Two-step wastewater surveillance reveals co-circulation of respiratory pathogens during the 2023–2024 influenza season in a low-resource setting

Highlights

	● Utility of wastewater monitoring for respiratory bacterial pathogen was confirmed.
	● We found strong link between the sewage concentration of pathogen & positivity rate.
	● A two-step solution was offered for early warning of multiple respiratory diseases.

Abstract

Clinical surveillance for respiratory pathogens has traditionally been challenging in low-resource settings, such as Western China. A low-cost wastewater monitoring network offers an alternative solution. To explore this, we first compared the sensitivity of a MeltArray-based qPCR assay, which detects 25 respiratory pathogens, with singleplex qPCR using both mock and real wastewater samples. We then employed this MeltArray assay to detect these respiratory pathogens in wastewater from a low-income region in Xi’an city from September 2023 to January 2024. Following this, qPCR and MLST were employed to quantify the dynamics of positive respiratory pathogens and confirm their genotypes. Results showed unusual surges in sewage influenza A virus (IAV) and adenovirus levels starting in October 2023, persisting until late December. Additionally, influenza B virus (IBV) outbreaks were identified beginning in late December. These findings matched the positivity rates reported by a sentinel hospital. For coronaviruses, HCoV-229E/OC43 were consistently detected in wastewater, while SARS-CoV-2 was occasionally found. The qPCR assays revealed continuous increases in sewage Mycoplasma pneumoniae and Hemophilus influenzae concentrations since September, both peaking in October. Genotyping confirmed the circulation of specific bacterial genotypes in the region. Therefore, to the best of our knowledge, this study is possibly the first to evaluate the efficacy of qPCR assays for wastewater monitoring of respiratory bacterial pathogens. Thus, these findings provide significant insights into the co-circulation of various respiratory pathogens during the autumn and winter of 2023, thereby suggesting that wastewater surveillance could be a powerful tool for the early warning of respiratory diseases.

Graphical abstract

Keywords

Respiratory bacterial pathogens / Wastewater surveillance / MeltArray / Mycoplasma pneumoniae

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CRediT Authorship Contribution Statement

Hui Li: Methodology, Experiment, Analysis, Writing–original draft. Haifeng Li: Methodology, Experiment, Analysis, Writing–original draft. Xin Du: Methodology, Experiment. Zhenyu Liu: Methodology, Experiment. Fenglan He: Methodology, Experiment. Xinyan Du: Methodology, Experiment, Analysis. Zengguo Wang: Conception, Resources, Writing–review and editing. Chunlong Zhu: Experiment, Resources, Writing–review and editing. Songzhe Fu: Writing–original draft. Resources, Writing–review and editing.

Conflicts of Interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

We thank volunteers for the sampling assistance. The authors would like to express their gratitude to EditSprings for the expert linguistic services provided. This study was supported by the National Natural Science Foundation of China (Nos. 81903372 and 82172312) and the Open fund of National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases (China) (No. 2024NITFID310).

Electronic Supplementary Material

Supplementary material is available in the online version of this article at https://doi.org/10.1007/s11783-025-1981-z and is accessible for authorized users.

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