Impact of reference design on estimating SARS-CoV-2 lineage abundances from wastewater sequencing data (preprint)

Sequencing of SARS-CoV-2 RNA from wastewater samples has emerged as a valuable tool for detecting the presence and relative abundances of SARS-CoV-2 variants in a community. By analyzing the viral genetic material present in wastewater, public health officials can gain early insights into the spread of the virus and inform timely intervention measures. The construction of reference datasets from known SARS-CoV-2 lineages and their mutation profiles has become state-of-the-art for assigning viral lineages and their relative abundances from wastewater sequencing data. However, the selection of reference sequences or mutations directly affects the predictive power. Here, we show the impact of a mutation- and sequence-based reference reconstruction for SARS-CoV-2 abundance estimation. We benchmark three data sets: 1) synthetic 'spike-in' mixtures, 2) German samples from early 2021, mainly comprising Alpha, and 3) samples obtained from wastewater at an international airport in Germany from the end of 2021, including first signals of Omicron. The two approaches differ in sub-lineage detection, with the marker-mutation-based method, in particular, being challenged by the increasing number of mutations and lineages. However, the estimations of both approaches depend on selecting representative references and optimized parameter settings. By performing parameter escalation experiments, we demonstrate the effects of reference size and alternative allele frequency cutoffs for abundance estimation. We show how different parameter settings can lead to different results for our test data sets, and illustrate the effects of virus lineage composition of wastewater samples and references. Here, we compare a mutation- and sequence-based reference construction and assignment for SARS-CoV-2 abundance estimation from wastewater samples. Our study highlights current computational challenges, focusing on the general reference design, which significantly and directly impacts abundance allocations. We illustrate advantages and disadvantages that may be relevant for further developments in the wastewater community and in the context of higher standardization.

Monitoring SARS-CoV-2 in sewage: toward sentinels with analytical accuracy (preprint)

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemia has been one of the most difficult challenges humankind has recently faced. Wastewater-based epidemiology has emerged as a tool for surveillance and mitigation of potential viral outbreaks, circumventing biases introduced by clinical patient testing. Due to the situation urgency, protocols followed for isolating viral RNA from sewage were not adapted for such sample matrices. In parallel to their implementation for fast collection of data to sustain surveillance and mitigation decisions, molecular protocols need to be harmonized to deliver accurate, reproducible, and comparable analytical outputs. Here we studied analytical variabilities linked to viral RNA isolation methods from sewage. Three different influent wastewater volumes were used to assess the effect of filtered volumes (50, 100 or 500 mL) for capturing viral particles. Three different concentration strategies were tested by electronegative membranes, polyethersulfone membranes, and anion-exchange diethylaminoethyl cellulose columns. To compare the number of viral particles, different RNA isolation methods (column-based vs. magnetic beads) were compared. The effect of extra RNA purification steps and different RT-qPCR strategies (one step vs. two-step) were also evaluated. Results showed that the combination of 500 mL filtration volume through electronegative membranes and without multiple RNA purification steps (using column-based RNA purification) using two-step RT-qPCR avoided false negatives when basal viral load in sewage are present and yielded more consistent results during the surveillance done during the second-wave in Delft (The Hague area, The Netherlands). By paving the way for standardization of methods for the sampling, concentration and molecular detection of SARS-CoV-2 viruses from sewage, these findings can help water and health surveillance authorities to use and trust results coming from wastewater based epidemiology studies in order to anticipate SARS-CoV-2 outbreaks. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=138 SRC="FIGDIR/small/21259751v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@c082acorg.highwire.dtl.DTLVardef@c2f9f9org.highwire.dtl.DTLVardef@a98445org.highwire.dtl.DTLVardef@12ad68e_HPS_FORMAT_FIGEXP M_FIG C_FIG

A pan-European study of SARS-CoV-2 variants in wastewater under the EU Sewage Sentinel System (preprint)

Wastewater based surveillance employing qPCR has already shown its utility for monitoring SARS-CoV-2 at community level, and consequently the European Commission has recommended the implementation of an EU Sewage Sentinel System. However, using sequencing for the determination of genomic variants in wastewater is not fully established yet. Therefore, we focused on the sequencing analysis of SARS-CoV-2 RNA in wastewater samples collected across 20 European countries including 54 municipalities. The results provide insight into the abundance and the profile of the mutations associated with the variants of concerns: B.1.1.7, P.1, B.1.351 and B.1.617.2, which were present in various wastewater samples. This study shows that integrating genomic and wastewater-based epidemiology (WBE) can support the identification of variants circulating in a city at community level.

Long-term monitoring of SARS-CoV-2 in wastewater of the Frankfurt metropolitan area in Southern Germany (preprint)

Wastewater-based epidemiology (WBE) is a great approach that enables us to comprehensively monitor the community to determine the scale and dynamics of infections in a city, particularly in metropolitan cities with a high population density. Therefore, we monitored the time course of the SARS-CoV-2 RNA concentration in raw sewage in the Frankfurt metropolitan area, the European financial center. To determine the SARS-CoV-2 concentration in sewage, we continuously collected samples from two wastewater treatment plant (WWTP) influents (Niederrad and Sindlingen) serving the Frankfurt metropolitan area and performed RT-qPCR analysis targeting three genes (N gene, S gene, and ORF1ab gene). In August, a resurgence in the SARS-CoV-2 RNA load was observed, reaching 3 x 1013 copies/day, which represents similar levels compared to April with approx. 2 x 1014 copies/day. This corresponds to an also continuous increase again in COVID-19 cases in Frankfurt since August, with an average of 28.6 incidences, compared to 28.7 incidences in April. Different temporal dynamics were observed between different sampling points, indicating local dynamics in COVID-19 cases within the Frankfurt metropolitan area. The SARS-CoV-2 load to the WWTP Niederrad ranged from approx. 4 x 1011 to 1 x 1015 copies/day, the load to the WWTP Sindlingen from approx. 1 x 1011 to 2 x 1014 copies/day, which resulted in a preceding increase in these loading in July ahead of the weekly averaged incidences. The study shows that WBE has the potential as early warning system for SARS-CoV-2 infections and as monitoring system to identify global hotspots of COVID-19.