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Volume 41 Issue 1
February 2026
    Citation: Peilan Wei, Lu Zhang, Qingtao Hu, Airu Zhu, Zhen Zhuang, Zhaoyong Zhang, Shengnan Zhang, Jiantao Chen, Xinyi Xiong, Bin Qu, Yuanyuan Zhang, Lei Chen, Zhiwei Xu, Zhao Chen, Qier Zhong, Xindan Xing, Xinxin Li, Jingjing Gao, Yifang He, Guifei Xie, Juan Shang, Xiaoke Guo, Jiaxin Jiang, Yongxia Shi, Jingxian Zhao, Yanqun Wang, Jincun Zhao, Yingkang Jin. Integrated multiplex PCR and metatranscriptomics reveal upper-lower airway microbial landscapes in pediatric respiratory infections .VIROLOGICA SINICA, 2026, 41(1) : 58-69.  http://dx.doi.org/10.1016/j.virs.2026.02.001
    shu

    Integrated multiplex PCR and metatranscriptomics reveal upper-lower airway microbial landscapes in pediatric respiratory infections

    • a. Pediatric Pulmonary Department, Guangzhou Women and Children's Medical Center, Guangzhou Medical University, Guangzhou 510623, China;

    • b. State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, National Center for Respiratory Medicine, Joint International Research Laboratory of Respiratory Health, Guangdong Basic Research Center of Excellence for Respiratory Medicine, Guangzhou Institute of Respiratory Health, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou 510182, China;

    • c. China-Portugal Artificial Intelligence and Public Health Technologies Joint Laboratory, Guangdong-Hong Kong-Macao Joint Laboratory of Respiratory Infectious Diseases, Guangdong Provincial Key Laboratory of Respiratory Disease Research, Guangzhou Medical University, China;

    • d. Guangzhou National Laboratory, Bio-Island, Guangzhou 510000, China;

    • e. Health and Quarantine Laboratory, State Key Laboratory of Respiratory Disease of Guangzhou Customs District Technology Center, Guangzhou 510623, China;

    • f. Shanghai Institute for Advanced Immunochemical Studies, School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China;

    • g. GMU-GIBH Joint School of Life Sciences, Guangzhou Medical University, Guangzhou 511436, China;

    • h. School of Basic Medical Sciences, Guangzhou Medical University, Guangzhou 511436, China

    • Corresponding author: Jingxian Zhao, zhaojingxian@gird.cn
      Yanqun Wang, wangyanqun@gird.cn
      Jincun Zhao, zhaojincun@gird.cn
      Yingkang Jin, yingkangjin@163.com
    • Received Date: 26 August 2025
      Accepted Date: 28 January 2026
      Available online: 05 February 2026

      Fund Project: This project was supported by grants from the National Key Research and Development Program of China (2024YFC2310205 to L.Z.), the National Natural Science Foundation of China (82025001, 82495200 and 82495203 to J.C.Z., 92369113 and 82172240 to Y.Q.W.), the Guangdong Medical Technology Research Foundation (B2022233 to Y.K.J.), the Guangzhou National Laboratory and State Key Laboratory of Respiratory Disease (GZNL2024B01001 to Y.Q.W., and GZNL2023A01004 to Y.K.J.), the Guangdong Basic and Applied Research Projects (2023B1515020040 to Y.Q.W.), the State Key Laboratory of Respiratory Disease (SKLRD-Z-202411 to L.Z., SKLRD-OP-202309 to Y.Q.W), the Science and Technology Planning Project of Guangzhou City (2024A03J1230 to J.C.Z., 2025B04J0006 to L.Z.), and the Science and Technology Project of General Administration of Customs, P.R. China (2025HK221, 2023HK065 to L.Z.). We thank the Biobank for Respiratory Disease in the National Clinical Research Center for Respiratory Disease (BRD-NCRCRD, Guangzhou, China).

    • Despite widespread use of multiple PCR, a substantial proportion of pediatric acute respiratory tract infections (ARTIs) lack identifiable pathogens and are classified as unknown etiology. The microbial characteristics and clinical relevance of these cases remain unclear. In this study, we compared the airway microbiomes of PCR-positive and PCR-negative ARTIs and examined their relationships with sampling site and disease severity. A total of 514 hospitalized children with ARTIs were enrolled. Nasopharyngeal swabs (NS) and bronchoalveolar lavage fluid (BALF) samples were tested using a 22-target multiplex PCR panel and subsequently stratified by pathogen status for pooled metatranscriptomic sequencing to profile active microbial communities, viral genotypes, and antibiotic resistance genes. PCR identified common respiratory pathogens in 77.0% of NS and 54.1% of BALF samples. Metatranscriptomic analysis showed that PCR-negative pools displayed markedly lower viral activity and comparatively higher bacterial transcript abundance, with notable enrichment of Pseudomonas. Microbial signatures differed between upper and lower airway samples and across clinical severity, with severe cases demonstrating increased bacterial burden and Pseudomonas enrichment, whereas mild infections exhibited relatively stronger viral signals. Under current thresholds, antibiotic resistance genes were detected in patient pools but not in healthy controls. Overall, PCR-negative pediatric ARTIs exhibited distinct, bacteria-enriched microbial profiles. Integrating metatranscriptomics with PCR enhances pathogen characterization and reveals site- and severity-related microbial patterns that may support diagnostic evaluation and clinical management.
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      4. Brealey JC, Leitao HG, Hofstede T, Kalthoff DC, Guschanski K., 2021. The oral microbiota of wild bears in Sweden reflects the history of antibiotic use by humans. Curr. Biol., 31, 4650-4658.

