Performance of Dual Immunoassay HIV Testing Strategy Using Bio Rad Genetic Systems and Multispot/OraQuick Advance

1. Performance of Bio-Rad Genetic Systems HIV-1/HIV-2 Plus O EIA Followed by Multispot or OraQuick Advance in a Dual Immunoassay HIV Testing Strategy Laura Wesolowski, PhD* S. Michele Owen, PhD Debra Candal, BS Susan Phillips, BS Steve Ethridge, BS, MT (ASCP) Duncan MacKellar (MA, MPH) *Diagnostic Applications Team, Behavioral and Clinical Surveillance Branch, Centers for Disease Control and Prevention

2. Objectives To assess the sensitivity and specificity of a dual immunoassay testing strategy using Bio-Rad HIV- 1/HIV-2 Plus O EIA (A1) followed by Multispot HIV- 1/HIV-2 rapid test (A2) for HIV-1 diagnosis To describe the concordance of A2 test (Multispot) when run in duplicate To assess the sensitivity of a dual immunoassay testing strategy using Bio-Rad EIA (A1) and OraQuick Advance (A2) rapid test for HIV-1 diagnosis

3. Background Lack of performance data for EIA followed by rapid test Concern about similarity of antigens in EIAs and rapid tests on market may contribute to dual false positives Bio-Rad EIA 3 rd generation EIA (detects IgG and IgM) Serum or plasma Sensitivity: 100% (99.84-100%) Specificity: 99.89% (99.83-99.96%)

4. Background: Multispot Serum or plasma Sensitivity Serum and plasma 100% (99.94-100%) Specificity Serum 99.93% (99.79-100%) Plasma 99.91% (99.77-100%)

5. Background: OraQuick Advance - Whole blood or plasma - Sensitivity Whole blood 99.6% (98.5 - 99.9) Plasma 99.6% (98.9-99.9) - Specificity Whole blood 100% (99.7-100%) Plasma 99.9% (99.6-99.9%)

6. Methods: HIV-1 uninfected specimens HIV-uninfected specimens: plasma samples from uninfected blood donors EIA negative (Abbott HIV AB HIV-1/HIV-2 (rDNA)) Pooled HIV-1 PCR-negative (Roche Ampliscreen) Uninfected specimens tested with Bio-Rad EIA and Multispot (in duplicate) at CDC. Not tested with OraQuick yet.

7. Methods: HIV-1 infected specimens HIV-1 infected specimens: Serum Western blot positive specimens from CDCs Validating Supplemental Testing to Confirm Preliminary Positive Rapid HIV Tests CDC IRB Approved: Feb. 2006 Enrollment began March to June 2006 Enrollees from 6 study sites AIDS Research Consortium of Atlanta, GA Denver Metro Health (STD) clinic Philadelphia FIGHT, Jonathan Lax HIV Clinic Howard Brown Health Center, Chicago, Gay & Lesbian Health Clinic Louisville Hospital, Louisville, KY, WINGS STD Clinic University of MD Baltimore, Evelyn Jordan Center (HIV Clinic) Reference laboratory: University of MD

8. Methods: HIV-1 infected specimens Inclusion criteria: Previously diagnosed with HIV infection No antiretrovirals within 3 months Not pregnant 18 - 55 years Understand English Infected specimens tested at UMD (Bio-Rad EIA) and CDC (Multispot in duplicate) OraQuick Advance conducted at study site using whole blood (not in duplicate)

9. Methods: Outcomes Specificity and sensitivity for Bio-Rad EIA (A1) followed by Multispot (A2) individually and together in dual test strategy Concordance of Multispot results run in duplicate Sensitivity for Bio-Rad EIA (A1) followed by OraQuick Advance (A2) individually and together in dual test strategy Specificity=True negative/(true negative + false positive) Sensitivity=True positive/(true positive + false negative)

10. Results: Specificity Bio-Rad EIA and Multispot Among 302 HIV-uninfected persons: 4 false positive on Bio-Rad EIA (specificity=298/302=98.7%) 7 false positive on Multispot (specificity=295/302=97.7%) 0 false positive on both Bio-Rad EIA and Multispot

