Advancing HIV Diagnostics: Comparative Evaluation of Multisure HIV-1/2 Rapid Confirmatory Test Against Geenius and Traditional Reference Assays Within a CDC-Aligned Diagnostic Framework

Human immunodeficiency virus (HIV) remains a major global health challenge, requiring accurate diagnostic testing for early detection. Chemiluminescent immunoassay screening, particularly the Architect HIV Ag/Ab Combo assay, followed by immunoblot confirmation using INNO-LIA™ has traditionally been used in many diagnostic workflows. To address these limitations, the U.S. Centers for Disease Control and Prevention (CDC) recommends the use of an HIV-1/2 antibody differentiation immunoassay, such as the Geenius HIV-1/2 Supplemental Assay, as part of the confirmatory testing algorithm. This study evaluates the performance of two rapid HIV-1/2 confirmatory assays-the Multisure HIV-1/2 Confirmatory Test and the Bio-Rad Geenius HIV-1/2 Supplemental Assay-within a CDC-aligned diagnostic framework, with the aim of assessing Multisure as a potential alternative differentiation assay. A total of 224 archived serum samples were analyzed, including true positives (<i>n</i> = 38), true negatives (<i>n</i> = 139), false positives (<i>n</i> = 20), and INNO-LIA™ indeterminate samples (<i>n</i> = 27), as defined by Architect HIV and INNO-LIA™ results. Samples were initially screened using the Architect HIV Ag/Ab Combo assay, confirmed by INNO-LIA™ and PCR, and subsequently re-tested using Multisure HIV-1/2 and Geenius HIV-1/2 assays. Diagnostic performance metrics were evaluated. Both rapid assays demonstrated 100% sensitivity and specificity when compared with INNO-LIA™. Among INNO-LIA™ indeterminate samples, Multisure HIV-1/2 classified 81.5% as negative compared with 55.6% using Geenius HIV-1/2. When compared with PCR, Multisure demonstrated higher specificity (89.2%) and positive predictive value (89.5%) than Geenius (82.9% and 84.6%). No confirmed HIV-2 infections were identified in the analyzed dataset, and HIV-1 subtype information was not available for the archived samples; therefore, conclusions regarding HIV-1/2 differentiation are based primarily on assay design and antigenic targets. Multisure HIV-1/2 demonstrated strong diagnostic performance comparable to established differentiation assays and may represent a practical alternative rapid confirmatory option within CDC-aligned HIV diagnostic workflows. Further studies including larger datasets and confirmed HIV-2 infections are warranted to further validate its clinical utility.