1. Limitations of HIV Antibody Testing in a Population with High Incidence of HIV Infection Joanne Stekler 1,2, Paul D. Swenson 1,2, Robert W. Coombs 1, Joan Dragavon 1, Catherine A. Brennan 3, Sushil G. Devare 3, Ana Vallari 3, Priscilla Swanson 3, Robert W. Wood 1,2, Matthew R. Golden 1,2 1 University of Washington, Seattle, WA, USA 2 Public Health - Seattle & King County, WA, USA 3 Abbott Diagnostics, Abbott Park, IL, USA METHODS CONCLUSIONS RESULTS BACKGROUND ● Recent Centers for Disease Control and Prevention recommendations aim to identify the estimated 21% of HIV-infected persons unaware of their HIV status. ● HIV antibody tests, antigen-antibody combination assays, and HIV nucleic acid amplification testing (HIV NAAT) have different abilities to detect HIV persons in the period after HIV acquisition. ● Public Health – Seattle & King County (PHSKC) began a pooled HIV NAAT program in 2003, targeted to men who have sex with men (MSM). OraQuick-negative/EIA-positive test results LIMITATIONS Poster #990 ABSTRACT Background The Centers for Disease Control and Prevention recently recommended expanding HIV antibody testing. However, HIV nucleic acid amplification tests (NAAT) are more sensitive than antibody tests in acute and early infection, when persons are highly infectious. Methods From 9/03-6/08, Public Health-Seattle & King County offered HIV antibody testing to men who have sex with men (MSM) using the OraQuick Advance Rapid HIV-1/2 Antibody Test (OraSure) on oral fluid or finger-stick blood specimens or using an enzyme immunoassay (EIA) [Vironostika HIV-1 Microelisa System (bioMerieux) or Genetic Systems rLAV EIA (Bio-Rad)]. An EIA was used to confirm reactive rapid tests and to screen specimens from OraQuick-negative MSM prior to pooling for HIV NAAT. We used frozen sera from 16 EIA-negative/NAAT-positive MSM to evaluate the ability of the 4th generation ARCHITECT HIV Ag/Ab Combo assay (Abbott Diagnostics, not available for sale in the US) to detect acute infection. HIV RNA levels were quantified using the Abbott RealTime HIV-1 assay. Results Overall, 328 (2.3%) of 14,005 specimens were HIV EIA-positive, and 36 (0.3%) EIA-negative MSM tested NAAT-positive. Among the 6811 OraQuick-negative specimens, 16 (0.2%) HIV-infected MSM were EIA-reactive, and 23 (0.3%) EIA-negative MSM were diagnosed with acute HIV infection through pooled HIV NAAT. 14 of the 16 OraQuick-negative/EIA-reactive testers had positive Western Blot results, and most had evidence of early HIV infection. Among rapid testers, OraQuick detected only 153 (80%) of 192 HIV-infected MSM, and OraQuick plus an EIA detected 169 (88%) infections detected by the program. The ARCHITECT HIV Ag/Ab Combo assay detected 15 (94%) of 16 MSM with acute HIV infection who had a median HIV RNA level of 6.6 (IQR 5.8-6.9) log10 copies/mL. The specimen with the non-reactive test had an HIV RNA level of 4.2 and 3.7 log10 copies/mL by the real-time RT-PCR and Abbott RealTime HIV-1 assays, respectively. Conclusions 1) OraQuick performed on oral fluids or finger-stick blood specimens may be less sensitive than early generation EIAs. 2) Our findings may not be generalizable to populations with lower HIV prevalence and incidence. 3) HIV NAAT should be integrated into HIV testing programs serving MSM and other high-incidence populations with frequent HIV testing, particularly when rapid antibody testing is employed. 