MEANS OF viral infection DIAGNOSIS

1. MEANS OF viral infectionDIAGNOSIS Claude MUVUNYI M.D., Ph.D.

2. Clinical diagnosis • The patient’s history and symptoms provide the first clues to the diagnosis of a viral infection, • but the diagnosis also includes the exclusion of other types of infection (e.g., bacterial, fungal) • Results from viral laboratory studies can confirm the clinical diagnosis by identifying the viral agent of the infection or detecting specific antigen antibodies

3. Viral laboratory studies The laboratory diagnosis of viral infection is based upon three general approaches: • Direct detection of viral antigens or structures, either in cells derived from infected tissues or free in fluid specimens; • Isolation and identification of viruses, usely accomplished in cell cultures; • Demonstration of a significant increase in serum antibodies to a etiological possible virus during the course of a illness; that’s by serological testing assays.

4. Specimens for virus isolation

5. Specimens for virus isolation

6. Specimen Collection

7. Specimen Collection

8. Cerebral Spinal Fluid

9. Throat and nasal Swabs

10. Ear and eye Swabs-

11. Wound Swabs

12. Poor sample quality from Young child

13. Urine specimen from young child

14. Genital Swabs

15. Lower respiratory Swabs Correct position Wrong position

16. Laboratory diagnosis of viral infection • A clinical diagnosis of a viral infection can be confirmed in laboratory through the observation of: • Virus-induced cytopathogenic effets (CPE) on inoculated permissive cells • Electron microscope detection of viral particles • Isolation and growth of the virus • Detection of viral components or antigens ( e.g., proteins, enzymes, nucleic acid) • Evaluation of the patient’s immune response to the virus that may be by serology detecting specifics antibodies against viral antigens

17. Laboratory diagnosis of viral infection • We currently used two kinds of settings in laboratory diagnostics: • the direct diagnostics • the indirect diagnostics or serological settings

18. Direct diagnostics • Direct diagnosis procedures concern : • Cytological examinations of CPE • Electron microscopy • Virus isolation and growth onto permissive cell’s culture • Detection of viral antigens: proteins, enzymes • Detection of viral genetic elememts , mainly genomic nucleic acid

19. Direct diagnostics • The “gold standard” for providing a viral etiology of a syndrome, infection or disease is the recovery and growth of infecting agent. • Isolation and growth studiesare very fastidious and mainly available only in referral laboratories. • CPEs can be detected by means of cytological examination. • Use of electronmicroscopyisn’t a standard clinical laboratory technique, but it can be used to detect some viruses if sufficient viral particles are presents, mainly in serum or feces such as new increminateRotavirus causative agents of children’s gastroenteritis

20. Picture of electron microscope picture of bright microcope of direct view of Calcivirusfluorescence

21. Molecular biology techniques • Viral genome detection often after been applified in PCR. We also can quantify and detect DNA or RNA sequences • PCR or polymerase chain reaction and reverse transcriptae PCR (RT-PCR) are more used and becoming very important for viral detection • Used of the appropriate primers can promote a million fold amplicaficationof a target genomic sequence in few hours. • Then we can qualify and/or quantify the genome structures

22. Fig : PCR or Polymerase Chain Reaction

23. Indirect diagnostics • Indirect diagnostic procedures mean: • Serological different testing of hemagglutination • Inhibitionof hemagglutination, • Neutralizing of cytopathologiceffect, • Indirect immunofluorescence, • ELISA, • Immunobotting, • Western blots.

24. Agglutination Tests Lattice Formation

25. Qualitative agglutination test • Ag or Ab Y + ↔ Y Y Agglutination/Hemagglutination • Definition - tests that have as their endpoint the agglutination of a particulate antigen • Agglutinin/hemagglutinin

26. 1/1024 1/256 1/512 1/128 1/16 1/64 1/32 Pos. 1/8 Neg. 1/4 1/2 Titer Patient 64 1 8 2 512 3 <2 4 32 5 128 6 32 7 4 8 Agglutination/Hemagglutination • Quantitative agglutination test • Titer • Prozone

27. 1/256 1/512 1/128 1/16 1/64 1/32 1/8 1/4 1/2 Agglutination/Hemagglutination • Definition • Qualitative test • Quantitative test • Practical considerations • Easy • Semi-quantitative

28. Y Y + ↔ Y Passive Agglutination/Hemagglutination • Definition - agglutination test done with a soluble antigen coated onto a particle • Applications • Measurement of antibodies to soluble antigens

29. Prior to Test Y Y + ↔ Y Y Test Y + + ↔ Y Patient’s sample Agglutination/Hemagglutination Inhibition • Definition - test based on the inhibition of agglutination due to competition with a soluble Ag

30. Radioimmuoassays (RIA)Enzyme-Linked Immunosorbent Assays (ELISA) Lattice formation not required

31. Prior to Test Y Y + ↔ Labeled Ag Test Y Y + + + ↔ Labeled Ag Patient’s sample Competitive RIA/ELISA for Ag • Method • Determine amount of Ab needed to bind to a known amount of labeled Ag • Use predetermined amounts of labeled Ag and Ab and add a sample containing unlabeled Ag as a competitor

32. Solid Phase Solid Phase Test Y Y + + + ↔ Labeled Ag Patient’s sample Competitive RIA/ELISA for Ag • Method cont. • Determine amount of labeled Ag bound to Ab • ↓NH4SO4 • ↓ anti-Ig • Immobilize the Ab • Concentration determined from a standard curve using known amounts of unlabeled Ag • Quantitative • Most sensitive test

33. Labeled Anti-Ig Ab in Patient’s sample Y Y Ag Immobilized Solid Phase Solid Phase Non-Competitive RIA/ELISA • Ab detection • Immobilize Ag • Incubate with sample • Add labeled anti-Ig • Amount of labeled Ab bound is proportional to amount of Ab in the sample • Quantitative

34. Labeled Ab Ag in Patient’s sample Y Ag Y Immobilized Solid Phase Solid Phase Non-Competitive RIA/ELISA • Ag detection • Immobilize Ab • Incubate with sample • Add labeled antibody • Amount of labeled Ab bound is proportional to the amount of Ag in the sample • Quantitative

35. Picture of ELISA results: colored are positive and colorless negative

36. Pictures of apparatus of distribution of specimens for ELISA

37. Assays Based on Complement Lattice formation not required

38. No Ag Ag Patient’s serum Y Y Y Y Y Y Y Y Y Y Complement Fixation • Methodology • Ag mixed with test serum to be assayed for Ab • Standard amount of complement is added • Erythrocytes coated with Abs is added • Amount of erythrocyte lysis is determined Ag Ag