The measurement of the enterovirus and the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in sewage water is relevant in the early detection of the introduction or disappearance of these viruses in the ecosystem. We evaluated the co-circulation of the enteroviruses and SARS-CoV-2 in 81 sewage water samples collected between September 2021 and April 2023 from different regions of north and southeast Romania, at the border with Ukraine. We used, for the molecular detection of the pathogens, the multiplex real-time polymerase chain reaction (PCR) assay produced for respiratory samples and the Respiratory 2.1 Plus panel Biofire Film array. The isolation of enteroviruses was performed on cell culture lines, in accordance with the World Health Organization (WHO) recommendations. By molecular investigations, we detected the SARS-CoV-2 in 22 (27%) samples, and the human rhinovirus/enterovirus in 64 (79%) samples. By isolation on cell culture lines, 27 samples (33,33%) were positive for non-polio enteroviruses, and no poliovirus strains were isolated, proving the maintenance of the polio-free status in Romania. In an emergency situation, the molecular detection of the pathogens in sewage water using a PCR system integrating sample preparation, amplification, detection, and analysis in 1 h could be implemented.

  • During the pandemic, the co-circulation of non-polio enteroviruses and SARS-CoV-2 was recorded in sewage water samples collected in Romania, at the border with Ukraine.

  • The surveillance of the pathogens in the sewage water can be a tool for mitigation of the potential outbreaks.

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