Comprehensive Pathogen Profiling in Adult Patients with Severe Acute Respiratory Infections Using Metagenomic Next-Generation Sequencing of Sputum Samples
Kham-Kjing, N.; Kawila, R.; Tariyo, P.; Puapun, K.; Ngo-Giang-Huong, N.; Hongjaisee, S.; Khamduang, W.
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Severe Acute Respiratory Infection (SARI) remains a major global health burden, yet conventional diagnostics frequently fail to identify the causative pathogens. This study aimed to comprehensively characterize the respiratory microbial landscape in adult SARI and to evaluate the clinical utility of an optimized long-read metagenomic next-generation sequencing (mNGS) workflow. A total of 101 respiratory specimens from hospitalized adults in northern Thailand (November 2023 to April 2024) were analyzed using SMART-9N-based cDNA synthesis, Oxford Nanopore sequencing, and a streamlined bioinformatics pipeline. Integrated clinical and laboratory data were used to assess associations with disease severity. Long-read mNGS demonstrated superior diagnostic yield compared to multiplex PCR and culture, detecting pathogens in 78% of cases and uniquely identifying etiologic agents in 18% of specimens negative by routine diagnostics, including Klebsiella pneumoniae, Haemophilus parainfluenzae, and SARS-CoV-2. High concordance was observed for respiratory viruses, with genome-wide coverage supporting accurate detection. Phylogenetic analysis revealed the co-circulation of HRV-A, HRV-B, and HRV-C, with HRV-C predominating. Multivariable analysis identified male sex and reduced oxygen saturation as independent predictors of severe disease. These findings underscore the diagnostic power of long-read mNGS for uncovering atypical, unculturable, and polymicrobial infections in adult SARI and support its integration into respiratory pathogen surveillance.
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