading to accumulationof squalene. Eg: terbinafine, naftifine


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Antifungals that Affect

Nucleic Acid synthesis

5-Flucytosine:an antimetabolite analog ofcytosine, inhibits RNA, DNA and proteinsynthesis.

ACTIVE on most YEASTS but not molds

Resistance develops so the use is combined withamphotericin B


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Antifungals that affect cell

wall synthesis

Agents acting on glucan and chitin synthesisare emerging

Echinocandinsable to block glucan synthesis;caspofungin may be used against Candidaand Aspergillus. Nicomycins that disruptchitin synthesis are being developed


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Other antifungal agents

Griseofulvin:for superficial mycoses(dermatophyte infection), oraladministration, concentrates in thekeratinized layers of the skin, slow response

Potassium Iodide: effective only forcutaneous sporotrichosis

A t M h f M h f i t R t Cli i l


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Agent Mech ofaction

Mech of resist Route Clinical use

POLYENES

Nystatin

Amph. B

Disrupt

membrane

Strerol modifcn

Topical

intravenous

Most fungi

AZOLES Blocks ergost.

synthesis

Active efflux,

demethylase

alteration, or

overproduction

Varies; Oral,

IV, topical

Candida

ALLYLAMINES

Terbinafine

Naftifine

Squalene

accumulation

? Active efflux

Oral

Topical

Dermatophytes

FLUCYTOSINE RNA & DNA

Synthesis

Permease or modifying

enzymes absent or

decrsd

Oral Candida,

Cryptococcus

ECHINOCANDIN

ES

Block glucan

synthesis

unknown IV Aspergillus,

Candida

GRISEOFULVIN Disrup

microtubules

unknown Oral Dermatophytes

POTASSIUMIODIDE

Unknown unknown Oral SporothrixSchenckii

TOLNAFTATE Unknown unknown oral Dermatophytes


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Further Readings

Sherris Medical Microbiology 4thed, anIntroduction to Infectious Diseases, Kenneth JRyan & C. George Ray (eds), 2004.

Bayley and Scotts Diagnostic Microbiology,Baron, Peterson, Finegold (eds).

basic diagnostic of fungal and viral infection

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