11. Strategy 3. HIV-1/2 dual immunoassay for 4 initially false positive on Bio-Rad EIA (truly uninfected) *Must be a different EIA, CIA, or non-waived rapid test depending on laboratory setting (for A2 only) If window period infection is suspected based on risk assessment or discordant testing, refer to Acute HIV Infection Testing, Strategy 4 HIV-2 Testing; Strategy 5, if applicable A1 BioRad EIA A1 (+) Negative for HIV-1 and HIV-2 antibodies A2 Multispot in duplicate Presumptive positive for HIV-1 or HIV-2 antibodies; requires medical follow-up for further evaluation and testing Inconclusive for HIV antibodies; request plasma redraw for NAAT Requires medical follow-up for further evaluation and testing Repeat A1 in duplicate A1 (-) A2 (- -) A2 (++ or + -) A1 (- -) A1 (++ or + -)

12. Results: Specificity Bio-Rad EIA and Multispot Strategy specificity excluding those with inconclusive results=300/300=100% 2 uninfected persons eventually get correct results, but in the interim will be told that their results are inconclusive and have to wait for correct results after NAAT testing

13. Results: Sensitivity Bio-Rad EIA and Multispot Among 1065 HIV-infected persons: 0 false negative on Bio-Rad EIA (sensitivity=1065/1065=100%) 1 false negative on Multispot (sensitivity=1064/1065=99.9%) 0 false negative on both Bio-Rad EIA and Multispot

14. Strategy 3. HIV-1/2 dual immunoassay for 1 person false negative on Multispot (truly infected) *Must be a different EIA, CIA, or non-waived rapid test depending on laboratory setting (for A2 only) If window period infection is suspected based on risk assessment or discordant testing, refer to Acute HIV Infection Testing, Strategy 4 HIV-2 Testing; Strategy 5, if applicable A1 BioRad EIA A1 (+) Negative for HIV-1 and HIV-2 antibodies A2 Multispot in duplicate Presumptive positive for HIV-1 or HIV-2 antibodies; requires medical follow-up for further evaluation and testing Inconclusive for HIV antibodies; request plasma redraw for NAAT Requires medical follow-up for further evaluation and testing Repeat A1 in duplicate A1 (-) A2 (- -) A2 (++ or + -) A1 (- -) A1 (++ or + -)

15. Results: Sensitivity Bio-Rad EIA and Multispot Strategy sensitivity excluding those with inconclusive results=1064/1064=100% 1 HIV-infected person likely have inconclusive results and would have to await NAAT results

16. Results: Multispot duplicates All Multispots same result on duplicate as when initially run (i.e. non-reactive on both test runs or reactive on both test runs) Testing in duplicate did not provide any additional information on infection status for those with false positive or false negative results

17. Results: Sensitivity Bio-Rad EIA and OraQuick Among 1065 HIV-infected persons: 0 false negative on Bio-Rad EIA (sensitivity=1065/1065=100%) 0 false negative on OraQuick (whole blood) (sensitivity=1065/1065=100%) 0 false negative on both Bio-Rad EIA and OraQuick Strategy sensitivity=1065/1065=100%

18. Conclusions Dual test strategy using Bio-Rad EIA and Multispot has high sensitivity and specificity. However, some persons would receive inconclusive results and require additional testing to get correct result. Conducting the secondary screening test in duplicate does not appear to be beneficial. Dual test strategy using Bio-Rad EIA and OraQuick has high sensitivity, though specificity should be evaluated.

19. Next steps Continue testing samples with Bio-Rad EIA, Multispot and OraQuick Obtain challenge specimens (false EIA positive, NAAT negative) to challenge dual immunoassay strategy

20. Acknowledgements PIs & staff from 6 study sites University of MD Laboratory of Viral Diagnostics MDC Associates, Joanne Lebrun CDC Bernard Branson Maxia Dong Tim Granade Dollene Hemmerlein Sheryl Lyss