Capturing the SARS-CoV-2 infection pyramid within the municipality of Rotterdam using longitudinal sewage surveillance (preprint)

Background: Despite high vaccination rates in the Netherlands, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to circulate. Longitudinal sewage monitoring was implemented along with the notification of cases as two parts of the surveillance pyramid to validate the use of sewage surveillance for monitoring SARS-CoV-2, as an early warning tool, and to measure the effect of interventions. Methods: Sewage samples were collected from nine neighborhoods from September 2020 to November 2021, and compared with reported cases. Comparative analysis and modeling were performed to understand the correlation between wastewater and case trends. Findings: Using high resolution sampling, normalization of wastewater SARS-CoV-2 concentrations and normalization of reported positive tests for testing delay and intensity, the incidence of reported positive tests could be modeled based on sewage data, and trends in both surveillance systems coincided. The high collinearity implied that high levels of viral shedding around the onset of disease largely determines SARS-CoV-2 levels in wastewater and the observed relation was independent of SARS-CoV-2 variants and vaccination levels. Interpretation: Wastewater surveillance can accurately display SARS-CoV-2 dynamics for small and large locations, and is sensitive enough to measure small variations in the number of infected individuals within or between neighborhoods. With the transition to a post-acute phase of the pandemic, continued sewage surveillance can help to keep sight on reemergence, but continued pyramid validation studies are needed to assess the predictive value of sewage surveillance with new variants. Funding: Horizon H2020, Adessium Foundation, STOWA, TKI, Ministry of Health, Welfare and Sport

Normalisation of SARS-CoV-2 concentrations in wastewater: the use of flow, conductivity and CrAssphages (preprint)

Over the course of the COVID-19 pandemic in 2020-2021, monitoring of SARS-CoV-2 RNA in wastewater has rapidly evolved into a supplementary surveillance instrument for public health. Short term trends (2 weeks) are used as a basis for policy and decision making on measures for dealing with the pandemic. Normalization is required to account for the varying dilution rates of the domestic wastewater, that contains the shedded virus RNA. The dilution rate varies due to runoff, industrial discharges and extraneous waters. Three normalization methods using flow, conductivity and CrAssphage, have been investigated on 9 monitoring locations between Sep 2020 and Aug 2021, rendering 1071 24-hour flow-proportional samples. In addition, 221 stool samples have been analyzed to determine the daily CrAssphage load per person. Results show that flow normalization supported by a quality check using conductivity monitoring is the advocated normalization method in case flow monitoring is or can be made available. Although Crassphage shedding rates per person vary greatly, the CrAssphage loads were very consistent over time and space and direct CrAssphage based normalization can be applied reliably for populations of 5600 and above.

Droplet Digital RT-PCR to detect SARS-CoV-2 variants of concern in wastewater (preprint)

Wastewater surveillance has shown to be a valuable and efficient tool to obtain information about the trends of COVID-19 in the community. Since the recent emergence of new variants, associated with increased transmissibility and/or antibody escape (variants of concern), there is an urgent need for methods that enable specific and timely detection and quantification of the occurrence of these variants in the community. In this study we demonstrate the use of RT-ddPCR on wastewater samples for specific detection of mutation N501Y. This assay enabled simultaneous enumeration of the concentration of variants with the 501Y mutation and Wild Type (WT, containing 501N) SARS-CoV-2 RNA. Detection of N501Y was possible in samples with mixtures of WT with low proportions of lineage B.1.351 (0.5%). The method could accurately determine the proportion of N501Y and WT in mixtures of SARS-CoV-2 RNA. The application to raw sewage samples from the cities of Amsterdam and Utrecht demonstrated that this method can be applied to determine the concentrations and the proportions of WT and N501Y containing SARS-CoV-2 RNA in wastewater samples. The emergence of N501Y in Amsterdam and Utrecht wastewater aligned with the emergence of B.1.1.7 as causative agent of COVID-19 in the Netherlands, indicating that RT-ddPCR of wastewater samples can be used to monitor the emergence of the N501Y mutation in the community. It also indicates that RT-ddPCR could be used for sensitive and accurate monitoring of current (like K417N, E484K) or future mutations present in SARS-CoV-2 variants of concern. Monitoring emergence of these mutations in the community via wastewater is rapid, efficient and valuable in supporting public health decision-making.

Monitoring SARS-CoV-2 circulation and diversity through community wastewater sequencing (preprint)

The current SARS-CoV-2 pandemic has rapidly become a major global health problem for which public health surveillance is crucial to monitor virus spread. Given the presence of viral RNA in feces in around 40% of infected persons, wastewater-based epidemiology has been proposed as an addition to disease-based surveillance to assess the spread of the virus at the community level. Here we have explored the possibility of using next-generation sequencing (NGS) of sewage samples to evaluate the diversity of SARS-CoV-2 at the community level from routine wastewater testing, and compared these results with the virus diversity in patients from the Netherlands and Belgium. Phylogenetic analysis revealed the presence of viruses belonging to the most prevalent clades (19A, 20A and 20B) in both countries. Clades 19B and 20C were not identified, while they were present in clinical samples during the same period. Low frequency variant (LFV) analysis showed that some known LFVs can be associated with particular clusters within a clade, different to those of their consensus sequences, suggesting the presence of at least 2 clades within a single sewage sample. Additionally, combining genome consensus and LFV analyses we found a total of 57 unique mutations in the SARS-CoV-2 genome which have not been described before. In conclusion, this work illustrates how NGS analysis of wastewater can be used to approximate the diversity of SARS-CoV-2 viruses circulating in a community.

Presence of SARS-Coronavirus-2 in sewage (preprint)

In the current COVID-19 pandemic, a significant proportion of cases shed SARS-Coronavirus-2 (SARS-CoV-2) with their faeces. To determine if SARS-CoV-2 is present in sewage during the emergence of COVID-19 in the Netherlands, sewage samples of 7 cities and the airport were tested using RT-PCR against three fragments of the nucleocapsid protein gene (N1-3) and one fragment of the envelope protein gene (E). No SARS-CoV-2 was detected in samples of February 6, three weeks before the first case was reported in the Netherlands on February 27. On March 5, the N1 fragment was detected in sewage of five sites. On March 15/16, the N1 fragment was detected in sewage of six sites, and the N3 and E fragment were detected at 5 and 4 sites respectively. This is the first report of detection of SARS-CoV-2 in sewage. The detection of the virus in sewage, even when the COVID-19 incidence is low, indicates that sewage surveillance could be a sensitive tool to monitor the circulation of the virus in the population.