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Diagnosis of Viral Infection

Diagnosis of Viral Infection. A. Specimen collection and transport. 1. Specimen for viral isolation Specimens for the detection of virus should be collected as early as possible following the onset of disease.

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Diagnosis of Viral Infection

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  1. Diagnosis of Viral Infection

  2. A. Specimen collection and transport 1. Specimen for viral isolation • Specimens for the detection of virus should be collected as early as possible following the onset of disease. • Specimens should be placed in ice and transported to the lab at once. <5 days, 4℃;>6 days, -20℃ or -70℃

  3. 2. Serum for antibody testing • Acute and convalescent serum specimens may be needed to detect antibody to specific viruses. • Acute specimens should be collected as soon as possible after the appearance of symptom. • The convalescent specimen is collected a minimum of 2 to 3 weeks after the acute specimen.

  4. B. Methods of viral isolation 1. Animal inoculation One of the earliest ways of detecting a virus was by inoculating a susceptible host animal with infectious material derived from a patient or sick animal and then observing that animal for signs of disease.

  5. 2. Chick embryo inoculation The chick embryo is very frequently employed in virology experiments. The egg has several cavities into which viruses may be inoculated and cultivated. Such as the chorioallantoic membrane (CAM); the allantoic sac; the yolk and amniotic sac;

  6. 3. Cell culture (1) Primary cultures: consist of cells obtained from normal tissue, which can not divide indefinitely.

  7. (2)Diploid cell cultures: are capable of being passed a large but finite number of times before they senesce, or undergo a significant change in their characteristics.

  8. (3) Continuous cultures: generally are obtained from malignant tissues, which can undergo unlimited number of division.

  9. Inoculation of susceptible animal cell cultures with many , but not all, animal viruses results in a pattern of cell destruction and death, which is often characteristic for a given host-virus combination. Lysis!

  10. CPE (cytopathic effects): the most recognizable of it is plaque. Plaque are clear, infectious centers in a monolayer resulting from the death of cells.

  11. C. Direct microscopic examination • Optical microscopy • A readily available technique for the detection of virus is cytologic or histologic examination for the presence of characteristic viral inclusion.

  12. Viral inclusions are intracellular structures formed by aggregates of virus or viral components within an infected cell or abnormal accumulations of cellular materials resulting from viral induced metabolic disruption.

  13. 2. Electron microscopy • Electron microscopy (EM) is most helpful for the detection of viruses that do not grow readily in cell culture and work best if the titer of virus is at least 10E6---10E7 particles per milliliter.

  14. Immune EM visualization of virus particles present in numbers too small for easy direct detection. • The addition of specific antiserum to the sample suspension causes the virus particles to form antibody-bound aggregates, which are more easily detected than are single virus particles.

  15. D. Detection of viral antigen and nucleic acid • Antibody can be used as sensitive and specific tools to detect, identify, and quantitate the virus and viral antigen in clinical specimens.

  16. Viral antigens on the cell surface or within the cell can be detected by immuno-fluorescence and enzyme immunoassay. (EIA)

  17. Virus or antigen released from infected cells can be detected : • enzyme-linked immunosorbent assay (ELISA) • radioimmunoassay (RIA)

  18. ELISA Anti -capture assay

  19. ELISA Anti -capture assay Anti-IgM

  20. ELISA Anti -capture assay Patient‘s serum Anti-IgM

  21. ELISA Anti -capture assay Antigen Patient‘s serum Anti-IgM

  22. ELISA Anti -capture assay E 2.Antibody E Antigen Patient‘s serum Anti-IgM

  23. ELISA Competition assay Antigen

  24. ELISA Competition assay Patient serum Antigen E E

  25. ELISA Competition assay E Marked Antibody E Patient serum Antigen

  26. ELISA Competition assay E E Marked Antibody Patient serum Antigen Antigen

  27. ELISA Competition assay E E Marked Antibody Patient serum Antigen Antigen

  28. ELISA Competition assay E E Marked Antibody Patient serum Antigen Antigen Positive Negative

  29. Molecular detection using probes and polymerase chain reaction assays • Nucleic acid probes are short segments of DNA that are designed to hybridize with complementary viral DNA or RNA segments. • The probe is labeled with a fluorescent, chromogenic, or radioactive tag that allows detection if hybridization occurs.

  30. Nucleic acid probes are most useful in situation in which the amount of virus is relatively abundant, viral culture is slow or not possible, and immunoassays lack sensitivity or specificity.

  31. Detection of Viral Nucleic Acid • For DNA virus the viral DNA can be detected by PCR, Southern blot, and ISH, et al. For RNA virus viral nucleic acid can be detected by RT-PCR, Northern blot, et al.

  32. PCR RT-PCR HBV HVC HIV HIV Herpes Group: FSME HSV Mumps VZV Masern CMV Rotavirus EBV Enterovirus/Polio (Coxsackie) HHV 6 HAV HHV 7 HDV HHV 8 Parvo B 19 RO/01/09Vor

  33. Serological Detection of Viral Infection • Serological testing is most helpful when attempts to demonstrate virus infection by other means are negative, impractical, or impossible. • Serology may be also aid in assessing the significance of a virus isolate.

  34. Antibody Class: IgM, IgG, IgA, IgE Function: - Neutralization - Hemagglutination - Complement binding - Immunopreciptation

  35. Specific IgG-Antibody Virus

  36. Specific IgG-Antibody Virus Patient serum

  37. Specific IgG-Antibody E E Anti-IgG Patient serum Virus

  38. IgM Antibodies: If paired sera are not available, demonstrating IgM antibodies to the virus, even in the first serum sample taken, can sometimes make a presumptive diagnosis.

  39. In congenital infections, IgM antibody detection is of singular value, because IgM does not cross the placenta as IgG does. Thus finding IgM antibodies to viruses in the serum of a newborn indicates that the child was infected in utero.

  40. Complement Fixation Test (CF): Because antiviral sera fix complement in the presence of the homologous antigens, CF tests are employed in the diagnosis of many viral infections.

  41. Neutralization Tests:Virus-neutralizing antibodies are measured by adding serum containing these antibodies to a suspension of virus and then inoculating the mixture into susceptible cell cultures. • The presence of neutralizing antibodies is demonstrated if the cell cultures fail to develop CPE.

  42. Hemagglutination Inhibition Test:Many viruses agglutinate erythrocytes, and this reaction may be specifically inhibited by immune or convalescent sera. • This reaction forms the basis of diagnostic tests for some viral infections, such as influenza, rubella, mumps, and measles.

  43. Hemagglutination test Virus suspension

  44. Hemagglutination test Patient serum

  45. Hemagglutination test Erythrocyte

  46. Hemagglutination test Positive Negative

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