Thirty years into the HIV pandemic, an estimated 60% of HIV-infected individuals worldwide and 21% of HIV-infected individuals in the United States remain undiagnosed [1,2]. Despite tremendous strides in HIV treatment, our ability to identify infected persons unaware of their status has lagged behind. Standard HIV diagnostic tools fail to identify most incident infections due to the window period following acquisition when HIV antibodies remain undetectable. As some recently infected persons present for medical care due to symptoms associated with acute HIV infection (AHI) or other sexually transmitted infections (STIs), screening HIV tests which narrow the window period could help address unacceptable levels of undiagnosed HIV, with substantial individual and public health benefits .
The detection of early and AHI has mainly relied on nucleic acid amplification testing (NAAT) of seronegative samples. However, this method is costly  and includes a substantial time-delay to confirmed diagnoses. Newer fourth generation HIV enzyme immunoassays (EIAs), which simultaneously detect p24 antigen and anti-HIV-1/2 antibodies used in third generation EIAs, reduce the window period of detecting early HIV by an average of 4 days (range, 2–14) [5–8] and produce confirmatory results within 24 h. Accordingly, the implementation of fourth generation EIAs into routine screening algorithms could detect more AHI cases due to increased sensitivity compared to third generation EIA assays, and considerably shorten delay to confirmed diagnosis.
To our knowledge, no fourth generation EIAs have been evaluated in a large, prospective study in the United States despite turn-around and sensitivity advantages over the current standard-of-care. We prospectively evaluated the performance of the ARCHITECT HIV Ag/Ab Combo (HIV Combo; Abbott Laboratories, Abbott Park, Illinois, USA) in HIV-tested persons at STI clinics and compared results to the North Carolina algorithm of third generation EIA testing with reflex NAAT pooling of seronegative samples (Fig. 1) . We selected the HIV Combo assay, given prior studies demonstrated the highest sensitivity among available fourth generation EIAs [8,10,11].
For this prospective, cross-sectional study, all individuals greater than 13 years of age who consented to HIV testing at two semi-urban public STI clinics in North Carolina from February 2010 to August 2010 were eligible to participate. At the time of phlebotomy for HIV testing, an additional 10 ml EDTA tube was drawn and labeled with a unique identification number. All samples were aliquoted into 2-ml tubes and stored and transported (2–8 °C/35.6–46.4 °F) the next business day from respective study sites to the University of North Carolina (UNC) Hospitals Clinical Microbiology/Immunology Laboratories. Samples were processed and tested per manufacturer's specifications and maintained at 2–8°C/35.6–46.4 °F until tested. Samples received after 13 April 2010 were frozen (−80 °C/−112 °F) per manufacturer guidelines due to temporary shortages of testing reagents and defrosted (2–8 °C/35.6–46.4 °F) for up to 24 h before postprocessing. HIV Combo results were compared to results from the NC State Laboratory of Public Health (NCSLPH), which uses a third generation EIA (Genetic Systems HIV-1/HIV-2 Plus 0 EIA; Bio-Rad Laboratories, Redmond, Washington, USA) with western blot confirmation (Genetic Systems HIV-1 Western Blot, Bio-Rad Laboratories), and NAAT (Aptima HIV-1 RNA Qualitative Assay; Gen-Probe Inc., San Diego, California, USA) pooling of all seronegative samples . As the HIV Combo assay was not yet Food and Drug Administration-approved in the United States, results were not utilized for clinical diagnosis. All testing results were entered into Microsoft Access 2010 (Microsoft, Redmond, Washington, USA). Sensitivity and specificity were calculated using standard procedures. Ninety-five percent confidence intervals (CIs) were calculated using exact methods. The study was approved by the UNC Institutional Review Board.
A total of 3345 persons consented to HIV testing at participating sites. Results were not available for 27 samples (20 specimens had no results on the HIV Combo assay due to freezing-related issues or missing samples; seven samples had no result at the NCSLPH for unspecified reasons). Twenty-seven of 31 (87%) samples testing positive by the HIV Combo also tested positive via third generation EIA/western blot testing. Among the remaining four samples, three were negative on third generation EIA and one was repeatedly reactive via rapid EIA and third generation EIA, but all were negative by NAAT. An additional four specimens negative on the HIV Combo assay were positive by third generation EIA, but negative on NAAT. Given these results, the sensitivity of the HIV Combo test was 100% (27/27; 95% CI 87.2–100). The specificity was 99.9% (3287/3291; 95% CI 99.7–99.97). No AHI cases were identified by HIV Combo testing or NCSLPH screening  during the study period. The median turn-around time for confirmed HIV Combo results prior to sample freezing was 26.1 h (interquartile range: 22.2–46.3) and included transportation time to the central laboratory.
The fourth generation HIV Combo assay evaluated in this study performed exceedingly well in detecting established HIV infection in one of the first prospective studies in the United States. The sensitivity and specificity were 100% and 99.9%, respectively, supporting results from retrospective studies [8,10]. Regrettably, we were unable to determine whether the four false-positives were due to the antigen or antibody component of the assay, and nor could we evaluate the assay's performance in detecting AHI, given limited study sites and study duration. Although the turn-around time can be improved by on-site testing, the median 26.1 h turn-around time from sample collection to HIV Combo results in our study versus a median of 12 days for AHI confirmation via NAAT pooling  could dramatically reduce delays to detection and notification of AHI cases. The latter is critical as persons with AHI are highly infectious [13–16] and may continue risky behaviors that lead to HIV infection if unaware of their status . The advantages of fourth generation assays warrant investigations into new testing algorithms, which incorporate rapid HIV antibody testing of fourth generation EIA-positive samples, followed by HIV RNA confirmation. Such algorithms have the potential to allow more widespread screening for AHI by reducing infrastructure and financial costs; however, the excellent performance of the HIV Combo assay in our study strongly supports its implementation as a routine HIV screening assay in all settings.
