Missed confirmatory HCV RNA testing in anti-HCV-reactive patients and the role of reflex testing in improving completion of the hepatitis C diagnostic care cascade: a laboratory-based retrospective study

Abstract Background Completion of confirmatory HCV RNA testing after anti-HCV reactivity is a critical step in the hepatitis C diagnostic care cascade. However, missed confirmatory testing and repeated anti-HCV testing within routine hospital laboratory workflows remain insufficiently characterized. This study aimed to evaluate the frequency of missed confirmatory HCV RNA testing among anti-HCV-reactive patients, investigate the presence of active infection using archived serum samples, and assess workflow-related diagnostic gaps that could potentially be addressed through laboratory-based reflex testing strategies. Methods This retrospective laboratory-based observational study included patients tested for anti-HCV antibodies at a tertiary-care university hospital between January 1 and December 31, 2025. Anti-HCV-reactive patients without confirmatory HCV RNA testing were identified, and retrospective HCV RNA testing was performed on available archived serum samples. Repeated anti-HCV testing patterns and clinic-based differences in confirmatory testing requests were also analyzed to evaluate potential diagnostic workflow gaps. Results Among 27,984 anti-HCV tests performed in 23,833 unique patients, the anti-HCV seroprevalence rate was 1.28%. Confirmatory HCV RNA testing was not requested in 32.3% of anti-HCV-reactive patients. Retrospective testing of archived serum samples from 79 patients without initial confirmatory testing identified active infection in 3.8% of cases (3/79). Confirmatory testing rates were significantly higher in specialist clinics than in non-specialist clinics (95.0% vs. 71.0%, p   Conclusions Missed confirmatory HCV RNA testing represents an important gap in the diagnostic care cascade, particularly in non-specialist clinical settings. Laboratory-based reflex HCV RNA testing strategies may improve completion of confirmatory testing and reduce unnecessary repeat serological testing, supporting more effective diagnostic stewardship and contributing to hepatitis C elimination efforts. Clinical trial number Not applicable.