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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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Isolation and Identification of
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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Isolation and Identification of
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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Isolation and Identification of
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).