1. Ricardo da Silva de Souza – August 2006 Rapid and Efficient Acute HIV Detection by 4th Generation Ag/Ab ELISA R. De Souza 1, C. Pilcher 2, S. Fiscus 2, A. Cachafeiro 2, M. Kerkau 2, L. Scherer 3, R.D. Sperhacke 4, M. Silva 4, S. Castro 3, L.F. Brigido 5, R. Ryder 2 1 Universidade de Caxias do Sul, Laboratorio de Pesquisa em HIV/AIDS, Caxias do Sul, Brazil, 2 University of North Carolina at Chapel Hill, Center for AIDS Research, Chapel Hill, United States, 3 Laboratório Central do Município de Porto Alegre, Porto Alegre, Brazil, 4 Fundação Estadual de Pesquisa e Produção em Saúde, Porto Alegre, Brazil, 5 Instituto Adolfo Lutz, Divisão de Biologia Médica, Sao Paulo, Brazil

2. Ricardo da Silva de Souza – August 2006 Acute HIV infections (first 2-3 months) are estimated to account for as much as half of all HIV transmission (Wawer at al JID 2005) They represent 0-10% of detectable infections presenting for HIV testing Real-time recognition of acute infections creates opportunities for highly targeted treatment, prevention and surveillance activities Detection of Acute HIV

3. Ricardo da Silva de Souza – August 2006 “Detuned” assays can identify recent seroconversion, but with a 1-2 month delay from infection. These also do not identify additional cases over routine antibody tests. Real-time diagnosis of acute HIV depends on the identification of HIV antigens (e.g., p24) or nucleic acids (NAAT) in the absence of HIV antibodies. Detection of Acute HIV

4. Ricardo da Silva de Souza – August 2006 HIV NegativeEstablished HIV Positive Ab screen NAAT screen + - + Possible Acute HIV - The Gold Standard for Acute Screening is RNA Group Testing of Ab - Specimens Ab confirm + - Pilcher, CD et al. JAMA 2002;288:216-221

5. Ricardo da Silva de Souza – August 2006 NAAT is highly sensitive and with pooling, may be made specific. However, even pooled NAAT may be inefficient in high prevalence areas (>5%) and is technically demanding. ‘Fourth generation’ HIV ELISAs detect both antigen and antibody simultaneously –Easy to perform –Equipment available in most HIV laboratories Testing to Identify Acute HIV

6. Ricardo da Silva de Souza – August 2006 Commercial Assays Comparative Timing of Detection of Acute HIV Infection = combined antigen-antibody= immunometric = Class specific antibody capture = antiglobulin / indirect Source HPA -UK

7. Ricardo da Silva de Souza – August 2006 The AMPLIAR Program Coordinated clinics using testing algorithm for the detection of subtype C and B acute HIV in south Brazil Long term objective is to reduce HIV transmission – real time behavioral intervention, contact tracing Development of a long term prospective cohort of recently infected patients Cleared by CONEP enrollment beginning August 2006 Present study is the pilot data evaluating laboratory alternatives for the identification of acute infection in the AMPLIAR Program

8. Ricardo da Silva de Souza – August 2006 Where we are Porto Alegre Brazil

9. Ricardo da Silva de Souza – August 2006 Primary Study Objective To evaluate an alternative laboratory algorithm for the detection of acute HIV infection suited to high prevalence, resource limited areas, we compared a fourth-generation EIA to HIV NAAT for detection of acute HIV infection

10. Ricardo da Silva de Souza – August 2006 Materials and Methods Study Design: Cross Sectional study Study population: 933 consecutive individuals seeking HIV testing at a public HIV clinic in southern Brazil between January and March 2004

11. Ricardo da Silva de Souza – August 2006 Study Definitions HIV infection – Ag/Ab + and one or more positive confirmatory tests (RNA or Ab +) Acute HIV Infection – Ag/Ab + and Ab - (one or more Ab negative test) or WB indeterminate

12. Ricardo da Silva de Souza – August 2006 Acute HIV Testing Strategy All subjects: Ag/Ab Screening (Genscreen Plus HIVAg-Ab Bio-rad) HIV Ag/Ab –: Confirmatory RNA Testing - 1:12 RNA pooling (Roche Amplicor 1.5) HIV Ag/Ab +: Confirmatory Ab Testing - Axsym HIV1/2 gO MEIA (Abbott) - IFA – (Biomanguinhos) - Determine Rapid test (Abbott) - WB (Genelabs) HIV Ag/Ab+, Ab-: RNA testing and Follow-up Ab testing

