1. 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.  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.  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. 3 US CDC, 2012 http://www.cdc.gov/hiv/resources/factsheets/us.htm

4.  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

5. SCREENING TEST  High Sensitivity CONFIRMATION TEST  High Specificity 5

6.  RNA virus  Surface envelope proteins  Matrix proteins  Capsule proteins  RNA 6

7. 7

8.  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

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

10.  Enzyme-linked immunosorbent assay (ELISA or EIA)  1 st generation—viral lysate  2 nd generation—synthetic antigen and HIV-2  3 rd generation—IgM and IgG and HIV-2  4 th generation—p24 + IgM and IgG and HIV-2  Western blot  Immunofluorescent assay 10

11.  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

12.  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. 12

13. 13

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

15. 15 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.  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

17.  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

18.  Western blot  Immunofluorescent assay  Qualitative RNA tests 18

19. 19 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. 20 Mandel, Principles & Practice of Infectious Diseases, 6th Edition

21. 21 HIV Immunoflourescent assay

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

23.  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

24.  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

25.  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

26.  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

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

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

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

30.  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

31.  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

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

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

34.  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

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

36. JDKlausner@mednet.ucla.edu 36