1. Alternatives for acute HIV testing: RNA, p24, heat dissociated p24 Christopher D. Pilcher, MD University of North Carolina at Chapel Hill Center for AIDS Research

2. Acknowledgements CIPRASUL-University of Caxias do Sul (UCS), Porto Alegre, Brazil Ricardo deSouza Emory University Frances Priddy Carlos del Rio The UNC Project- Lilongwe, Malawi Francis E.A. Martinson Peter N. Kazembe David Chilongozi Clement Mapanje Phyllita Kumwera Syze Gama Dave Namakwa NC DHHS Evelyn Foust J. Todd McPherson Rhonda Ashby Del Williams UNC-Chapel Hill Myron S. Cohen Peter A. Leone Irving F. Hoffman Susan A. Fiscus JoAnn Kuruc Lisa Hightow Matthew A. Price Joseph J. Eron, Jr William C. Miller Melissa Kerkau Priya Joshi Trang Q. Nguyen Kristen Hampton Marc Serre Paul Alabanza Amy James Brant Stalzer NIMH, NIDDK, HPTN, UNC Fogarty Center, UNC STD CRC, UNC CFAR

3. Acute HIV infections (first 2-3 months) are estimated to account for as much as half of all HIV transmission (Wawer JID in press) They represent 0-10% of detectable infections presenting for HIV testing, depending on the population Recognition of the acutely infected population creates opportunities for highly targeted treatment, prevention and surveillance activities Diagnosis requires modification of “standard” treatment algorithms Testing to Identify Acute HIV

4. HIV NegativeEstablished HIV Positive Ab screen NAAT screen + -+ HIV Indeterminate, Possible Acute HIV - 1.Ab testing up front— reflexed to NAAT if negative Ab confirm + -

5. HIV NegativeEstablished HIV Positive NAAT or Ag and Ab screen + HIV Indeterminate, Possible Acute HIV - 2. Simultaneous acute and Ab screening— reflexed to Ab confirmation if positive Ab confirm + -

6. Algorithms must include: A screening assay for acute HIV infection –EARLY sensitivity for viral nucleic acid or proteins –High specificity to reduce “false alarms” Screening and confirmatory testing for established HIV –LATER sensitivity will allow one to discern more acute infections from established infections Testing to Identify Acute HIV

7. Individual specimens Pools of 10 The gold standard for acute screening: RNA group testing of Ab- specimens Pilcher, CD et al. JAMA 2002;288:216-221

8. Pooling schema A B C D E Individual specimens N=100 10 Pools of 10 F G H I J A B C D E F G H I J

9. 1 Screening Pool 2-Stage Pooling Individual specimens N=100 10 Pools of 10 A B C D E F G H I J A B C D E F G H I J

10. A Individual testing on 10 specimens 10 pools of 10 screened 20 Screening Pools Tested N=2000 Resolution Testing

12. HIV Negative Established HIV Positive + + - HIV Indeterminate, Possible Acute HIV - NC DHHS STAT Program: all publicly funded VCT in NC (N~120,000 per yr) 90:10:1 pooling NucliSens NAAT Vironostika 1 st gen EIA WB

13. Established HIV Positive NAAT + + - F/U Testing (Ab+NAAT) - + Acute HIVHIV Negative - STAT Confirmatory Testing Vironostika 1 st gen EIA WB

14. Specimen Pooling/RNA Testing: NC’s STAT Program Of 109,250 previously negative clients tested 11/02- 10/03, 23/606 (4%) of HIV infections were acute 16/23 (67%) acutes were identified in STD clinic testing sites 1 pregnant female was identified NAAT PPV 90%; Specificity >99.99%

15. Pooling and NAAT: Logistical and Cost Issues Ideally suited to high throughput, centralized testing Most efficient at low prevalences of acute HIV Turnaround time: 10-14 d Cost reasonable –$3.63 per HIV test performed –$17,000 per acute index case identified

16. Evaluating Alternative Algorithms 3 studies conducted in high HIV incidence testing populations RNA group testing as the gold standard Evaluate different algorithms

17. Established HIV Positive NAAT + + - F/U Testing (Ab+NAAT) - + Acute HIVHIV Negative - STAT Testing Vironostika 1 st gen EIA WB

18. Established HIV Positive + + - + Acute HIVHIV Negative - p24 vs. Up24 vs. NAAT: n=1440 STD clients in Lilongwe, Malawi Determine RT Unigold RT - or discordant NAAT p24 Up24 F/U Testing EIA/WB

