Risk assessment and mitigation of hepatitis C virus RNA carryover contamination in a reflex testing algorithm

Laboratory-based diagnosis of active hepatitis C virus (HCV) infection currently requires a positive antibody test and a confirmatory RNA test. Many institutions in the United States continue to require two separate sample collections, partially due to concerns for carryover contamination. Reflexive RNA testing of antibody-positive samples reduces loss to follow-up, improves linkage to care, and is a recommended operational strategy by the Centers for Disease Control and Prevention. This study describes false positivity rates of reflexive RNA testing in a reference laboratory before and after introduction of a risk mitigation strategy that requires medical director review for reflexed HCV viral loads < 1,000 IU/mL (defined as low-positive). Initial HCV antibody testing was performed at one of five chemistry laboratories; reflex HCV RNA testing was performed using the cobas HCV assay on cobas 8800 (Roche) at a central reference laboratory. A total of 4,079 consecutive positive RNA results were included pre-intervention, and 4,433 were included post-intervention. All low-positive results ( N = 157) were retrospectively adjudicated as true-positive ( N = 66), false-positive ( N = 38), or unknown ( N = 53), based on clinical history and redraw results. The pre-intervention false-positive rate was 0.44%, which was significantly reduced to 0.14% post-intervention, P = 0.008. Median viral load for those clinically adjudicated as true-positive was significantly higher at 97 IU/mL (IQR: 24, 330 IU/mL) compared to 17 IU/mL (IQR: 14, 58 IU/mL) classified as false-positive or 16 IU/mL (IQR: 14, 62 IU/mL) classified as unknown. This study supports wider implementation of reflexive RNA testing for antibody-positive samples and provides a blueprint for a risk mitigation strategy.