Clinical Impact of Polymicrobial Interactions in Human-Metapneumovirus-Infected Children: A Targeted Next-Generation Sequencing Based Retrospective Study

Purpose: Human metapneumovirus ( hMPV ) is a significant pathogen of acute respiratory infection in children. While previous studies have relied on conventional diagnostics that underestimate complex polymicrobial interactions, this study aimed to utilize targeted next-generation sequencing (tNGS) to comprehensively profile the dynamics of its co-colonization or co-infection pathogen spectrum and their impact on disease severity, thereby informing clinical risk stratification. Patients and Methods: This retrospective study enrolled 526 hospitalized children with hMPV infection. Targeted next-generation sequencing (tNGS) was used to comprehensively analyze the co-colonization or co-infection spectrum stratified by age, gender, sample origin, immune deficiency, and immunization status. Multivariable logistic regression was employed to assess independent associations with host factors and severe clinical outcomes (ICU intensive care unit admission, mechanical ventilation, septic shock, pneumonia on imaging). Results: The median age of the inpatients was 36 months, and 15.6% required ICU admission. The overall prevalence of co-detection among the cohort was 100% since we identified at least one pathogen in the existing results from tNGS. The co-colonization or co-infection pathogen spectrum demonstrated significant age specificity, temporal dynamics, and sample type dependency. Multivariable analysis identified several pathogens independently associated with severe outcomes. Specifically, opportunistic and atypical pathogens (such as Cytomegalovirus prevalence: 7.22%, Herpes Simplex Virus type 1 1.71%, Klebsiella aerogenes 0.76%, and Tropheryma whipplei 1.90%) were strong risk factors for ICU admission, invasive ventilation, and septic shock (adjusted Odds Ratios aOR ranging from 4.57 to 28.69). Conversely, Fusobacterium nucleatum (25.67%) consistently exhibited a protective effect (eg, aOR=0.43 for ICU admission). Mycoplasma pneumoniae (12.93%) was an independent risk factor for pneumonia on imaging (aOR=2.25), while Human RSV A (1.14%) showed a significant protective association (aOR=0.09). Conclusion: Despite limitations including a single-center retrospective design and the absence of an hMPV-negative control group, the co-colonization or co-infection pathogen spectrum in children with hMPV infection is complex and closely related to host characteristics. Early identification of high-risk co-pathogens (eg, CMV or K. aerogenes ) via tNGS should trigger intensified clinical monitoring. Future prospective controlled studies are needed to validate these findings and confirm causality. Keywords: human metapneumovirus, pediatrics, co-colonizations, co-infection, targeted NGS diagnostics, bacterial-viral interactions