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      12. Hou M, Liu G, Meng C, Dong L, Fang Y, Wang L, Wang N, Cai C, Wang H., 2024. Circulation patterns and molecular characteristics of respiratory syncytial virus among hospitalized children in tianjin, China, before and during the covid-19 pandemic (2017-2022). Virol. Sin., 39, 719-726.

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      Integrated multiplex PCR and metatranscriptomics reveal upper-lower airway microbial landscapes in pediatric respiratory infections

        Corresponding author: Jingxian Zhao, zhaojingxian@gird.cn
        Corresponding author: Yanqun Wang, wangyanqun@gird.cn
        Corresponding author: Jincun Zhao, zhaojincun@gird.cn
        Corresponding author: Yingkang Jin, yingkangjin@163.com
      • a. Pediatric Pulmonary Department, Guangzhou Women and Children's Medical Center, Guangzhou Medical University, Guangzhou 510623, China;
      • b. State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, National Center for Respiratory Medicine, Joint International Research Laboratory of Respiratory Health, Guangdong Basic Research Center of Excellence for Respiratory Medicine, Guangzhou Institute of Respiratory Health, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou 510182, China;
      • c. China-Portugal Artificial Intelligence and Public Health Technologies Joint Laboratory, Guangdong-Hong Kong-Macao Joint Laboratory of Respiratory Infectious Diseases, Guangdong Provincial Key Laboratory of Respiratory Disease Research, Guangzhou Medical University, China;
      • d. Guangzhou National Laboratory, Bio-Island, Guangzhou 510000, China;
      • e. Health and Quarantine Laboratory, State Key Laboratory of Respiratory Disease of Guangzhou Customs District Technology Center, Guangzhou 510623, China;
      • f. Shanghai Institute for Advanced Immunochemical Studies, School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China;
      • g. GMU-GIBH Joint School of Life Sciences, Guangzhou Medical University, Guangzhou 511436, China;
      • h. School of Basic Medical Sciences, Guangzhou Medical University, Guangzhou 511436, China
      • Received Date: 26 August 2025
      • Accepted Date: 28 January 2026
      Fund Project:  This project was supported by grants from the National Key Research and Development Program of China (2024YFC2310205 to L.Z.), the National Natural Science Foundation of China (82025001, 82495200 and 82495203 to J.C.Z., 92369113 and 82172240 to Y.Q.W.), the Guangdong Medical Technology Research Foundation (B2022233 to Y.K.J.), the Guangzhou National Laboratory and State Key Laboratory of Respiratory Disease (GZNL2024B01001 to Y.Q.W., and GZNL2023A01004 to Y.K.J.), the Guangdong Basic and Applied Research Projects (2023B1515020040 to Y.Q.W.), the State Key Laboratory of Respiratory Disease (SKLRD-Z-202411 to L.Z., SKLRD-OP-202309 to Y.Q.W), the Science and Technology Planning Project of Guangzhou City (2024A03J1230 to J.C.Z., 2025B04J0006 to L.Z.), and the Science and Technology Project of General Administration of Customs, P.R. China (2025HK221, 2023HK065 to L.Z.). We thank the Biobank for Respiratory Disease in the National Clinical Research Center for Respiratory Disease (BRD-NCRCRD, Guangzhou, China).

      Abstract: Despite widespread use of multiple PCR, a substantial proportion of pediatric acute respiratory tract infections (ARTIs) lack identifiable pathogens and are classified as unknown etiology. The microbial characteristics and clinical relevance of these cases remain unclear. In this study, we compared the airway microbiomes of PCR-positive and PCR-negative ARTIs and examined their relationships with sampling site and disease severity. A total of 514 hospitalized children with ARTIs were enrolled. Nasopharyngeal swabs (NS) and bronchoalveolar lavage fluid (BALF) samples were tested using a 22-target multiplex PCR panel and subsequently stratified by pathogen status for pooled metatranscriptomic sequencing to profile active microbial communities, viral genotypes, and antibiotic resistance genes. PCR identified common respiratory pathogens in 77.0% of NS and 54.1% of BALF samples. Metatranscriptomic analysis showed that PCR-negative pools displayed markedly lower viral activity and comparatively higher bacterial transcript abundance, with notable enrichment of Pseudomonas. Microbial signatures differed between upper and lower airway samples and across clinical severity, with severe cases demonstrating increased bacterial burden and Pseudomonas enrichment, whereas mild infections exhibited relatively stronger viral signals. Under current thresholds, antibiotic resistance genes were detected in patient pools but not in healthy controls. Overall, PCR-negative pediatric ARTIs exhibited distinct, bacteria-enriched microbial profiles. Integrating metatranscriptomics with PCR enhances pathogen characterization and reveals site- and severity-related microbial patterns that may support diagnostic evaluation and clinical management.

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