1. CDC HIV Diagnostics Conference December 5-7, 2007 Sensitivity of OraQuick and Early Generation Enzyme Immunoassay (EIA) within a Pooled HIV Nucleic Acid Amplification Testing (HIV NAAT) Program J Stekler 1,2, PD Swenson 2, RW Coombs 1, J Dragavon 1, RW Wood 1,2, MR Golden 1,2 From the 1 University of Washington, Seattle, WA and 2 Public Health - Seattle & King County (PHSKC), WA

2. Objective ●To describe the sensitivity of different HIV antibody tests within an HIV NAAT program ● To understand that HIV test sensitivity may vary in populations with different rates of HIV acquisition and testing intervals

3. Background ● 9/2003: pooled NAAT testing began ● Targeted HIV NAAT: men who have sex with men (MSM) prevalence = 16% incidence = 2.7 per 100 person-years ● Sites: rapid Ab testing: CBO, bathhouses, or high-risk at STD 1) UAI w/ HIV-positive or unknown serostatus partner, 2) bacterial STD, or 3) methamphetamine/popper use in last year standard Ab testing (EIA): mostly “low-risk” STD clients

4. Methods: HIV testing ● Rapid testing OraQuick (Orasure Technologies, Inc) fingerstick whole blood or oral fluids ● EIA Vironostika HIV-1 Microelisa System (Organon Teknika Corp) Genetic Systems rLAV EIA (Bio-Rad Laboratories) ● HIV NAAT: 30 person pools (manually combined) 9/03-1/05: Procleix HIV-1 Discriminatory Assay (Gen-Probe Inc) Individual NAAT also performed 2/05-present: independently validated real-time RT-PCR assay Individual NAAT when acute HIV suspected

5. Methods: Retrospective testing ● Serum stored for confirmatory testing and quality control ● First OraQuick-neg/EIA-pos tester identified in 11/05 by NAAT ● After 11/05, OraQuick-neg MSM tested by EIA before NAAT to reduce time/costs for pooled HIV NAAT to decrease time to receive HIV-positive results Other retesting 3rd gen. EIA (HIVAB HIV-1/HIV-2 (rDNA) EIA, Abbott Laboratories) Other rapid tests Clearview HIV 1/2 StatPak (Inverness Medical Prof. Diagnostics) Uni-Gold Recombigen HIV-1 Test (Trinity BioTech)

6. False negative EIA and OraQuick results: Data Needs for Strategy #3-1 Standard testers (MSM) N= 7437 # HIV-positiveCumulative “Sensitivity”* EIA148 (2.0%)14893% NAAT10 (0.1%)15899% Rapid testers (MSM) N= 5460 # HIV-positiveCumulative “Sensitivity” OraQuick123 (2.3%)12383% EIA10 (0.2%)13389% NAAT16 (0.3%)149100% *False-positive HIV NAAT = 0, False-negative HIV NAAT = 1 Individual NAAT: no additional cases in first 16 months of program

7. False positive test results (EIA): Data Needs for Strategy #3-2 Standard testers N= 7437 HIV-positiveHIV-negative Sensitivity = 93.2% Specificity = 99.9% PPV = 97.4% NPV = 99.8% EIA-positive1514 EIA-negative117271 EIA-positive and WB “indeterminate” 1) Multiple “faint” bands, known HIV-pos = true positive 2) “Faint” gp 120/160 – later confirmed =true positive 3) “Faint” p24, gp55, gp160 – confirmed = true positive 4) p40, RNA negative = false positive 5) p66, RNA negative = false positive 6) gp160, RNA negative (vaccine study) = false positive 7) p18, RNA negative, 3 month f/u negative =false positive

8. False positive test results (OraQuick): Data Needs for Strategy #3-2 Rapid testers N= 5455 HIV-positiveHIV-negative Sensitivity = 82.8% Specificity = 99.9% PPV = 97.7% NPV = 99.5% OraQuick-positive1253 OraQuick-negative265301 Unconfirmed OraQuick results 1) Refused blood draw (n=5) 2) Confirmatory test elsewhere = true positive 3) Indeterminate WB (p18, p55, gp160)= true positive 4) Indeterminate rapid test = false positive (n=2) 5) Negative EIA/NAAT = false positive

9. Results: retesting frozen sera from MSM with acute HIV infection # with acute HIV infection HIV-positive on retesting by 3 rd generation EIA3 of 11 (27%) by OraQuick0 of 6

10. Number and % with discordant Ab test results: Data Needs for Strategy #3-3 Rapid testers N= 5455 EIA-posEIA-neg HIV-infected 0/3 (0%) EIA-/OQ+ 10/11 (90.9%) EIA+/OQ- (vaccine study participant) OraQuick-pos1253 OraQuick-neg115317

11. Results: OraQuick-negative/EIA-positive testers EIAS/CO WB 1)Vironostika6.2p24, p51, gp160 2) Vironostika 2.4p24, p55, gp160 3) rLAV EIA4.7 p24, p55, gp160 4) rLAV EIA 5.2p18, p24, p55, gp160 5) rLAV EIA 6.4p24, p65, gp160 6) rLAV EIA 4.7p24, faint gp160 (indet) 7) rLAV EIA 4.7p24, gp160 8) rLAV EIA 4.3p24, p55, gp160 9) rLAV EIA 6.6p24, p55, gp120/160 10) rLAV EIA6.0p24, p32, gp41, p51, p55, gp120/160

12. Results of rapid HIV antibody tests on frozen sera from MSM with early HIV infection “weak positive”: barely visible (specimen test line < positive control) “positive”: visible (test line ≥ positive control but < external control) “strongly positive”: easily visible (test line reactivity ≥ external control) ClearviewOraQuickUni-Gold Clinical Cases OraQuick-neg/EIA-pos (n=5) Positive (5) Weak positive (2) Positive (2) Strong positive (1) Weak positive (2) Positive (3) EIA-pos/gp41-neg (n=5) Positive (4) Strong positive (1) Positive (4) Strong positive (1) Weak positive (2) Positive (3) EIA-pos/gp41-pos (n=5) Positive (1) Strong positive (4)Strong positive (5)

13. Results (incidence/time) Test “sensitivity” by test frequency HIV TestQuarterlySemi-annuallyAnnually HIV Ab72%84%91% HIV NAAT86%93%96% Assumptions: 1) Timing of testing is uniform 2) Testing is independent of risk EIA window period = 35 days NAAT window period = 14 days

14. Conclusions 1) No HIV test has a sensitivity of 100%. HIV NAAT will appear to be more effective with less sensitive Ab tests. 2) OraQuick may be less sensitive than 1 st or 2 nd generation EIAs in early HIV infection. Other rapid tests did not clearly perform any differently on stored sera. 3) As testing increases, sensitivity decreases because of a greater likelihood of testing during the “ window period ”. It is relatively more important to use the most sensitive tests in populations with frequent HIV testing and high rates of HIV acquisition.

15. Acknowledgements Clients and providers at the Gay City Health Project, PHSKC STD Clinic, and other PHSKC testing sites. Mentors Ann Collier Matt Golden Funding: The HIV NAAT pilot study was partially supported by GenProbe Incorporated from 9/03-2/05. Grants: NIH K23 AI-65243 UW CFAR: NIH AI-27757