LABORATORY DIAGNOSIS OF VIRAL INFECTIONS

1. LABORATORY DIAGNOSIS OF VIRAL INFECTIONS Level 8 LECTURE 3

2. serology • The branch of medical immunology concerned with antigen-antibody reactions in vitro is serology [serum and -ology]. The usefulness of serological test is dependent on its sensitivity and specificity. • False Negatives/Positives • False negatives occurs when there is no reaction when the Ag or Ab is present. • High sensitivity prevents false negatives. • False positives occurs when there is cross reaction with another molecule • High specificity prevents false positives.

3. SEROLOGY detection of rising titres of antibody between acute and convalescent stages of infection, or the detection of IgM.

4. SEROLOGY Serology forms the mainstay of viral diagnosis. • Following exposure, the first antibody to appear is IgM, which is followed by a much higher titres of IgG. • In cases of reinfection, the level of specific IgM either remain the same or rises slightly. But IgG shoots up rapidly and far more earlier than in a primary infection. • The sensitivity and specificity of the assays depend greatly on the antigen used (Assays that use synthetic peptide antigens tend to be more specific than those using whole or disrupted virus particles).

5. Seroconversion is the development of detectable specific antibodies to microorganisms in the serum as a result of infection or immunization. • Before the seroconversion point, the antigen is prevalent and the corresponding antibody is not detectable in sera. • After seroconversion, the antibody becomes prevalent and the corresponding antigen is not any more detectable in sera. • Antigen-antibody complexes (immune complexes) formed in organism are quickly removed by several immune mechanisms.

6. Indirect methods for viral diagnosis based on detection of viral specific Ab response • Some test measure total Ab (mainly IgG) 1.CFT (complement fixation test) 2.HAI (haemagglutination inhibition) 3.Neutralization test 4.Radial haemolysis test • Test that can detect IgM 1.RIA (radioimmunoassay) 2.EIA (enzyme immunoassay

7. Advantages of Viral serology • Determine immune status – person or population • Verify immune response to vaccination • Diagnosis of chronic infection/ reactivation ? • Diagnosis of recent infection ? (IgM & IgA) • Wide range of tests available • Uses serum – easily collected and transported • (compared with biopsy tissue or DNA ) : 􀂃

8. Viral markers • Some common viral markers detected by serology : • HAV • HBV • HCV • CMV • OTHERS

9. HEPATITIS A • Serology: • Acute – IgM – lasts 3-6 months . • Chronic – IgG – lifetime .

10. IN HEPATITIS A VIRUS INFECTION Clinical illness Infection ALT IgM IgG Viremia Response HAV in stool 0 1 2 3 4 5 6 7 8 9 10 11 12 13 Week

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12. Complement Fixation Test The complement fixation assay can be used to look for the presence of i) specific antibody or ii) specific antigen in a patient's serum. The test utilizes sheep red blood cells (SRBC), anti-SRBC antibody and complement, along with specific antigen (if looking for antibody in serum) or specific antibody (if looking for antigen in serum). If antibody (or antigen) is present in the patient's serum, then the complement is completely utilized and SRBC lysis is minimal. However, if the antibody (or antigen) is not present in the patient's serum, then the complement binds anti-SRBC antibody and lysis of the SRBCs ensues. The following graphics illustrate the complement fixation assay.

13. Pppo xxx Detection of Ab

14. Hemagglutinationusually results from antibodies cross linking red blood cells through attachment to surface antigens and is routinely used in blood typing. The hemagglutination inhibition test is widely used to diagnose influenza, measles, mumps, mononucleosis, and other viral infections

15. Viral Hemagglutination. • Certain viruses can bind to red blood cells causing hemagglutination. • If serum containing specific antibodies to the virus is mixed with the red blood cells, the antibodies will neutralize the virus and inhibit hemagglutination (a positive test).

16. Immuno/Western Blot • Immunoblot detects for a specific protein associated with specific organism. The procedure involves: • Separationof the proteins on polyacrylamide gel. • Transfer (blotting) of proteins from the gel to a membrane (nitrocellulose or nylon) and identification of the protein with a specific Ab. • The method is sensitive for detecting proteins in complex mixtures. • Immunoblot is laborious, time consuming and less sensitive than ELISA. Immunoblot

17. Immuno/Western Blot • Immunoblot is used as a confirmatory test for HIV. • The ELISA test for HIV often yields false positive and the immunoblot test is used to confirm a positive ELISA results. • To perform the HIV immunoblot purified HIV is treated with SDS to solubilize the proteins and inactivate the virus. • The proteins (at least 7) are resolved by polyacrylamide gel electrophoresis and the proteins are blotted unto a membrane and incubated with the test serum.

19. HIV Immunoblot Test • The test is considered positive if bands occur at, 2 locations e.g. gp160 and gp 120 or p24 and gp 41-45.

20. HIV Immunoblot Test • Test was done at 6 different times (after the suspected exposure). • Test is positive if bands occur at two locations e.g. gp160 or gp 120 and p31 or p24. • Test is negative if no bands are present for any HIV antigen. • SRC is positive control

21. CSF antibodies • Used mainly for the diagnosis of herpes simplex and VZV encephalitis • CSF normally contain little or no antibodies • presence of antibodies suggest meningitis or meningoencephalitis Diagnosis depends on the presence of an intact blood-brain barrier

22. Rapid Diagnosis Based on the Detection of Viral Antigens Nasopharyngeal Aspirate Influenza A and B Parainfluenza Adenovirus Faeces Rotaviruses Adenoviruses Astrovirus Skin HSV VZV Blood CMV (pp65 antigenaemia test)

23. Molecular Methods • Methods based on the detection of viral genome are also commonly known as molecular methods. It is often said that molecular methods is the future direction of viral diagnosis. • However in practice, although the use of these methods is indeed increasing, the role played by molecular methods in a routine diagnostic virus laboratory is still small compared to conventional methods. • It is certain though that the role of molecular methods will increase rapidly in the near future.

24. Classical Molecular Techniques • Dot-blot, Southern blot, in-situ hydridization are examples of classical techniques. They depend on the use of specific DNA/RNA probes for hybridization. • The specificity of the reaction depends on the conditions used for hybridization. However, the sensitivity of these techniques is not better than conventional viral diagnostic methods. • However, since they are usually more tedious and expensive than conventional techniques, they never found widespread acceptance.

25. Polymerase Chain Reaction (1) • PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique. • It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest. • These oligonucleotides act as primers for the thermostable Taq polymerase. Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC), annealing of primers to their complementary sequences (50oC), and primer extension (72oC) result in the exponential production of the specific target fragment. • Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis, hybridization with a specific oligonucleotide probe, restriction enzyme analysis, or DNA sequencing.

26. Polymerase Chain Reaction (2) • Advantages of PCR: • Extremely high sensitivity, may detect down to one viral genome per sample volume • Easy to set up • Fast turnaround time • Disadvantages of PCR • Extremely liable to contamination • High degree of operator skill required • Not easy to set up a quantitative assay.

27. Schematic of PCR Each cycle doubles the copy number of the target