1. Bernard M. Branson, M.D. Associate Director for Laboratory Diagnostics Divisions of HIV/AIDS Prevention National Center for HIVAIDS, Viral Hepatitis, STD, and TB Prevention Centers for Disease Control and Prevention Introduction: 2007 HIV Diagnostics Conference The findings and conclusions in this presentation are those of the author and do not necessarily represent the views of the Centers for Disease Control and Prevention

2. HIV Viremia and Antibody Response HIV RNA (plasma) HIV Antibody 11 0102030405060708090100 HIV p24 Ag 1622 Ramp-up viremia DT = 21.5 hrs 1 st gen 2 nd gen 3 rd gen p24 Ag EIA - HIV MP-NAT - HIV ID-NAT - Peak viremia: 10 6 -10 8 gEq/mL “blip” viremia Viral set-point: 10 2 - 10 5 gEq/mL Acute HIV Infection

3. 1989: State of the Art EIA Western blot

5. Organization Criteria APHL/CDCAny two: p24, gp41, gp120/160 ARCThree or more bands, one from each gene product group: gag, pol, env CRSSTwo or more bands: p24 or p31 and gp41 or gp120/160 WHOTwo env bands with or without gag or pol Indeterminate: Presence of any bands

6. Diagnostic Algorithm: 1989 The Public Health Service recommends that no positive test results be given to clients/patients until a screening test has been repeatedly reactive (i.e., greater than or equal to two tests) on the same specimen and a supplemental, more specific test such as the Western blot has been used to validate those results

7. 1989 State of the Art

8. WHO HIV Diagnostic Testing: Strategy III Asymptomatic; prevalence < 10% A1 A2 Negative A1+A1- A1+A2+ A1+A2- Positive Indeterminate A3 A1+A2-A3+ Positive A1+A2-A3-

9. SUDS: Single Use Diagnostic System for HIV-1

10. Blue color with reactive HIV test

12. Recommendation …and a Promise Health-care providers should provide preliminary positive test results before confirmatory results are available in situations where tested persons benefit. When additional rapid tests become available for use in the United States, the PHS will re-evaluate algorithms using specific combinations of two or more rapid tests for screening and confirming HIV infection.

13. 1 st and 2 nd Generation EIA Plasma/serum (1 h/37 o C) Plasma/serum Color reagent Enzyme- detection Antigen coated well 1 st - Viral lysate 2 nd – Recombinant proteins or synthetic peptides IgG HIV antibody enzyme anti-human IgG Detects HIV IgG if present

14. 3 rd Generation “Sandwich” EIA Antigen coated well: Recombinant proteins or synthetic peptides Enzyme- detection Color reagent Plasma/serum HIV antibody enzyme HIV antigen Detects HIV IgM or IgG if present IgG IgM

15. 4 th Generation Combo EIA Color reagent Enzyme- detection HIV antigen …Or 2 Fluorescent labels allow independent detection of antigen or antibody p24 antibody Plasma/serum HIV antibody p24 antigen HIV antigen p24 antibody Detects HIV antibody or p24 antigen if present Coated well:

16. Detection of HIV by Diagnostic Tests 0 1 2 3 4 5 6 7 8 9 10 Symptoms p24 Antigen HIV RNA HIV EIA* Western blot Weeks Since Infection *3 rd generation, IgM-sensitive EIA After Fiebig et al, AIDS 2003; 17(13):1871-9 *2 nd generation EIA *viral lysate EIA

17. EIAs Used by Public Health Labs FDA approval date % used by PHL labs, 2004 Vironostika HIV-1 Microelisa 198763% Abbott HIVAB HIV-1/2 199220% Genetic Systems rLAV 199820% Gen Sys HIV-1/HIV-2 200018% Gen Sys HIV-1/2 Plus O 200310% Vironostika HIV-1 Plus O 20032% viral lysate EIA

18. EIAs Used by Public Health Labs FDA approval date % used by PHL labs, 2004 Vironostika HIV-1 Microelisa 198763% Abbott HIVAB HIV-1/2 199220% Genetic Systems rLAV 199820% Gen Sys HIV-1/HIV-2 200018% Gen Sys HIV-1/2 Plus O 200310% Siemens 1/O/2 eHIV 2006 viral lysate EIA2 nd generation EIA 3 rd generation, IgM-sensitive EIA

19. Changes in Availability of EIAs FDA approval date % used by PHL labs, 2004 Vironostika HIV-1 Microelisa 198763% Abbott HIVAB HIV-1/2 199220% Genetic Systems rLAV 199820% Gen Sys HIV-1/HIV-2 200018% Gen Sys HIV-1/2 Plus O 200310% Bayer 1/O/2 eHIV 2006 viral lysate EIA2 nd generation EIA 3 rd generation, IgM-sensitive EIA

