This document summarizes the various methods used to diagnose HIV infection in a laboratory setting. It discusses screening tests like ELISA and rapid tests that detect HIV antibodies, supplemental tests like Western Blot for antibody confirmation, and confirmatory tests including viral RNA detection by PCR and p24 antigen detection. It also mentions nonspecific immunological markers and stresses the importance of pre-test and post-test counseling.

1. HIV-LAB DIAGNOSIS

2. LAB DIAGNOSIS
• A number of ethical, legal& psychosocial
issues are associated with a positive HIV status
• The following care should be taken while
performing the test for HIV
*Consent-in written format
*Confidentiality
*Counselling-pre test&post test counselling to
educate the
patient

3. Lab diagnosis
Specific tests for HIV infection
• Screening tests
• Supplemental tests
• Confirmatory tests
Nonspecific methods
• Low CD4 cell count
• Altered CD4:CD8 ratio
• Hypergammaglobulinemia
*neopterin
*beta 2-microglobulin

4. Antibody detection
• It is the mainstay of diagnosis
• Tests can be classified into
*Screening assays- ELISA & Rapid/Simple tests
*Supplemental tests- western blot

5. Screening assays
• Usually takes less time
• High sensitivity: must detect all positive sera
• Should be confirmed : results of screening test
should never be used as final interpretation. It is
always subjected to confirmatory tests
• Antigens used in most of screening tests-
*HIV-1 specific (p24,gp 120, gp 160, gp 41)
*HIV-2 specific gp 36
They detect HIV-1&2 seperately or together

6. ELISA (Enzyme Linked immunosorbent
assay)
• Most commonly performed test at blood banks &tertiary care
centres
• It is easy to perform, sensitive , specific& cost effective
• Types
*1st generation ELISA- uses crude preparation of HIV
antigens
*2nd generation ELISA- uses recombinant antigens of
HIV
*3rd generation ELISA- uses synthetic oligopeptide
antigens of HIV
*4th generation ELISA- detects both HIV antibodies&
antigen p24 by using combination of recombinant/ synthetic
peptides as well as monoclonal antibodies respectively

7. Rapid/ simple tests
• Developed for ease of performance& quick
results
• Requires less than 30 minutes to perform
• They work on various principles such as
*Dot blot assays
*Immunochromatography
*Particle agglutination assays
* Dip stick/ comb tests

8. Supplemental tests
• Highly specific antibody detection methods
• Used for validation of positive results of
screening tests
• They are expensive, labor intensive, need
expertise to interpret
• It may also give indeterminate results

9. Western blot
• Most commonly used supplimentary test
available & recommended by NACO
• It works on principle of immunoblot technique
• It detects individual antibodies in serum
seperately against various antigenic fragments of
HIV such as
*Antibody to gag gene products(gp55,gp40)
*Antibody to pol gene products (gp 68,gp53)
*Antibody to env gene products (gp120,gp 160)

10. Western blot…
• The antigen-antibody complexes appear as
distinct bands on nitrocellulose strip
• Reactive results are interpreted as per
*WHO criteria-presence of atleaast two env
bands with or without gag or pol bands
*CDC criteria- presence of any two out of p24,
gp 120,gp 160,gp41 bands

11. Confirmatory tests
• Detection of p 24 antigen
• Viral culture by co-cultivation technique
• HIV RNA
*RT PCR
*Branched DNA assay
*NASBA(nucleic acid sequence based
amplification)
*Real timeRT PCR
• HIV DNA detection –diagnosis of paediatric HIV

12. Detection of p24 core antigen
• It becomes detectable after 1-2 weeks of infection and
lasts for 3-4 weeks thereafter. Again it is elelvated
during late advanced stages.
• It is less sensitive (Ag-Ab complex elimination from
circulation)
• Uses of p24 antigen detection test
*for confirmation of diagnosis of HIV
*Diagnosis of HIV during window period
*To diagnose late stage of HIV
*Diagnosis of HIV in infants
*Monitoring the progress of HIV infection
*To resolve equivocal western blot results

13. Viral RNA detection
• It is the gold standard method for confirmation
of HIV diagnosis
• It is the most sensitive & specific method
• Various formats available-
Reverse Trancriptase PCR
Branched DNA assay
Nucleic Acid Sequence Based Amplification
Real Time RT PCR : for estimating viral load

14. Isolation of the virus from blood or
tissues
• It is time consuming, expensive, takes longer time& not
sensitive
• Two methods are being used for virus isolation
1.Indirect method : peripheral blood mononuclear cells
(PBMCs)are cultured invitro in presence of mitogen
phytohaemoagglutinogen(PHA)
2.Co-cultivation method : PBMCsfrom heterologous
HIV uninfected donor are stimulated with PHA, & after
48-72 hrs, the stimulated cells are cultured along with
the PBMCs from the patient

15. Nonspecific/immunological tests
• CD4 T cell count : carried out by flow cytometry method
• It is useful in-
*Assessing the risk of opportunistic infections
*Monitoring the response to antiretroviral therapy
*Initiation of antiretroviral therapy-if CD4 Tcell count is
less than 350/mm3
• Abnormal proteins-neopterin, beta2-
microglobulin&soluble
IL-2 receptors produced by PBMCs, stimulated by
interferon-gamma which in turn are produced by HIV
activated TH-1 cells

16. Diagnosis of HIV in window period
• Window period: Initial time interval between
the exposure &appearance of detectable levels
of antibodies in the serum
• Detection of p24 antigen : detected by 1-2
weeks
• HIV RNA detection : It is the best method,
detects as
early as 12th day

17. THANK YOU