AbstractIntroductionScreening for hepatitis C virus (HCV) is performed by testing for anti‐HCV antibodies, which may yield false‐positive results leading to additional testing and other downstream consequences for the patient. We report our experience in a low prevalence population (<0.05%) using a two‐assay algorithm aimed at testing specimens with borderline or weak positive anti‐HCV reactivity in the screening assay by a second anti‐HCV assay prior to confirming positive anti‐HCV results with RT‐PCR.Materials and MethodsRetrospective analysis of 58,908 plasma samples was obtained over a 5‐year period. Samples were initially tested using the Elecsys Anti‐HCV II assay (Roche Diagnostics), with borderline or weakly positive results (defined in our algorithm as a Roche cutoff index of 0.9–19.99) reflexively analyzed using the Architect Anti‐HCV assay (Abbott Diagnostics). The Abbott anti‐HCV results dictated the final anti‐HCV interpretation for reflexed samples.ResultsOur testing algorithm resulted in 180 samples requiring second‐line testing, with final anti‐HCV results interpreted as 9% positive, 87% negative, and 4% indeterminate. The positive predictive value (PPV) of a weakly positive Roche result was 12%, which was significantly lower than the PPV using our two‐assay approach (65%).ConclusionsThe incorporation of a two‐assay serological testing algorithm in a low prevalence population provides a cost‐effective method of improving the PPV of HCV screening in specimens with borderline or weakly positive anti‐HCV results.