1. Incorporation of the Aptima® HIV-1 RNA Assay into Serodiagnostic and Rapid Test Confirmation Testing Algorithms to Resolve Discordant Serological Results M. Parker 1, R. Boromisa 1, T. Sullivan 1, M. San Antonio-Gaddy 2, A. Richardson-Moore 2 New York State Department of Health 1 Wadsworth Center, 2 AIDS Institute

2. Issue Confirmatory testing of EIA RR and Rapid test reactive specimens For neg. or indeterminate WB results: Follow-up specimens are requested In some cases, no follow-up specimen is sent, or HIV infection status cannot be resolved by follow-up specimen

3. NAAT Supplemental Test Testing strategies 1/2 addressed Two lab-based algorithms evaluated: –Serodiagnostic (EIA screening) –Rapid test referral for confirmation Data from 2006 and 2007 presented

5. Screening Assays A1 Serodiagnostic algorithm: –Genetic Systems HIV-1/HIV-2 + O (Bio-Rad) A1 Rapid test referral algorithm: –OraQuick Rapid Test (OraSure) –Uni-Gold Rapid Test (Trinity Biotech) Additional screening test: Multispot

6. Supplemental Tests B1 HIV-1 Western Blot –Genetic Systems HIV-1 WB (Bio-Rad) –Cambridge Biotech HIV-1 WB (Maxim) B2 Individual HIV-1 NAAT –Procleix HIV-1 RNA Qualitative Assay (’06) –Aptima HIV-1 RNA Qualitative Assay (’07) (both Gen-Probe)

7. Serodiagnostic Test Data

8. Serodiagnostic Algorithm: Initial Request Follow-up EIA - - Report: Negative Report: Positive EIA + EIA ++/+- Multispot Reactive Neg or Ind WB

9. Not detectedDetected NAAT Reactive Report: Positive Neg or IndWB Revised Algorithm: NAAT Added Report: RNA – Follow-up only if risk Report: RNA + Possible AHS

10. Serodiagnostic Testing 20062007Total Specimens tested52,66510,90163,556 EIA RR565149714 WB POS467 (82.7%)129 (86.6%)596 (83.5%) WB IND20 (3.5%)6 (4.0%)26 (3.6%) WB NEG78 (13.8%)14 (9.4%)92 (12.9%)

11. Data needs 7: # and % of false positive supplemental tests for HIV-1 as confirmed by additional follow-up testing of original or follow-up specimens 8: # and % of false-negative and false indeterminate supplemental tests for HIV-1 as confirmed by additional follow-up testing (including NAAT) of original or follow-up specimens

12. Serodiagnostic Testing 20062007Total Specimens tested52,66510,90163,556 EIA RR565149714 WB POS467 (82.7%)129 (86.6%)596 (83.5%) WB IND20 (3.5%)6 (4.0%)26 (3.6%) IND NAAT tested186 24 WB NEG78 (13.8%)14 (9.4%)92 (12.9%) NEG NAAT tested7214 86

13. Serodiagnostic/NAAT Data 20062007TOTAL RNA +7 (38.9%)0 (0%)7 (29.2%) RNA -10 (55.6%)6 (100%)16 (66.7%) Invalid1 (5.6%)0 (0%)1 (4.2%) TOTAL18624 WB Indeterminate

14. WB Indeterminate/NAAT Positive 7 RNA reactive: –EIA s/co mean 7.75 (range = 3.1-10.0; sd 2.12) –6 MS HIV-1 + –1MS neg (EIA = 9.0, WB bands = 24+/-) –RNA NR : EIA s/co mean 1.56 (1.1-3.1; sd 0.5) 20062007TOTAL RNA +7 (38.9%)0 (0%)7 (29.2%) RNA -10 (55.6%)6 (100%)16 (66.7%) Invalid1 (5.6%)0 (0%)1 (4.2%) TOTAL18624

15. Serodiagnostic/NAAT Data WB Negative 20062007TOTAL RNA +1 (1.4%)0 (0%)1 (1.2%) RNA -65 (90.3%)13 (92.9%)78 (90.7%) Invalid6 (8.3%)1 (7.1%)7 (8.1%) TOTAL721486

16. WB False Negative or RNA False Positive? 1 RNA detected: –EIA s/co 1.4; MS neg; No bands –Specimen 30 days prior: EIA +, MS -, WB -, RNA – –Follow-up rapid test referral: MS -, WB neg 20062007TOTAL RNA +1 (1.4%)0 (0%)1 (1.2%) RNA -65 (90.3%)13 (92.9%)78 (90.7%) Invalid6 (8.3%)1 (7.1%)7 (8.1%) TOTAL721486

