tNGS Reveals Predominant Mixed Viral Infections in a Multicenter Retrospective Study

Xiaoqi Zhang,1,* Rui Tang,2,* Xiaoyan Li,3,* Lijuan Li4 1Department of Pulmonary and Critical Care Medicine, Second People’s Hospital of Weifang, Weifang, 261041, People’s Republic of China; 2Department of Pulmonary and Critical Care Medicine, Tangshan Gongren Hospital, Tangshan, 063003, People’s Republic of China; 3Department of Pulmonary and Critical Care Medicine, Renqiu Friendship Hospital, Cangzhou, 062550, People’s Republic of China; 4Department of Pulmonary and Critical Care Medicine, National Centre for Clinical Research on Respiratory Diseases, China-Japan Friendship Hos-Pital, Beijing, 100029, People’s Republic of China*These authors contributed equally to this workCorrespondence: Lijuan Li, Department of Pulmonary and Critical Care Medicine, National Centre for Clinical Research on Respiratory Diseases, China-Japan Friendship Hospital, Beijing, 100029, People’s Republic of China, Email [email protected]: 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.Plain Language Summary: Respiratory viruses can damage the airways and weaken the immune system, making people more likely to develop bacterial or fungal infections. Identifying these additional “co-infections” is important for treatment but can be difficult with standard lab tests. In this study, researchers compared two testing approaches: targeted next-generation sequencing (tNGS) and conventional diagnostic methods. They reviewed data from 834 patients tested with tNGS and 2263 patients tested with conventional methods across multiple centers. The results showed that tNGS detected a much wider range of infections. It identified not only common respiratory viruses but also multiple simultaneous viral infections, herpesviruses, bacteria, and fungi that were often missed by conventional testing. Frequent viruses found included Epstein–Barr virus, SARS-CoV-2, herpes simplex virus type 1, influenza A, and rhinovirus. Bacteria, such as Klebsiella, Fusobacterium nucleatum, and Streptococcus mitis, were commonly seen, along with fungal pathogens like Aspergillus and Mucor. tNGS also had higher detection rates for Mycoplasma, Mycobacterium tuberculosis, and nontuberculous mycobacteria. Overall, tNGS provided a more complete picture of mixed respiratory infections than conventional methods, suggesting it may be a valuable tool for improving diagnosis and patient care in the post–COVID-19 era.Keywords: Aspergillus, mixed viral infections, Mucor, post-COVID-19, tNGS, virus