19. Lilongwe, Malawi; N=1,440 Overall, 575 cases of HIV detected 20 (3.5%) were WB-/indeterminate and seroconverted –12/16 (75%) detected by standard p24 Ag –16/19 (84%) detected by Up24 Ag –20/20 (100%) detected by NAAT

20. Performance of p24 and Up24? Fiscus, et al 12 th CROI Estimate(95% CI) Sensitivity p240.750(0.476, 0.927) Sensitivity Up240.842(0.604, 0.966) Specificity0.995(0.988, 0.999) LR+ p24161.8(58.5, 447.8) LR+ Up24181.7(67.0, 492.3) LR-0.25(0.11, 0.59)

21. p24 and post-test probability: Malawi STD clinic 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 00.0050.010.0150.030.0350.04 Prevalence Post-test Probability p24 Up24 Negative 0.0226 Expected post-test prob for p24+ (prev.023): 0.79

22. p24 and post-test probability: Urban US STD Clinic 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0 0.001 0.010.0150.030.0350.04 Prevalence Post-test Probability p24 Up24 Negative 0.0226 Expected post-test prob for p24+ (prev 0.001): 0.15

23. + - + - The 4 th generation EIA vs. NAAT N=933 VCT clients in Porto Alegre, Brazil Genscreen Biorad HIV Ag/Ab EIA + + HIV Negative 183 ESTABLISHED HIV 97.3% all HIV+ 5 ACUTE HIV 2.7 % all HIV+ NAAT - Biomanguinhos IF Abbott MEIA

24. 4 th generation EIA performance: preliminary observations 5/5 (100%) acutes detected consistent with previous observations of p24 sensitivity Specificity for acute HIV needs further evaluation

25. Summary: p24 performance Algorithms based on p24-based screening (std p24, Up24, 4 th generation EIA) have adequate sensitivity and specificity for acute HIV screening in settings with ~1% or greater prevalence of acute HIV infection (e.g., African STD clinics) However, the risk of false positives and costs of individual re-testing all Ab negative specimens may be unacceptable in most US routine testing populations

26. Laboratory Detection of HIV from Pilcher CD, et al. JCI 2004 0 1 2 3 4 5 6 7 8 9 10 Symptoms (~60%) p24 Antigen HIV RNA HIV Ab Tests Weeks Since Infection 1 st generation (viral lysate) EIAs

27. Laboratory Detection of HIV from Pilcher CD, et al. JCI 2004 0 1 2 3 4 5 6 7 8 9 10 Symptoms (~60%) p24 Antigen HIV RNA HIV Ab Tests Weeks Since Infection 3 rd generation EIA, MEIA, rapid tests 1 st generation (viral lysate) EIAs

28. + - + - deSouza, et al unpublished N=933 VCT clients in Porto Alegre, Brazil Genscreen Biorad HIV Ag/Ab EIA + + HIV Negative 183 ESTABLISHED HIV 97.3% all HIV+ 5 ACUTE HIV 2.7 % all HIV+ NAAT - Biomanguinhos IF Abbott MEIA

29. 68 ESTABLISHED HIV (1 false- rLAV assay) 95.8% all HIV+ NAAT + + - or indeterm. F/U Testing EIA/WB - + HIV negative - Priddy, et al. 12 th CROI n=2,202 VCT and STD clients in Atlanta, GA Gen Systems HIV ½+O EIA WB 3 ACUTE HIV 4.2 % all HIV+

30. 555 ESTABLISHED HIV (2 false – on RTs) 96.5% all HIV+ + 20 ACUTE HIV 3.5% all HIV+ HIV Negative Fiscus, et al. 12 th CROI n=1440 STD clients in Lilongwe, Malawi Determine RT Unigold RT - or discordant (n=22) + - + - NAAT F/U Testing EIA/WB

31. Rapid Test Sensitivity In a Malawi STD clinic with a high ratio of incidence-to- prevalence, the sensitivity of rapid tests for HIV infection varied with their sensitivity for acute HIV –Determine: detected 559 of 575 (97.2%) –Unigold: detected 554 of 575 (96.3%) Performance should be similar in a US STD clinic or MSM testing population 6 (30%) of 20 WB -/indeterminate acute HIV infections were Determine+ but Unigold – Choice of the rapid test, or series of rapid tests used, can significantly affect the ability to detect early infections

32. Summary: Ab Screening Choices Even maximum-sensitivity antibody tests will miss between 2-5% of detectable HIV infections in high risk testing populations If acute screening is to be done, maximum early- sensitivity Ab tests are neither necessary nor desirable If acute screening is not to be done, choice of Ab assay can significantly affect HIV detection in testing populations with high proportion of incident cases