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LAB. DIAGNOSIS OF VIRUSES

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1 LAB. DIAGNOSIS OF VIRUSES
5 methods are used for diagnosis in the virology laboratory: Direct microscopy Cultivation of viruses Serology 4. Detection of viral antigens 5. Detection of viral nucleic acids

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3 DIRECT MICROSCOPY @ Three procedures can be used :
1. Light microscopy: to reveal inclusion bodies or multinucleated giant cells e.g. herpesvirus 2. Immunofluorescence microscopy: to read viral fluorescent antibodies 3. Electron microscopy: to detect virus particles

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5 Cultivation of Viruses
Inoculation of fertile: Specimen is injected in day-old eggs. Three days later eggs are opened to see spots on membranes and fluids. Inoculation of lab. Animals: Specimen is injected in 2 day-old suckling mice, and look for clinical signs in mice. 3. Viral tissue culture

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7 VIRAL TISSUE CULTURE @ Transport specimen in Hank’s medium
or store at 4 °C immediately @ Virus growth in living cells produces a Cytopathic Effect (CPE) . @ Identification depends on : * time taken for the CPE to appear * type of cell present in CPE

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9 If no CPE, virus is detected by:
Hemadsorption Test: For viruses with hemagglutination antigens, eg mumps, and influenza viruses 2. Interference Test: Formation of CPE by another virus, eg Rubella which does not cause CPE, but it can cause CPE when ECHO virus is added to the cell culture

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11 3. Phenol red indicator changes yellow: This is due to decrease in acid production when the infected cells die in medium 4. Phenol red indicator remains red (alkaline): This is used to detect Eenteroviruses

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13 5. Use of a known antibody and made to react with the virus antigen in the cell culture by one of the following tests : 1) Complement Fixation Test . 2) Hemagglutination Inhibition Test 3) Neutralization Test. 4) Fluorescent-Antibody Test

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15 5) Radioimmunoassay Test.
6) Enzyme-Linked Immunosorbent Assay (ELlSA) Test. 7) Immunoelectron microscopy Test

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17 SEROLOGY @ Blood is collected twice: at the
acute stage and days later @ If the antibody titer in second sample is at least 4-fold higher than the titer in the acute stage sample, patient is considered to be infected.

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19 @ IgM antibody only is used to diagnose
acute infection, e g infection by hepatitis B virus. @ Tests used are: Complement fixation, Hemagglutination-inhibition, ELlSA, Neutralization, Radioimmunoassay, and Fluorescent-antibody tests @ Heterophile non-specific agglutination antibody test: can be used to detect Epstein-Bar virus

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21 DETECTION OF VIRAL ANTIGENS
@ Detected in patient's blood or body fluids by ELISA. @ Examples : * p24 antigen of HIV * HBs antigen of Hepatitis B

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23 DETECTION OF VIRAL NUCLEIC ACIDS
@ DNA,RNA, & mRNA are detected in blood or tissues by polymerase chain reaction (PCR), using DNA or RNA probe @ For small amounts of N.A., amplify these N.A. by PCR.

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