tNGS Reveals Predominant Mixed Viral Infections in a Multicenter Retrospective Study.

Purpose: Viral respiratory infections can damage epithelial cells and suppress host immunity, thereby predisposing patients to secondary bacterial or fungal infections. Accurate and timely identification of co-pathogens remains a clinical challenge. We aimed to examine whether tNGS can more comprehensively identify viral and mixed infections in comparison to conventional methods. Patients and Methods: We retrospectively analyzed 834 patients tested using targeted next-generation sequencing (tNGS) and 2263 patients tested using conventional methods across multiple centers. Pathogen profiles-including viral, bacterial, fungal, and atypical microorganisms-were compared between the groups. Results: Compared with conventional diagnostic methods, tNGS detected significantly higher proportions of viral co-infections, including fungal pathogens (eg, Aspergillus and Mucor), bacterial pathogens, Mycobacterium spp., herpesviruses, and multiple viral combinations (such as two distinct respiratory viruses, respiratory viruses with herpesviruses, or two non-herpesviruses). The most frequently identified viruses by tNGS were Epstein-Barr virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), herpes simplex virus type 1, influenza A virus, and rhinovirus. Commonly detected bacterial pathogens included Klebsiella spp. Fusobacterium nucleatum, and Streptococcus mitis, while fungal pathogens frequently identified included Aspergillus spp. and Mucor spp. In addition, the detection rates of Mycoplasma spp., Mycobacterium tuberculosis, and nontuberculous mycobacteria were significantly higher with tNGS than with conventional testing (all P < 0.05). Conclusion: Targeted next-generation sequencing identified a higher prevalence and broader spectrum of viral co-infections and secondary bacterial/fungal infections compared with conventional methods. These findings highlight the complexity of respiratory infections in the post-COVID-19 era and support the potential of tNGS as a comprehensive diagnostic approach in clinical practice.