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 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 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 HIVinfected. US CDC, 2012 http://www.cdc.gov/hiv/resources/factsheets/us.htm 3 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 4 SCREENING TEST High Sensitivity CONFIRMATION TEST High Specificity 5 RNA virus Surface envelope proteins Matrix proteins Capsule proteins RNA 6 7 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 8 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 9 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 10 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 11 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 antiHIV antibodies, the liquid in the microwell will turn red. 12 13 Detects anti-HIV IgG Includes HIV-2 antigen Uses synthetic antigens Window period 6+ weeks 14 • 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 15 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 16 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 17 Western blot Immunofluorescent assay Qualitative RNA tests 18 • 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. 19 Mandel, Principles & Practice of Infectious Diseases, 6th Edition 20 HIV Immunoflourescent assay 21 Used to diagnose HIV infection Resolve indeterminate HIV-antibody results Manual Lower limit of detection > 30 RNA copies/ ml 22 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 23 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. 24 Culture is an extremely sensitive virologic method for documenting HIV infection, especially in neonates whose serologies are complicated by the presence of maternal antibody 25 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. 26 End; patient is considered negative non-reactive Enzyme Immunoassay (EIA) (run singly) Repeat EIA in Duplicate reactive 2 nonreactive results 1 or 2 reactive results Must perform one of the following confirmation tests IFA immunofluorescence assay Western Blot Patient is considered negative TMA transcriptionmediated amplification Indeterminate Positive: patient is HIV+ Negative: patient is HIV-negative Result = inconclusive Re-draw in 2-4 weeks Most sensitive EIA available Negative HIV Negative Positive 4th Generation Strongly recommended (run singly) Repeat In Duplicate 2 neg 1 or 2 pos Pos for HIV-1 Ab HIV-1 Positive HIV Negative An HIV-1 / HIV-2 Differentiating Test Pos for HIV-2 Ab Neg HIV RNA Test HIV-2 Positive No RNA RNA Detected HIV-1 Acute Infection HIV Negative (follow-up for HIV-2) 30 year old bisexual man comes into clinic He has had 15 lifetime partners, never been HIV-tested What test is appropriate? 29 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? 30 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? 31 17 year old girl had a rapid HIV test that was positive She comes to clinic for testing What test is appropriate? 32 17 year old girl had a rapid HIV test that was positive The ELISA test was indeterminate What test is appropriate? 33 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? 34 6 week baby had an HIV-infected mother The mother receive treatment during pregnancy What test is appropriate for the baby? 35 JDKlausner@mednet.ucla.edu 36