Diagnosis of Viral infections

2. Diagnosis of Viral infections • Clinical: • Symptoms (History). • Signs (Physical Examination). • Investigation: • Non laboratory: (e.g X-rays, CT scan, MRI, US) • Laboratory: • Non Virological. (CBC, LFT, …) • Virological.

3. Laboratory Diagnosis of viral infections • Viral Isolation. • Electron Microscopy. • Antigen detection. • Antibody detection. • Nucleic acid detection. • Cytology. • Histopathology.

4. Viral Isolation • Methods Used; • Cell Culture (tissue ,organ culture). • Embryonated egg. • Animal inoculation.

5. Conventional Cell Culture • Viruses are cultivated in cell cultures derived from animal tissues.

6. Conventional Cell Culture • Maintaining cells in culture; • Provide suitable temperature, pH, glucose concentration. • Provide essential nutrients for the cell medium(amino acids, growth factors ..etc). • Addition of antibiotics and antifungal.

7. Conventional Cell Culture Cell lines:  population of cells obtained from multicellular organism (animal)  which can be maintained to keep undergoing division. • Human cell lines examples: • HeLa (cervical cancer). • Human neuroblastoma cells, • Primate cell lines example: • Vero (African green monkey kidney epithelial cell line initiated 1962).

8. Cytopathic Effect (CPE) Observable morphologic changes in the cell line which can be tested by light microscope or other techniques.

9. Cytopathic Effect (CPE) • Giemsa-stained HeLa cells 7 days postinfection with human adenovirus showing pronounced cell enlargement, rounding, and distinctive “grape-like" clusters (ballooning).

10. Cytopathic Effect (CPE) Giemsa-stained Vero cells (from an African green monkey kidney) 30 hours postinfection with herpes simplex virus-1 showing cell rounding and clustering

11. Cytopathic Effect (CPE) The small syncytia, or multinucleated giant cells, result from fusion of cell membranes formed by Vaccinia virus

12. Cytopathic Effect (CPE) rounding, bridging, cell lysis, and syncytium formation with respiratory syncitial virus

13. Shell Vial Culture (SVC) • Mixture of conventional cell culture and antigen detection (IF) method (CMV). • To obtain rapid results (24 – 48 hrs)

14. Cell Culture: Haemadsorption Phenomenon • Ability of the virus (influenza) in the clinicall specimen to adhere and clump erythrocyte due to expression of haemagglutinin on the cell surface. • Identification can then be confirmed by haemagglutination inhibition, haemadsorption inhibition, or, immunofluorescence.

15. Cell culture: Interference Phenomenon Rubella Rubella ECHO 17 + ECHO 17 + + + Cell Cell Cell CPE No CPE No CPE • Seen Rubella virus: • do not produce CPE. • Make the infected cell resistant to normally infected viruses

16. Cell culture • Definitive diagnosis to the virus grown in cell culture: • Complement fixation. • Hemagglutination inhibition. • Neutralization. • Radioimmunoassay. • Immuno-electron microscopy.

17. Electron Microscope • Principle: • Production of “Electron beam” focuses on the image. • Electrons have Wave length 100,000 time shorter than Photons • Illuminationof the specimen. • Production of magnified image (10 millions times more than light microscope.

18. Electron Micrograph Uses negative stain by contrasting the specimen with an optically opaque fluid. Characterization of virus morphology by using different specimens:

19. Electron Micrograph Rotavirus Stool:

20. Electron Micrograph Adenovirus Stool:

21. Electron Micrograph Herpesvirus Vesicular fluid

22. Electron Micrograph Ebola virus Tissue specimen:

23. Electron Micrograph Rabies Brain (animals)

24. Antigen detection • Diagnosis of viral infections by detecting viral specific antigens. • Examples: • Hepatitis B Surface antigens. • HIV p24 antigens. • Sandwich ELISA. Immunofuorescence

25. Antibody detection ELISA Compliment Fixation • Using Serological tests for: • Screening for viral infections. • Diagnosis of acute infection: • Sero-conversion. • Significant rising titer. • Detection of specific IgM. • Main problems: • False positive e.g. past infection. • False negatives e.g. infection in immunosuppressed,

26. Nucleic Acid detection • Most sensitive and most specific method for: • Diagnosis of viral infection e,g. herpes encephalitis. • Demonstration of the disease progression e.g. HIV. • Genotyping of virus e.g. Hepatitis C. • E.g. • PCR. • PCR. • RT – PCR. • Real time – PCR • NASBA. • bDNA. • Other.

27. Cytopathology • studying and diagnosing diseases on the cellular level. • Some cytopathologic features are highly associated with certain viral infections: e.g. • Tzanck’s smear test. • Pap smear test.

28. Cytopathology • Tzanck’s Smear: • Scrapping the Herpetic ulcer or vesicle. • Looking for “multinucleated Giant cells”. • Diagnostic for Herpes Simplex virus infection and chicken pox

29. Cytopathology • Pap smear test: • Obtaining a smear from Cervical Canal. • Stained by Papanicolaoustain. • Screening test detect pre-cancerous changes called cervical intraepithelial neoplasia (CIN) caused by Human papilloma virus.

30. Immunohistochemistry (IHC) Spinal cord: Immunoperoxidase-staining of WN viral antigen in glial cells Process of detecting viral antigens within cells of a tissue section (biopsy). IHC uses the principle of antibodies binding specifically to antigens in biological tissues. E.g. Immunoperoxidase, immunofluorescence

31. Postmortem examination • Negri bodies: • eosinophiliccytoplasmic inclusion (mass of nucleocapsids) pathognomonic for rabies. •  examination of tissue obtained from a corpse to recognize pathologic features of certain viral infections  that may be present. E.g. • Rabies. • Ebola.