Quantifying the impact of pathogen interactions on vaccine effectiveness estimates in the test-negative design

The test-negative design (TND) has been widely adopted for real-world vaccine effectiveness (VE) assessment, assuming that control selection is independent of vaccination status. However, pathogen interactions may contradict this assumption, posing challenges to VE evaluations. We developed a theoretical framework to quantify the pathogen-dependence-related biases in VE estimates. The framework was further empirically evaluated using data from 66,117 pediatric patients tested for multiple respiratory pathogens during 2023–2024, comparing influenza VE estimates using standard influenza-negative controls with those using specific non-influenza pathogen positive or negative controls. The theoretical framework indicated that the TND may overestimate VE even without pathogen dependence. Pathogen dependence further modified influenza VE estimates in a pathogen-specific direction, i.e., positive pathogen dependence with influenza tended to overestimate influenza VE, whereas negative pathogen dependence biased VE estimates downward. However, the magnitude of dependence-related bias was generally smaller than the bias attributable to the baseline model-specific discrepancy. The use of all-pathogen-negative controls minimized estimation bias. These findings suggest that pathogen dependence and control selection should be considered when using TND for VE estimation.