OBJECTIVE(S): To determine the effect of viral suppression on cross-sectional incidence testing. METHODS: In 2001 and 2003, patients entering the Johns Hopkins Hospital (JHH) Emergency Department (ED) were enrolled into an interview-based identity-unlinked serosurvey. All HIV-positive samples were tested by the Vironostika-less sensitive (LS) enzyme immunoassay (EIA) (Organon-Teknika, Charteston, SC) and an avidity assay to determine recent HIV infection. Additionally 16 samples from 8 previously characterized elite suppressors (ES) were tested by cross-sectional incidence assays. RESULTS: HIV prevalence was 12% for the 2001 survey and 11% for the 2003 survey. Of the HIV-infected subjects, 18% did not know they were infected. The Vironostika-LS EIA determined that 6% (11 of 183) and 7% (17 of 243) of HIV-positive individuals in 2001and 2003, respectively, were recently infected. Avidity testing confirmed that 6 of 11 in 2001 and 5 of 17 in 2003 were newly infected, leaving 17 discrepant samples. All 17 discrepant samples were Western blot-positive and viral load undetectable, and 7 of 17 had antiretroviral drugs (ARVs) in their serum. Ten individuals were virally suppressed without ARVs and seemed incident by the Vironostika-LS EIA but chronic by avidity testing. These 10 subjects had similar testing profiles to the known 16 ES samples, because 9 of 16 were incident by the Vironostika-LS EIA and 0 of 16 were incident by avidity testing. CONCLUSIONS: By removing the viral load-negative individuals and confirming the initial Vironostika-LS EIA results by avidity testing, the incidence estimate was lowered from 1.73% to 0.94% per year in 2001 and from 1.90% to 0.56% per year in 2003. Viral suppression affects the performance of the cross-sectional incidence tests, which rely on antibody titer. In addition, 2% (10 of 426) of all HIV-infected individuals who use the JHH ED for medical care seem to suppress HIV to undetectable levels without ARVs.