The authors greatly appreciate the support of all study staff, the UNC CFAR Immunology Core Laboratory, Arlene C. Sena, MD, MPH, Cedar Eagle, Eddie Watkins, Cindy Toler, Susan Krawczyk, all staff at the Durham County and Guilford County Health Departments who contributed to this study, and especially the individuals who participated in this study. All authors approved this article. J.J.B., C.L.G., J.D.K., J.L.S., F.A.A., P.A.L., and W.C.M. were responsible for study design. J.E.H., F.A.A., and J.L.S. performed testing of samples. J.J.B., C.L.G., J.D.K., and J.A.E. were responsible for sample collection. J.J.B., J.A.E., and W.C.M. performed statistical analysis. J.J.B. and C.L.G. wrote and edited the article, which was reviewed and approved by all authors.
Conflicts of interest
C.G. has received research support from Bristol Myers Squibb, Gilead Sciences, Abbott Laboratories, and Tibotec Therapeutics. J.S. has received grant support from the Alere Scarborough National Marrow Donor Program. P.L. has received grant support from Abbott Diagnostics and is on the speaker's bureau for Abbott Diagnostics, GlaxoSmithKline, and Novartis. For the remaining authors, none were declared. This study was supported by the generous contributions of Abbott Laboratories and the NIH funded UNC Center for AIDS Research (CFAR) (1P30AI 50410–04).
1. Organization WH. Towards universal access: scaling up priority HIV/AIDS interventions in the health sector Progress Report 2010; 2010.
2. Campsmith ML, Rhodes PH, Hall HI, Green TA. Undiagnosed HIV prevalence among adults and adolescents in the United States at the end of 2006
. J Acquir Immune Defic Syndr
3. Cohen MS, Gay CL, Busch MP, Hecht FM. The detection of acute HIV infection
. J Infect Dis
2010; 202 (Suppl 2):S270–S277.
4. Hutchinson AB, Patel P, Sansom SL, Farnham PG, Sullivan TJ, Bennett B, et al. Cost-effectiveness of pooled nucleic acid amplification testing for acute HIV infection after third-generation HIV antibody screening and rapid testing in the United States: a comparison of three public health settings
. PLoS Med
5. Weber B, Orazi B, Raineri A, Thorstensson R, Burgisser P, Muhlbacher A, et al. Multicenter evaluation of a new 4th generation HIV screening assay Elecsys HIV combi
. Clin Lab
6. Weber B, Fall EH, Berger A, Doerr HW. Reduction of diagnostic window by new fourth-generation human immunodeficiency virus screening assays
. J Clin Microbiol
7. van Binsbergen J, Keur W, Siebelink A, van de Graaf M, Jacobs A, de Rijk D, et al. Strongly enhanced sensitivity of a direct anti-HIV-1/-2 assay in seroconversion by incorporation of HIV p24 ag detection: a new generation vironostika HIV Uni-Form II
. J Virol Methods
8. Ly TD, Laperche S, Brennan C, Vallari A, Ebel A, Hunt J, et al. Evaluation of the sensitivity and specificity of six HIV combined p24 antigen and antibody assays
. J Virol Methods
9. Pilcher CD, Fiscus SA, Nguyen TQ, Foust E, Wolf L, Williams D, et al. Detection of acute infections during HIV testing in North Carolina
. N Engl J Med
10. Owen M, Patel P, Wesolowski L, Vallan A, Brennan C, Devare S, et al.Evaluation of the Abbott ARCHITECT Ag/Ab Combo assay, an antigen/antibody combination test: implications for US HIV testing programs
[abstract 991]. 16th Conference on Retroviruses and Opportunistic Infections
. Montreal, Canada; 2009.
11. Ly TD, Martin L, Daghfal D, Sandridge A, West D, Bristow R, et al. Seven human immunodeficiency virus (HIV) antigen-antibody combination assays: evaluation of HIV seroconversion sensitivity and subtype detection
. J Clin Microbiol
12. Gay C, Dibben O, Anderson JA, Stacey A, Mayo AJ, Norris PJ, et al. Cross-sectional detection of acute HIV infection: timing of transmission, inflammation and antiretroviral therapy
. PLoS One
13. Brenner BG, Roger M, Routy JP, Moisi D, Ntemgwa M, Matte C, et al. High rates of forward transmission events after acute/early HIV-1 infection
. J Infect Dis
14. Wawer MJ, Gray RH, Sewankambo NK, Serwadda D, Li X, Laeyendecker O, et al. Rates of HIV-1 transmission per coital act, by stage of HIV-1 infection, in Rakai, Uganda
. J Infect Dis
15. Pilcher CD, Tien HC, Eron JJ Jr, Vernazza PL, Leu SY, Stewart PW, et al. Brief but efficient: acute HIV infection and the sexual transmission of HIV
. J Infect Dis
16. Yerly S, Vora S, Rizzardi P, Chave JP, Vernazza PL, Flepp M, et al. Acute HIV infection: impact on the spread of HIV and transmission of drug resistance
17. Colfax GN, Buchbinder SP, Cornelisse PG, Vittinghoff E, Mayer K, Celum C. Sexual risk behaviors and implications for secondary HIV transmission during and after HIV seroconversion