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

ABSTRACT 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. IMPORTANCE Most patients infected with hepatitis C virus are unable to spontaneously clear the virus and develop chronic infection. Chronic hepatitis C virus (HCV) infection can lead to life-threatening complications including liver cirrhosis, liver failure, and liver cancer. Therefore, accurate diagnosis of hepatitis C infection and linkage to treatment is paramount. To increase linkage to care, current Centers for Disease Control and Prevention (CDC) guidelines recommend single-visit sample collection to test for antibodies and, if antibody positive, viral RNA. However, almost half of the laboratories do not use reflexive testing strategies, in part due to concerns for carryover contamination. Our study describes a low risk of false-positive RNA results and suggests a risk mitigation intervention to further minimize false positives. Our work reaffirms CDC guidelines and provides real-world data to encourage other facilities to follow a reflex testing algorithm to increase linkage to care.