Comparative Performance of Digital PCR and Real-Time RT-PCR in Respiratory Virus Diagnostics

<i>Background</i>: Respiratory viral infections pose a major global health burden, and molecular diagnostics such as Real-Time RT-PCR have revealed frequent co-infections. However, precise quantification of viral RNA remains challenging. Digital PCR (dPCR) offers absolute quantification without standard curves and may improve diagnostic accuracy. This study compares dPCR and Real-Time RT-PCR in detecting and quantifying influenza A, influenza B, respiratory syncytial virus (RSV), and SARS-CoV-2 during the 2023-2024 tripledemic. <i>Methods</i>: A total of 123 respiratory samples were analysed and stratified by cycle threshold (Ct) values into high, medium, and low viral load categories. Both dPCR and Real-Time RT-PCR were used to quantify and compare viral loads across these categories. <i>Results</i>: dPCR demonstrated superior accuracy, particularly for high viral loads of influenza A, influenza B, and SARS-CoV-2, and for medium loads of RSV. It showed greater consistency and precision than Real-Time RT-PCR, especially in quantifying intermediate viral levels. <i>Conclusions</i>: These findings highlight the potential of dPCR to enhance respiratory virus diagnostics and support a better understanding of co-infection dynamics. Nonetheless, its routine implementation is currently limited by higher costs and reduced automation compared to Real-Time RT-PCR.