Quantitative relationship between cycle threshold values and infectious influenza virus in patient nasopharyngeal samples

Real-time reverse transcription polymerase chain reaction (rRT-PCR) is nowadays commonly used to diagnose viral infections. This assay provides cycle threshold (Ct) values, which inversely correlate with viral load but do not directly indicate infectivity. The relationship between Ct values and infectivity remains unclear. As a first step, we assessed this relationship in nasopharyngeal samples from 97 seasonal influenza A (IAV) and 95 influenza B (IBV) virus-rRT-PCR-positive patients. Samples were cultured, viral RNA was quantified using reverse transcriptase digital PCR, and antibody titers against hemagglutinin (HA) were evaluated. We found a robust inverse correlation between sample Ct value and infectious virus titer and virus cultures are consistently positive at Ct < 22 with a 10% positive culture probability near Ct 36, which corresponds to 5.37 × 10 2 and 1.57 × 10 3 RNA copies mL −1 for seasonal IAV and IBV, respectively. Nearly all available serum samples showed HA-specific antibodies, with no correlation between HA titer and Ct value or virus titer. In conclusion, these results support the use of Ct values as a semiquantitative proxy for infectious viral load and underscore their value in interpreting molecular diagnostics and informing infection prevention and control strategies.