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HIV Testing

HIV Testing. Jeffrey D. Klausner, MD, MPH Professor of Medicine and Public Health Attending Physician Ronald Reagan Medical Center Center for World Health and Division of Infectious Diseases David Geffen School of Medicine Department of Epidemiology

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HIV Testing

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  1. HIV Testing Jeffrey D. Klausner, MD, MPH Professor of Medicine and Public Health Attending Physician Ronald Reagan Medical Center Center for World Health and Division of Infectious Diseases David Geffen School of Medicine Department of Epidemiology Karin and Jonathan Fielding School of Public Health African-American HIV University MRL 1-441 August 13, 2013

  2. Disclosures • Dr. Klausner is a faculty member of the University of California Los Angeles • Dr. Klausner is a guest researcher with the US CDC Mycotics Diseases Branch • Dr. Klausner is a member of the WHO Congenital Syphilis Elimination advisory group • Dr. Klausner is medical director, APLA Health and Wellness Center • Dr. Klausner is a board member of Isis-Inc. • In the past 36 months, Dr. Klausner has received: • Travel support for meeting attendance from WHO, London School of Hygiene and Tropical Medicine and Council of Scientific Industrial Research, South Africa • Research grant funding, supplies or unrestricted gifts from the NIH, CDC, California HIV Research Program, Hologic Gen-Probe, Inc., Gilead Sciences, Inc., Cepheid, Inc., APLA, Public Health Foundation Enterprises, American Jewish World Service and Qpid.me JDKlausner@mednet.ucla.edu

  3. HIV in the U.S. • An estimated 1,148,200 persons in the United States live with HIV • About 50,000 new HIV infections occur in the United States each year • Each year, approximately 16-22 million persons are tested for HIV • An estimated 38%-44% of all adults had been tested for HIV • About 1 in 5 (18% or 207,600 persons) do not know they are HIV-infected. US CDC, 2012 http://www.cdc.gov/hiv/resources/factsheets/us.htm

  4. Why provide HIV testing? • Earlier detection leads to improved treatment outcomes • Knowledge of HIV-infection status is associated with less transmission risk behavior • HIV-infected persons on treatment are less infectious

  5. Principle Screening test Confirmation Test High Specificity • High Sensitivity

  6. Human Immunodeficiency Virus • Proteins— • Surface envelope • Matrix • Capsule • Fatty membrane • RNA

  7. Targets of HIV tests • Human antibodies to various viral proteins • HIV antigen (P24 capsule protein) • HIV RNA in blood

  8. Generations of HIV antibody tests • 1st generation—viral lysate • 2nd generation—synthetic antigen + HIV-2 antigen • 3rdgeneration—anti-IgMand anti-IgG+ HIV-2 antigen • 4th generation—p24 + anti-IgMand anti-IgG+ HIV-2

  9. Evolution of HIV infection

  10. HIV antibody test formats • Enzyme-linked immunosorbent assay (ELISA or EIA) • 1st generation—viral lysate • 2nd generation—synthetic antigen and HIV-2 • 3rd generation—IgM and IgG and HIV-2 • 4th generation—p24 + IgM and IgG and HIV-2 • Rapid tests • 2nd generation—synthetic antigen and HIV-2 • 3rd generation—IgM and IgG and HIV-2 • 4th generation—p24 + IgM and IgG and HIV-2 • Western blot • Immunofluorescentassay • RNA tests

  11. What is an ELISA test? • ELISA = Enzyme-Linked Immuno Sorbent Assay, AKA EIA • This technique is based on the lock and key theory of antibodies • Antibodies and antigens work like locks and keys • One antibody fits one antigen • Having the antibody means the antigen is also present • ELISA technique involves placing HIV antigens (locks) on the bottom of a microwell cup • The microwell is then filled with the serum to be tested • If the right anti-HIV antibodies are present (keys), antibodies stick to the antigens (locks) • High throughput, automated, sensitive

  12. What is an ELISA test? • Since antibodies are proteins too, they themselves are also antigens. • Use anti-HIV antibody to capture the first antibody. • This second antibody has an enzyme is attached to it. • When a reactive chemical is added to the mix, the enzyme will turn the chemical a different color (usually red). • If the serum to be tested contains anti-HIV antibodies, the liquid in the test well will turn red.

