Mycobacterium tuberculosis and SARS-CoV-2 Co-Infection: An Efficient Cell Model to Study Lung Host-Pathogen Interactions.

In less than a year, SARS-CoV-2 managed to displace Mycobacterium tuberculosis (Mtb) as the leading cause of death worldwide due to a single infectious agent. Both pathogens affect the respiratory tract, mainly the lungs. However, the impact that a possible Mtb + SARS-CoV-2 co-infection can have on the host response is still unknown. Herein we depict a rigorous system to evaluate the complex interaction between two simultaneous infections in vitro in a lung epithelial cell line (A549). Overall, the process includes eight steps: (1) Mtb culture, (2) cell maintenance, (3) preparation of viral stocks, (4) determination of infectious titers, (5) Mtb and SARS-CoV-2 co-infection, (6) determination of intracellular bacterial load, (7) SARS-CoV-2 viability test, and (8) optional UV-C validation assay for infectivity reduction. This comprehensive protocol will allow experimentalists to study the pathogenesis of co-infection in vitro and facilitate collaborative work in the literature. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: In vitro coinfection model using Mtb and SARS-CoV-2 in human lung epithelial cells.