4) 4th generation antigen-antibody combination assays may be a reasonably sensitive alternative to HIV NAAT. Summary of confirmed HIV test results (9/03-6/08) Study Population ● MSM tested for HIV through PHSKC: 1) PHSKC Sexually Transmitted Diseases (STD) Clinic (~3500 tests/yr) 2) Gay City Health Project Wellness Center (~1800 tests/yr) 3) Seattle bathhouses (3 sites, ~400 tests/yr) ● HIV screening recommended quarterly for high risk MSM, annually for other MSM High risk MSM = report of unprotected anal intercourse with partners of unknown or discordant serostatus, methamphetamine or popper use, or bacterial STD in last year. Data collection and statistical analyses ● Electronic medical records and detailed chart review ● Program certified as exempt from informed consent requirements other than verbal consent required for HIV testing. Laboratory testing ● HIV antibody testing by: - 1 st generation Vironostika HIV-1 Microelisa System (2003-2006) - 2 nd generation Genetic Systems rLAV EIA (2006-2008) ● Rapid HIV testing by OraQuick Advance Rapid HIV-1/2 Antibody Test Offered to: high risk MSM at PHSKC STD Clinic, all MSM at Gay City/bathhouses Both oral fluid testing and fingerstick whole blood ● Pooled HIV NAAT by Procleix HIV-1 Discriminatory Assay (2003-2005) and independently- validated, quantitative real-time RT-PCR amplification assay (TaqMan, 2005-2008). Pooling algorithm: 30 MSM → 3 intermediate pools → 1 master pool METHODS (continued) 1 False-negative HIV NAAT result 4 days after exposure to partner with acute HIV infection ● Median interval between testing and receipt of results was 19 (IQR 15-21) days for anonymous testers and 16 (IQR 14-19) days for confidential testers (p=.2). STANDARD ANTIBODY TESTING N=7041 #HIV positiveCumulative # HIV positiveCumulative sensitivity EIA159 (2.0%)15992% HIV NAAT13 (0.2%)17299% 1 RAPID ANTIBODY TESTING N=6964 OraQuick153 (2.2%)15380% EIA16 (0.2%)16988% HIV NAAT23 (0.3%)192100% Results of specimens tested by ARCHITECT ® HIV Ag/Ab Combo ● All 16 specimens from HIV-uninfected individuals tested HIV-negative ● All specimens from EIA-positive individuals were HIV-reactive ● The 4 th generation ARCHITECT HIV Ag/Ab Combo detected 15 (94%) of 16 EIA-negative/NAAT-positive MSM with acute HIV infection and median HIV RNA of 6.6 (IQR 5.8-6.9) log 10 copies/mL. Frequency of testing among persons with recent HIV infection changes test sensitivity ● Findings may not be generalizable to populations with lower HIV prevalence and incidence. 1)OraQuick performed on oral fluids or finger-stick blood specimens may be less sensitive than early generation EIAs. 2) HIV NAAT should be integrated into HIV testing programs serving MSM and other high-incidence populations with frequent HIV testing, particularly when rapid antibody testing is employed. 3)4th generation antigen-antibody combination assays may be a reasonably sensitive alternative to HIV NAAT. Contact Information: Joanne Stekler, MD, MPH Box 359931, Harborview Medical Center 325 Ninth Avenue, Seattle, WA 98104 Phone: (206) 731-8312, Fax: (206) 731-5109 Email: jstekler@u.washington.edu Bands Present on Western Blot WBLast negative IDspecimenEIAResultp18p24p32gp41p51p55p65gp120gp160HIV test (days) 1FS1 st ++++39 2OF1 st ++++374 a 3FS2 nd ++++516 4FS2 nd +++++324 5OF2 nd ++++161 a 6FS2 nd Ind++ b 290 7OF2 nd +++78 c 8FS2 nd ++++559 9OF2 nd +++++345 10OF2 nd +++NA 11OF2 nd ++++++++1209 12FS2 nd +++++++201 13OF2 nd Ind+NA d 14FS2 nd +++++251 15OF2 nd +++++98 16OF2 nd +++++++++387 e WB: Western Blot; OF: oral fluids; FS: fingerstick; Ind: Indeterminate; NA: not available. a IDs #2 and #5 reported seroconversion symptoms 23 and 42 days prior to testing, respectively. b Faint gp160. c ID #7 had a positive EIA and WB six days prior to his false-negative rapid test result through PHSKC. e ID #13 had a CD4 count of 4 cells/mm3, no Western Blot evolution, and was likely OQ-negative due to late stage HIV/AIDS and seroreversion e ID #16 had a positive FS OraQuick one day prior to his false-negative rapid test result through PHSKC. ACKNOWLEDGEMENTS We would like to thank clients and providers at the Gay City Health Project Wellness Center, PHSKC STD Clinic and other PHSKC testing sites as well as PHSKC for database management and data support. Acute HIV infectionPossible early HIV infectionEstablished HIV infection (EIA-neg/RNA-pos)(EIA-positive/gp41-negative)(gp41-positive) S/CO a : resultLog 10 copies/mL S/CO a : resultLog 10 copies/mL S/CO a : result Log 10 copies/mL EIAComboTaqManAbbottEIAComboAbbottEIAComboAbbott 10.4 b : -2.2: +5.55.117 c 5.2: +14.5: +4.1338.3: +328.6: +2.8 2NA2.7: +5.65.4183.4: +10.0: +<2.6347.5: +240.8: +4.0 30.3 b : - 9.8: +>66.019 c 4.3: +58.5: +<2.6357.5: +293.3: +<2.6 4NA5.1: +5.85.720 c 6.6: +51.0: +4.4367.3: +301.8: +<2.6 50.5 b : -108.9: +>66.8214.8: +24.9: +2.6375.3: +82.2: +3.9 60.6 b : -101.6: +>67.4226.2: +15.3: +4.5387.6: +437.7: +3.0 70.4 b : -463.4: +>6>8235.6: +27.1: +2.9396.4: +57.4: +3.9 80.08: -21.3: +5.66.2245.2: +23.5: +4.2407.2: +251.4: +<2.6 90.6: -36.4 d : +>66.6253.2: +9.0: +4.7416.8: +174.2: +<2.9 100.02: -36.3 d : +>66.8265.9: +29.2: +5.4427.9: +410.4: +3.8 110.1: -42.8 d : +>66.8276.0: +16.5: +3.7437.9: +399.2: +<2.8 120.3: -45.9 d : +>66.6285.8: +35.9: +4.5448.1: +200.3: +4.1 130.2: -10.4: +5.95.8296.0: +90.6: +4.9456.8: +149.5: +<2.9 140.2: -80.7: +>67.3305.9: +36.2: +4.6468.3: +339.9: +3.5 150.2: -0.2: -4.23.7316.6: +94.6: +5.5477.6: +276.4: +4.1 160.1: -54.6: +>66.9323.3: +100.7: +<2.4487.8: +334.7: +2.8 EIA: enzyme immunosorbent assay; NA: not available. a A signal to cut-off (S/CO) ratio greater or equal to 1.0 was considered reactive. b IDs #1, 3, and 5-7 were screened by the 1 st generation EIA. All others were screened by the 2 nd generation EIA. c Early infection IDs #17, 19, and 20 in this table are samples from IDs #4, 8, and 9, respectively, from OQ-neg/EIA-pos table. d Acute infection IDs # 9-12 were previously tested using the Abbott HIVAB HIV-1/HIV-2 (rDNA) 3rd generation EIA, and all tested HIV-negative with S/CO ratios of 0.18, 0.36, 0.13, and 0.18, respectively. ● Assumptions: - timing of testing is independent of the risk for HIV acquisition - HIV antibody and NAAT detect all infections after the "window period“ ● Annual testing by HIV antibody and NAAT detect 91% and 96% of infections ● Semi-annual testing intervals detect 84% and 93% of infections ● Quarterly testing intervals detect 72% and 86% of infections ● Increased testing frequency will increase frequency of false-negative HIV tests ● Retrospective antibody testing - 4 th generation ARCHITECT® HIV Ag/Ab Combo - Abbott RealTime HIV-1 assay quantified HIV RNA levels in stored serum HIV NAAT Negative EIA Negative rapid test HIV NAAT EIA Negative rapid test Negative EIA 2003-2005 2005-2008