CH34:LABORATORY DIAGNOSIS

1. Islamic University Gaza PREPARD BY: Basel Auda PRESENTED TO: Dr.Abdelraouf El Manamma 23-11-2008 CH34:LABORATORY DIAGNOSIS

2. Introduction • There are five approaches to the diagnosis of viral infection: • (1) identification of the virus in cell culture • (2) microscopic identification directly in the specimen • (3) serologic procedures to detect a rise in antibody titer or the presence of IgM antibody • (4) detection of viral antigens in blood or body fluids • (5) detection of viral nucleic acids in blood or the patient's cells

3. Identification in Cell Culture The growth of viruses requires cell cultures, because viruses replicate only in living cells. Because many viruses are inactivated at room temperature, it is important to inoculate the specimen into the cell culture as soon as possible; brief transport or storage at 4°C is acceptable Virus growth in cell culture frequently produces a characteristic cytopathic effect (CPE) that can provide a presumptive identification. CPE is a change in the appearance of the virus-infected cells. This change can be in such features as size, shape, and the fusion of cells to form multinucleated giant cells (syncytia). CPE is usually a manifestation of virus-infected cells that are dying or dead. The time taken for the CPE to appear and the type of cell in which the virus produces the CPE are important clues in the presumptive identification.

4. Cont. If the virus does not produce a CPE, its presence can be detected by several other techniques: Hemadsorption,i.e., attachment of erythrocytes to the surface of virus-infected cells. This technique is limited to viruses with a hemagglutinin protein on their envelope, such as mumps, parainfluenza, and influenza viruses. Interference with the formation of a CPE by a second virus. For example, rubella virus, which does not cause a CPE, can be detected by interference with the formation of a CPE by certain enteroviruses such as echovirus or coxsackievirus. A decrease in acid production by infected, dying cells. This can be detected visually by a color change in the phenol red (a pH indicator) in the culture medium. The indicator remains red (alkaline) in the presence of virus-infected cells but turns yellow in the presence of metabolizing normal cells as a result of the acid produced. This

5. definitive identification • A definitive identification of the virus grown in cell culture is made by using known antibody in one of several tests. Complement fixation, hemagglutinationinhibition, and neutralization of the CPE are the most frequently used tests. • Other procedures such as fluorescent antibody, radioimmunoassay,enzyme-linked immunosorbent assay (ELISA), and immunoelectron microscopy are also used in special instances.

6. Complement Fixation • If the antigen (the unknown virus in the culture fluid) and the known antibody are homologous, complement will be fixed to the antigen–antibody complex. This makes it unavailable to lyse the "indicator" system, which is composed of sensitized red blood cells.

7. If the virus and antibody are homologous, the virus is blocked from attaching to the erythrocytes and no hemagglutination occurs. Only viruses that agglutinate red blood cells can be identified by this method. Hemagglutination Inhibition

8. Neutralization If the virus and antibody are homologous, the antibody bound to the surface of the virus blocks its entry into the cell. This neutralizes viral infectivity, because it prevents viral replication and subsequent CPE formation or animal infection

9. Fluorescent-Antibody Assay If the virus-infected cells and the fluorescein-tagged antibody are homologous, the typical apple-green color of fluorescein is seen in the cells by ultraviolet (UV) microscopy.

10. Radioimmunoassay If the virus and the antibody are homologous, there is less antibody remaining to bind to the known radiolabeled virus.

11. Enzyme-Linked Immunosorbent Assay In the ELISA test to identify a virus, known antibody is bound to a surface. If the virus is present in the patient's specimen, it will bind to the antibody. A sample of the antibody linked to an enzyme is added, which will attach to the bound virus. The substrate of the enzyme is added and the amount of the bound enzyme is determined

12. Immunoelectron Microscopy If the antibody is homologous to the virus, aggregates of virus–antibody complexes are seen in the electron microscope

13. Microscopic Identification Viruses can be detected and identified by direct microscopic examination of clinical specimens such as biopsy material or skin lesions. Three different procedures can be used. Light microscopy can reveal characteristic inclusion bodies or multinucleated giant cells. The Tzanck smear, which shows herpesvirus-induced multinucleated giant cells in vesicular skin lesions, is a good example. (2) UV microscopy is used for fluorescent-antibody staining of the virus in infected cells. (3) Electron microscopy detects virus particles, which can be characterized by their size and morphology.

14. Serologic Procedures A rise in the titer of antibody to the virus can be used to diagnose current infection. Seroconversion is the term used to describe the finding of antibody to a virus (or any microbe) in a patient's serum who previously had no antibody. Stated another way, the patient's serum has converted from antibody-negative to antibody-positive.

15. Cont. A serum sample is obtained as soon as a viral etiology is suspected (acute-phase), and a second sample is obtained 10–14 days later (convalescent-phase). If the antibody titer in the convalescent-phase serum sample is at least fourfold higher than the titer in the acute-phase serum sample, the patient is considered to be infected If, however, the titer in the convalescent-phase serum sample is less than two folds this is not a significant rise and should not be interpreted as a sign of recent infection.

16. Cont. It is important to realize that an antibody titer on a single sample does not distinguish between a previous infection and a current one. The antibody titer can be determined by many of the immunologic tests mentioned above. These serologic diagnoses are usually made retrospectively, because the disease has frequently run its course by the time the results are obtaied. In certain viral diseases, the presence ofIgMantibody is used to diagnose current infection. For example, the presence of IgM antibody to core antigen indicates infection by hepatitis B virus. Other nonspecific serologic tests are available. For example, the heterophil antibody test (Monospot) can be used to diagnose infectious mononucleosis

17. Detection of Viral Antigens Viral antigens can be detected in the patient's blood or body fluids by various tests but most often by an ELISA. Tests for the p24 antigen of human immunodeficiency virus (HIV) and the surface antigen of hepatitis B virus are common examples of this approach

18. Detection of Viral Nucleic Acids Viral nucleic acids, i.e., either the viral genome or viral mRNA, can be detected in the patient's blood or tissues with complementary DNA or RNA (cDNA or cRNA) as a probe. If only small amounts of viral nucleic acids are present in the patient, the PCR can be used to amplify the viral nucleic acids. Assays for the RNA of HIV in the patient's blood (viral load) are commonly used to monitor the course of the disease and to evaluate the

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