13. Ricardo da Silva de Souza – August 2006 Results Characterists of Study Population N (%) Age MeanMinMax Male560 (61)31,9564 Female373 (39)31,51157 Total933 (100)31,52

14. Ricardo da Silva de Souza – August 2006 FU 11 - (False Positive) LTF NAAT 1:12 0750 + - + - 933 - HIV Ag/Ab 183 750 32 NAAT 1:1 + QNS IF Biomanguinhos, Abbott MEIA, Determine, WB 1785 + -

15. Ricardo da Silva de Souza – August 2006 IF Biomanguinhos, Abbott MEIA, Determine, WB 1785 + NAAT 1:1 32 + QNS FU 11 NAAT 1:12 0750 + - + - HIV Ag/Ab Genscreen Biorad EIA 183 750 - - (False Positive) LTF

16. Ricardo da Silva de Souza – August 2006 182 IF Biomanguinhos, Abbott MEIA, Determine, WB 1785 + NAAT 1:1 32 + QNS FU 11 NAAT 1:12 0750 + - + - HIV Ag/Ab Genscreen Biorad EIA 183 750 - - (False Positive) LTF

17. Ricardo da Silva de Souza – August 2006 Five Ag/Ab + but Ab - Cases # FGEIA OD MEIAIFRTWB HIV NAAT Subtype Acute HIV FU data 1Pos (3.136) PosNegPos CBYes 2Pos (2.897) Neg PosNDQNS No 3Pos (0.442) Neg PosNDPosQNSYesLTF 4Pos (0.950) Pos IndetPosBYes 5Pos (1.2) Pos Neg QNS YesLTF

18. Ricardo da Silva de Souza – August 2006 + - + - Summary: 933 Consecutive Clients HIV Ag/Ab Genscreen Biorad EIA 182 confirmed 5 discordants 750 negatives Established HIV 4 acutes All NAAT- 1 false + IF Biomanguinhos Abbott MEIA Determine WB

19. Ricardo da Silva de Souza – August 2006 Summary I HIV prevalence: 19.5% Five discordant cases of Ab/Ag + but Ab- negative infection – 3 confirmed by NAAT – 1 QNS and lost to follow up – 1 false positive (seronegative during follow-up) Four cases of acute HIV –Acute HIV prevalence: 0.42%, all detected by FGEIA

20. Ricardo da Silva de Souza – August 2006 Summary II Pooled NAAT detected no additional cases in Ag/Ab – specimens Ag/Ab Performance: –Sensitivity: 100% (95CI;98.3,100.0) –Specificity: 99.8% (95CI; 99.2,100.0) FGEIA had one false positive –PPV: 99.4% (95CI; 96.9,100.0) –NPV: 100% (95CI; 99.6,100.0)

21. Ricardo da Silva de Souza – August 2006 Additional Cases Detected Using Fourth Generation EIA Additional Cases Detected n% Overall 42.18 MEIA21.09 IFA31.63 Determine20.5 WB21.09

22. Ricardo da Silva de Souza – August 2006 Conclusions In a clinic setting with high HIV prevalence and mixed subtypes in Southern Brazil, HIV antibody tests failed to detect 2% of confirmed HIV infections. Pooled NAAT offered no advantage over 4th generation EIA screening for detection of AHI. Simpler FGEIA screening detects AHI with sufficient accuracy to make acute HIV based prevention strategies feasible in resource limited settings.

23. Ricardo da Silva de Souza – August 2006 Estimated Number of Missed Cases Brazil 2000 - 2005 Year 200020012002200320042005 # HIV Tests paid by SUS2.999.5143.653.2524.104.7713.761.6533.706.0293.965.195 Prevalence ( 0,6%) 17.99721.92024.62922.57022.23623.791 Estimated missed cases ( 2%) 360438493451445476 Cumulative 7981.2911.7422.1872.663 Source: DATASUS – SIS 2006

24. Ricardo da Silva de Souza – August 2006 Acknowledgments Clinic Vila dos Comerciários VCT Simone Castro Luciene Scherer Gisele Preussler University of Caxias do Sul HIV/AIDS Research Lab Carla de Souza Vera Crippa Leonardo Motta Gustavo Borges Machiline Pagnella Daiana Pellenz Instituto Adolfo Lutz Luis Brigido University of North Carolina-Chapel Hill Chris Pilcher Susan Fiscus Ron Swanstrom Robert Ryder Support National DST AIDS Program NAID IRID NIAID CIPRA Fogarty International University of Miami Dr. Gail Shor Possner Special thanks Volunteer patients

25. Ricardo da Silva de Souza – August 2006 Obrigado Thank you