20. FDA approval date % used by PHL labs, 2004 Vironostika HIV-1 Microelisa 198763% Abbott HIVAB HIV-1/2 199220% Genetic Systems rLAV 199820% Gen Sys HIV-1/HIV-2 200018% Gen Sys HIV-1/2 Plus O 200310% Siemens 1/O/2 eHIV 2006 viral lysate EIA2 nd generation EIA 3 rd generation, IgM-sensitive EIA Changes in Availability of EIAs

21. 2007 State of the Art

22. Uni-Gold Recombigen Multispot HIV-1/HIV-2 Reveal G3 OraQuick Advance Clearview Complete HIV 1/2 Clearview HIV ½ Stat Pak

23. Uni-Gold Recombigen Multispot HIV-1/HIV-2 Reveal G3 OraQuick Advance Clearview Complete HIV 1/2 Clearview HIV ½ Stat Pak

24. Negative HIV-1 & HIV-2 Positive Peptide HIV-2 Recombinant HIV-1 Peptide HIV-1 Reactive Control

25. The ADVIA ® Centaur™ Random Access HIV 1/O/2 Enhanced (EHIV)

26. HIV 1/O/2 Enhanced (EHIV) Multiple epitopes 3 rd generation “sandwich” format Random access EIA FDA-approved July 2006

27. On-board Refrigeration of 30 Different Assays

28. STAT sample requests without pausing Results in ~60 minutes

29. APTIMA HIV-1 RNA Qualitative Assay Gen-Probe Incorporated  Target capture specimen processing  Transcription-Mediated Amplification (TMA)  Dual Kinetic Assay  FDA approved September 2006

30. Qualitative RNA Assay: Intended Use Aid to HIV-1 diagnosis Diagnosis of acute HIV-1 infection in antibody- negative persons Confirmation of HIV-1 infection in antibody- positive persons when it is reactive

31. Clinical Syndrome of Acute HIV 40-90% develop symptoms of Acute HIV 50%-90% with symptoms seek medical care Of those diagnosed with Acute HIV, 50% of patients seen at least 3 times before diagnosis - Kahn et al, NEJM 1998 - Weintrob et al, Arch Int Med 2003

32. Pooled RNA Screening for Early HIV Infection

33. 1 Screening Pool Individual specimens 10 Pools of 10 A B C D E F G H I J A B C D E F G H I J 100 (HIV antibody negative) Pooled NAAT Screening for Early HIV Infection

34. A Individual NAAT testing on 10 specimens 10 pools of 10 tested with NAAT Screening Pools of 100 specimens tested with NAAT Resolution Testing

35. NAAT + - F/U Testing (Ab+NAAT) - + Acute HIVHIV Negative - EIA/WB + HIV antibody positive NC Pooled RNA Screening Program 109,250 clients Nov 02 – Oct 03 583 (0.5%) 23 (0.02%) 25 108,632 (99.5%)

36. 3-Test Algorithm: Antenatal Women Determine n=1179 HIV- HIV+ n=999 (85%) Confirm w/Genie II n=177 (15%) n=169 (95.5%) Capillus n=8 (4.5%) n=8 (100%)n=0 Koblavi-Deme et al, J Clin Micro 2001

37. 3-Test Algorithm, Uganda EIA/WB Positive EIA/WB Negative Rapid Test Positive 295166129 Rapid Test Negative 122041218 (Determine – Unigold – Stat Pak) Sensitivity: 97.6% Specificity: 90.4% - Gray et al, BMJ 2007

38. Current Assays with 15 Seroconverter Panels 185 specimens from 15 seroconverters - Owens et al, CDC unpublished data

39. WB positive Procleix RNA Abbott HIVAB ½ (3 rd ) BioRad HIV-1/2+O (3 rd ) GS rLav (2 nd ) Multi-Spot Reveal G2 Vironostika HIV-1 (1 st ) OraQuick Days before Western blot positive when 50% of Specimens Reactive Uni-Gold WB Indeterminate 0 510 152025 Current Assays with 15 Seroconverter Panels 185 specimens from 15 seroconverters - Owens et al, CDC unpublished data

40. Dilemmas Screening in low prevalence False-positive Western blots? What metric?

41. Pediatric Diagnosis

42. Last false negative= 7 weeks (43-49 days)

43. Last False Negative = 11 weeks 11 (71-77 days)

44. Interpreting Test Results Negative likelihood ratio: likelihood of negative test when disease is present likelihood of negative test when disease is absent - Ratio of false negative rate specificity { true negative rate}

45. Interpreting Test Results Negative likelihood ratio: - Does not depend on prevalence - Ratio < 0.1 = convincing diagnostic evidence Ratio < 0.2 = strong diagnostic evidence - Can be used with continuous variables (viral load)