Simultaneous detection of influenza A, B and respiratory syncytial virus in wastewater samples by one-step multiplex RT-ddPCR assay.

BACKGROUND: After the occurrence of the COVID-19 pandemic, detection of other disseminated respiratory viruses using highly sensitive molecular methods was declared essential for monitoring the spread of health-threatening viruses in communities. The development of multiplex molecular assays are essential for the simultaneous detection of such viruses even at low concentrations. In the present study, a highly sensitive and specific multiplex one-step droplet digital PCR (RT-ddPCR) assay was developed for the simultaneous detection and absolute quantification of influenza A (IAV), influenza B (IBV), respiratory syncytial virus (RSV), and beta-2-microglobulin transcript as an endogenous internal control (IC B2M). RESULTS: The assay was first evaluated for analytical sensitivity and specificity, linearity, reproducibility, and recovery rates with excellent performance characteristics and then applied to 37 wastewater samples previously evaluated with commercially available and in-house quantitative real-time reverse transcription PCR (RT-qPCR) assays. IAV was detected in 16/37 (43%), IBV in 19/37 (51%), and RSV in 10/37 (27%) of the wastewater samples. Direct comparison of the developed assay with real-time RT-qPCR assays showed statistically significant high agreement in the detection of IAV (kappa Cohen's correlation coefficient: 0.834, p = 0.001) and RSV (kappa: 0.773, p = 0.001) viruses between the two assays, while the results for the detection of IBV (kappa: 0.355, p = 0.27) showed good agreement without statistical significance. CONCLUSIONS: Overall, the developed one-step multiplex ddPCR assay is cost-effective, highly sensitive and specific, and can simultaneously detect three common respiratory viruses in the complex matrix of wastewater samples even at low concentrations. Due to its high sensitivity and resistance to PCR inhibitors, the developed assay could be further used as an early warning system for wastewater monitoring.

Wastewater surveillance of the most common circulating respiratory viruses in Athens: The impact of COVID-19 on their seasonality.

Due to governments' actions to contain the spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the activity of common circulating respiratory viruses was significantly disrupted after the COVID-19 pandemic and thorough surveillance of respiratory pathogens was considered essential worldwide. Wastewater-based epidemiology has proven to be a valuable tool, that provides complementary information on disease outbreaks and is increasingly used to study the infection dynamics of other viruses, apart from SARS-CoV-2. The aims of the present study were the detection of four commonly circulating respiratory viruses: SARS-CoV-2, influenza A, B and Human Respiratory Syncytial Virus (RSV), the evaluation of the COVID-19 pandemic impact on their seasonality and the determination of the possible common trends in the viral load of these viruses in the wastewater of the Attica region. A standardized and validated concentration and extraction protocol was used, generic for all four viruses, followed by Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR) assays. The study proved that there was a prolonged period when all four viruses circulated in the population and an early outbreak of seasonal influenza and RSV in 2022-2023, compared to data from the pre-COVID-19 period. SARS-CoV-2, influenza A and RSV concentrations showed peak levels during December, followed by a slight decline in influenza A concentrations, followed by steady increase of influenza B concentrations in January 2023. SARS-CoV-2 was the dominant virus throughout the whole study period. This is the first study in Greece that investigated the most common circulating viruses simultaneously and in one of the largest timelines, providing crucial information about their infection dynamics during a period when an outbreak of respiratory diseases was declared by the National Public Health Organization. Presented results highlight the establishment of environmental surveillance as a non-invasive and complementary virus outbreak monitoring tool and the importance of influenza A, B and RSV integration into a wastewater-based surveillance system to help in disease management.