Abstract Advances in HIV diagnostic testing have improved detection of HIV infections. HIV can be detected earlier so patients benefit from treatment and negative results reassure uninfected patients. Because first line immunoassays lack specificity, positive results need to be confirmed. The aim of this study was to evaluate the algorithm used at The Ohio State Wexner Medical Center for the diagnosis of HIV infection. The algorithm begins with a combination immunoassay that detects HIV-1 and HIV-2 antibodies and HIV-1 p24 antigen [ADVIA Centaur® HIV Ag/Ab Combo (CHIV), Siemens]. All positive specimens are confirmed with an immunoassay that differentiates HIV-1 from HIV-2 antibodies [Geenius HIV-1/HIV-2 Confirmatory assay (GHIV), Bio-Rad]. Specimens that are positive by CHIV and negative or indeterminate by GHIV proceed to HIV-1 nucleic acid testing for resolution. A total of 14,994 patients were evaluated using CHIV assay, of these 99.7% were negative (n= 14,950) and 0.3% were positive (n=44). The 44 positive specimens were tested by GHIV, only 4 confirmed positive and 40 were negative. Of the 40 that tested negative, only 32 had enough serum left to be tested by RNA HIV molecular test and all tested negative. Of the 8 that there was not enough serum left, 6 patients had a follow up viral load and they were all negative. In summary, of the 44 CHIV positive, only 4 were confirmed by GHIV, and 38 confirmed negative by the molecular tests, questioning the utility of the additional molecular step.