17. Rapid Test Referral Data

18. Rapid test referral algorithm: 2006 Report: Positive Reactive HIV-1 Reactive HIV-1 WB Multispot Neg or Ind Request Follow-up HIV-1 R/NR

19. Rapid test referral algorithm: 2007 EIAMultispotWB Follow-up only if risk factors Non-reactive Report: Positive Reactive Rapid reactive ReactiveNeg or Ind RNA test scheduled R or NR

20. Not detectedDetected NAAT Reactive Report: Positive Neg or IndWB Revised Algorithm: NAAT Added Report: RNA – Follow-up only if risk Report: RNA + Possible AHS

21. Rapid Test Referral Data 20062007Total Referrals253291544 WB POS162 (64.0%)202 (69.4%)364 (66.9%) WB IND14 (5.5%)10 (3.4%)24 (4.4%) WB NEG77 (30.4%)79 (27.1%)156 (28.7%)

22. Rapid Test Referral Data 20062007Total Referrals253291544 WB POS162 (64.0%)202 (69.4%)364 (66.9%) WB IND14 (5.5%)10 (3.4%)24 (4.4%) IND NAAT tested11718 WB NEG77 (30.4%)*79 (27.1%)156 (28.7%) NEG NAAT tested481058 *In 2007, EIA test added Only EIA reactives rec’d NAAT (10 of 79)

23. Rapid Test Referral Data 20062007TOTAL RNA +7 (63.6%)7 (100%)14 (77.8%) RNA -3 (27.3%)0 (0%)3 (16.7%) Invalid1 (9.2%)0 (0%)1 (5.6%) TOTAL11718 WB Indeterminate

24. WB Indeterminate/NAAT Positive 20062007TOTAL RNA +7 (63.6%)7 (100%)14 (77.8%) RNA -3 (27.3%)0 (0%)3 (16.7%) Invalid1 (9.2%)0 (0%)1 (5.6%) TOTAL11718 7 RNA detected in 2007: –EIA mean s/co 10.0 (range 9.4-10.1; sd 0.45) 7 MS + 7 RNA detected in 2006; EIA on 4 retrospectively –Mean s/co 9.9 (sd = 0) 7 MS +

25. Rapid Test Referral Data WB Negative 20062007TOTAL RNA +1 (2.1%)1 (10.0%)2 (3.5%) RNA -45 (93.7%)9 (90.0%)54 (93.1%) Invalid2 (4.2%)0 (0%)2 (3.5%) TOTAL481058

26. WB False Negative or RNA False Positive? 1 RNA detected in 2007: –EIA s/co 10.7; MS + (no further follow-up) 1 RNA detected in 2006 –EIA s/co 0.227; MS – (no further follow-up) 20062007TOTAL RNA +1 (2.1%)1 (10.0%)2 (3.5%) RNA -45 (93.7%)9 (90.0%)54 (93.1%) Invalid2 (4.2)0 (0%)2 (3.5%) TOTAL481058

27. Considerations Expense of the RNA test (Aptima™) –Consistent specimen load from ongoing PHI study ensured frequent Aptima™ runs Plasma specimens required for Aptima™ EIA screening of rapid reactive specimens greatly reduced # of RNA tests run due to negative or indeterminate WB

28. Concluding Remarks NAAT is being used to resolve discordant serological test results at NYSDOH –More data are needed to assess the costs/benefits of NAAT supplemental testing Of the RNA reactive results we obtained, at least one was a probable false positive –More data are needed to fully assess the specificity of NAAT in this setting

29. Acknowledgements Diagnostic HIV Lab Judith Wethers Joe Schwendemann The technical staff CDC/PHI Study Team –Pragna Patel –Duncan Mackellar –Steve Etheridge

30. Western Blot WB interpretation: –Consortium for Retrovirus Serology Standardization 2 bands: –p24 or p31, plus –gp41 or gp120/gp160

31. Description of Multispot (BioRad) HIV-1/HIV-2 Reactive Control Spot and Test Spots 1.Procedural control:Anti-human IgG (goat) 2.HIV-2 Peptide: Peptide representing the HIV-2 virus gp36 3.Recombinant HIV-1: Recombinant gp41 (gp41rDNA) 4.HIV-1 Peptide: Peptide representing the HIV-1 virus gp41