  13. 1stGeneration Antibody test

  14. 2ndGeneration Antibody test Detects anti-HIV IgG Includes HIV-2 antigen Uses synthetic antigens Window period 6+ weeks

  15. 2ndGeneration Antibody testRapid test • OraQuick Rapid HIV-1/2 Antibody Test • Reveal G3 Rapid HIV-1 Antibody Test • Uni-Gold Recombigen HIV Test • Multispot HIV-1/HIV-2 Rapid Test • Clearview HIV 1/2 Stat Pak • Clearview Complete HIV 1/2

  16. 3rdGeneration Antibody test • The method of 3rd generation of HIV ELISA test is double antigen sandwich • Detects IgM and IgG antibody to HIV • Antibodies to HIV-2, HIV-1 group O • Improved sensitivity and specificity • The main laboratory-based diagnostic ELISA test worldwide now • Window period 3 weeks

  17. 4thGeneration Antibody test • Detects antibodies to HIV -1, HIV-2 and HIV-2 O type AND p24 antigen • Used for detection of infection within window period which shows positive in HIV P24 antigen tests but negative for antibodies to HIV • Alere Determine rapid HIV ½ Ag/AbFDA-approved August 9, 2013

  18. HIV Confirmation Tests • Western blot • Immunofluorescent assay • HIV RNA tests

  19. HIV Western blot • Identifies antibodies against eight HIV-1 encoded proteins: p18, p24, p31, gp41, p51, p55, p65/66, gp120/p160. • Criteria require antibodies against any two of the following HIV-1 proteins: p24, gp41, or gp120/160. • Specimens showing reactivity to HIV-1 protein(s), but not fulfilling the criteria for a positive result, are reported as Indeterminate. • All indeterminate Western blots are further tested in supplemental HIV-1 and HIV-2 specific assays. • A negative Western blot has no detectable bands, i.e. no antibodies reacting to either HIV-1 or non-HIV-1 proteins.

  20. HIV antigen is mixed with a fluorescent compound and then with a sample of the patient's blood. If HIV antibody is present, the mixture will fluoresce when examined under ultraviolet light. • HIV Immunoflourescent assay

  21. HIV RNA Test • Used to confirm HIV infection • Resolve indeterminate HIV-antibody results • Manual • Lower limit of detection > 30 RNA copies/ ml

  22. HIV RNA Quantitation • To monitor antiviral therapy and to predict HIV disease progression • With a reactive EIA, the RNA test may be diagnostic • High levels of RNA are found during acute infection and in patients who are more likely to have disease progression. • Inhibition of cell-free HIV, as reflected by RNA copy number, is associated with better CD4 response and clinical response in some patient populations. • The range for HIV RNA detection by PCR is 30 to 1,000,000 copies/mL • Often used in newborns and infants for early diagnosis

  23. HIV-1 Culture • Culture is an extremely sensitive virologic method for documenting HIV infection, especially in neonates whose serologies are complicated by the presence of maternal antibody

  24. Case 1 • 30 year old bisexual man comes into clinic • He has had 15 lifetime partners, never been HIV-tested • What test is appropriate?

  25. Case 2 • 22 year old man who has sex with men, methamphetamine user • Last tested HIV-negative 6 months ago • History of syphilis • What test is appropriate?

  26. Case 3 • 46 year old man diagnosed with HIV infection 6 years ago • Has been on treatment for 3 years but has not had a check up in a year • What test is appropriate?

  27. Case 4 • 17 year old girl had a rapid HIV test that was positive • She comes to clinic for testing • What test is appropriate?

  28. Case 5 • 17 year old girl had a rapid HIV test that was positive • The ELISA test was indeterminate • What test is appropriate?

  29. Thank You JDKlausner@mednet.ucla.edu

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