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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 AAHIVU August 2014

  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 STD Guidelines group • Dr. Klausner is a board member of YTH, Inc, non-profit • Dr. Klausner is medical advisor for Healthvana.com • In the past 12 months, Dr. Klausner has received: • Travel support for meeting coordination and speaking from Standard Diagnostics, Inc. • Research funding or donated supplies from the NIH, CDC, Hologic, Inc., Alere, Inc., Chembio, Inc. Cepheid, Standard Diagnostics, Inc., and MedMira, Inc. 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 in the United States are tested for HIV • An estimated 38%-44% of all adults had been tested for HIV • Approximately 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 • HIV-infection status awareness is associated with reduced 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 • RNA virus • Surface envelope proteins • Matrix proteins • Capsule proteins • RNA

  7. Evolution of HIV infection

  8. HIV tests • Detect human antibodies to surface proteins • Detect human antibodies to surface, matrix and capsule proteins • Detect HIV antigen (P24 capsule protein) • Detect RNA • Detect DNA, integrated within cells

  9. HIV antibody tests • 1st generation—viral lysate • 2nd generation—synthetic antigen + HIV-2 • 3rd generation—IgM and IgG + HIV-2 • 4th generation—p24 + IgM and IgG + HIV-2

  10. HIV antibody tests • 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 • Western blot • Immunofluorescent assay

  11. What is an ELISA test? • ELISA = Enzyme-Linked Immuno Sorbent Assay. • 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 appropriate anti-HIV antibodies are present (keys), they will 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 antibody to capture back of the first antibody. • This second antibody has an enzyme is attached to it. • When a reactive substrate is added to the mix, the enzyme will turn the substrate a different color (usually red). • If the serum to be tested contains anti-HIV antibodies, the liquid in the microwell will turn red.

  13. HIV 1st Generation Antibody test

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

  15. HIV 2nd Generation 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. HIV 3rd Generation 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. HIV 4th Generation 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

  18. Confirmation Tests • Western blot • Immunofluorescent assay • Qualitative 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 Western blot Mandel, Principles & Practice of Infectious Diseases, 6th Edition

  21. 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

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

  23. HIV-RNA Quantitation • Used to monitor antiviral therapy and to predict disease progression in HIV infected persons. • In conjunction with a positive DNA PCR or a reactive EIA, the RNA quantitation 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 dynamic range for HIV RNA detection by Real-Time PCR is 30 to 1,000,000 copies/mL of plasma. • Often use in newborns and infants for early diagnosis

  24. HIV-1 Proviral DNA Detection • The detection of cell associated Human Immunodeficiency Proviral DNA by polymerase chain reaction (PCR) amplification is one of the most sensitive non-serologic methods for confirming HIV infection. • This assay is recommended for confirming HIV infection in the neonate. • HIV DNA PCR may also be used as a supplemental test to determine the significance of an indeterminate HIV Western Blot serology result.

  25. 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

  26. HIV-1 Genotypic Resistance Assay • The assay involves sequencing of the HIV pol gene, after which mutations in the gene can be compared to sequences known to confer resistance to different classes of antiretroviral drugs. • The assay is most useful in patients who lose viral suppression on antiretroviral therapy and should be performed before switches in therapy are entertained.

  27. Repeat EIA in Duplicate Enzyme Immunoassay (EIA) (run singly) End; patient is considered negative non-reactive reactive 2 non- reactive results 1 or 2 reactive results Patient is considered negative Must perform one of the following confirmation tests TMA transcription-mediated amplification Western Blot IFA immunofluorescence assay Indeterminate Positive: patient is HIV+ Negative: patient is HIV-negative Result = inconclusive Re-draw in 2-4 weeks

  28. Most sensitive EIA available 4th Generation Strongly recommended (run singly) Negative Positive Repeat In Duplicate HIV Negative 2 neg HIV Negative 1 or 2 pos An HIV-1 / HIV-2 Differentiating Test Pos for HIV-1 Ab Pos for HIV-2 Ab Neg HIV RNA Test HIV-1 Positive No RNA HIV-2 Positive RNA Detected HIV Negative HIV-1 Acute Infection (follow-up for HIV-2)

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

  30. 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?

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

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

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

  34. Case 6 • 47 year old man has been on treatment for years but ran out of meds 1 year ago. • About 6 months ago he restarted 2 medications he obtained from his partner. • He has been losing weight and complains of fatigue and fevers • What test is appropriate?

  35. Case 7 • 6 week baby had an HIV-infected mother • The mother receive treatment during pregnancy • What test is appropriate for the baby?

  36. Thank You JDKlausner@mednet.ucla.edu

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