In April 2020, ICRA was commissioned by ACA and ASPCAT to design and implement a surveillance network to track the circulation of SARS-CoV-2 in wastewater across Catalonia. The main objective was to generate quantitative data on the spread of SARS-CoV-2 in the Catalan population that could be used by health authorities to complement clinical epidemiological indicators in their decision-making and management of the COVID pandemic. -19. SARSAIGUA was approved by the Catalan government on June 20, 2020, based on the state of alert decreed by the Spanish authority (RD 463/2020) resulting from the health crisis caused by the pandemic. Sample collection and analysis began on July 6, 2020, approximately four months after the detection of the first clinical case of COVID-19 in Catalonia (February 25, 2020). The SARSAIGUA roadmap, from its conception to its final implementation, is described in the following sections.
Selection of treatment plants
The current number of urban wastewater treatment plants in Catalonia is 532, serving 97% of the Catalan population. The first step in SARSAIGUA was to select which wastewater treatment plants to monitor and how often, taking into account the maximum number of weekly samples that could be analyzed per laboratory (see “Laboratory analyses”). The selection was made by the coordination team, the ACA and the ASPCAT, and it was based on two criteria: (i) reaching the maximum percentage of the assisted population, and (ii) uniformly covering the entire Catalan territory. The operational and technical capabilities of each treatment plant in terms of sampling equipment (i.e. autosamplers capable of collecting composite samples over 24 h) and the availability of flow data were also taken into account to refine the final selection.
The sewage treatment plant selection procedure was a step-by-step process taking into account the served population of each sewage treatment plant as reported by the ACA and the territorial balance calculated as the average distance from any point in Catalonia to the nearest sewage treatment plant included in the network. Calculations were based on Euclidean distances and performed in R software19 version 4.1 using packages sp20 and raster21. First, we selected wastewater treatment plants serving more than 150,000 inhabitants, resulting in 7 wastewater treatment plants that cover 50% of the population (threshold A in Suppl. Fig. S1). These larger wastewater treatment plants are mainly located in the metropolitan area of Barcelona (Fig. 1). Second, we added the largest sewage treatment plant in each Catalan county that was not included in the previous step. This selection was made for 41 of the 42 Catalan departments fulfilling the technical conditions for good sample collection (see “Sample collection and delivery”). In this second stage, 42 treatment plants (including the 7 treatment plants from the first stage) were selected, resulting in a population coverage of 61.92% (threshold B in Suppl. Fig. S1 ). Third, the largest remaining treatment plants were finally added to the list until reaching a satisfactory coverage of up to 80% of the Catalan population without compromising the analytical and budgetary limits (threshold C in Suppl. Fig. S1, 56 treatment plants). As a result, we obtained a uniform distribution of the selected treatment plants on the territory while maintaining a high population coverage (80%) (Suppl. Fig. S1). Data above threshold C in Suppl. Figure S1 shows the cumulative incorporation of most of the remaining wastewater treatment plants in Catalonia (422 out of 532) until reaching almost total population coverage (nearly 97%), a scenario that far exceeded the capacity of analysis and the network budget. Figure 1 shows the distribution of the treatment plants finally selected. Overall, the total cost (including laboratory analyses, coordination costs, web design and sending of samples) for the monitoring of the 56 selected treatment plants was budgeted at €396,836 for the first 6 months (July-December 2020). In December 2020, the Catalan government extended the surveillance program until December 2022 with an additional cost of €947,430/year22.
The sampling frequency was set at one sample per week in 36 of the 56 selected treatment plants and every two weeks for the other 18, which resulted in the collection and analysis of 45 samples per week. Remarkably, some sewage treatment plants are monitored only during the summer season to better monitor municipalities receiving high tourism (eg Castell-Platja d’Aro, Vilaseca-Salou). Details of sample collection and sampling frequency for all monitored WWTPs are presented in Suppl. Table S1.
Collection and delivery of samples
Despite controversy over the best wastewater sample collection strategy for WBE studies (i.e., grab or composite samples)24, SARSAIGUA chose to take composite samples based on the flows at the entrance of the selected treatment plants, using refrigerated automatic samplers. This strategy was adopted based on studies demonstrating that composite samples provide more representative and less variable results than grab samples for viral particle quantification.24,25,26 and micropollutants27and also aligned with the recommendations subsequently proposed by the European Commission6. However, several technical constraints regarding the number of autosamplers available, the limitations of installing sampling equipment and the presence of sampling points with discharge waters hampered the use of this approach at all stations. supervised purification. In this respect, 91% (51 out of 56) of the treatment plants monitored carry out composite sampling proportional to the flow (supplementary table S1). In addition, 48 of the 56 factories (85%) collect sample fractions at 20-minute intervals and the remaining 8 (15%) collect hourly fractions. Sampling is carried out from 8:00 a.m. Monday to 7:40 a.m. Tuesday and composite samples are then prepared, either automatically or manually, from the available fractions. To facilitate the whole process and sample handling during collection and storage, we have prepared a detailed sampling protocol which has been distributed and explained to WWTP staff through online training sessions. before the start of the first sampling campaign (July 6, 2020; sampling protocol available on request).
After collection and preparation, weekly composite samples are sent on Tuesday morning to the laboratories in charge of molecular analyzes (see next section). To optimize time and resources, we have mapped the best shipping routes taking into account: (i) delivery start time (Tuesday, 8:00 am); (ii) the deadline for delivery to the laboratories (Tuesday, 3:00 p.m.), and (iii) the distances and routes between the various treatment plants. Due to the varying frequency of sewage treatment plants being monitored (weekly or bi-weekly) and the changing seasonal schedule for some sewage treatment plants, we ultimately calculated eight optimized sailing routes using packages. mapapi28 in the R software (see “Code availability” section).
The 45 weekly samples are distributed to the three reference laboratories with broad expertise in molecular diagnostics and environmental virology, two of which are at the University of Barcelona, namely: the Enteric Virus Laboratory (https://is.gd/GFSH7W ) and the Water and Food Contaminating Virus Laboratory (https://is.gd/rNmGr1); and the third in Reus, at EURECAT (https://eurecat.org/es/) composed by the Omic Sciences Unit and the Nutrition and Health Unit. The three laboratories fully comply with all the necessities required for the planned analyses, including internationally recognized expertise in environmental virology and optimized and validated protocols for quantifying viruses in complex environmental matrices. Additionally, all samples are handled and processed in a Biosafety Level 2 laboratory to minimize risk to the user and the environment, and to mitigate sample cross-contamination. Each lab receives 15 samples per week which are analyzed for SARS-CoV-2 genome abundance using optimized protocols. In particular, the wastewater samples are concentrated using the aluminum hydroxide adsorption-precipitation concentration method.29ultrafiltration using Centricon apparatus (Millipore Corp.)5or the CP-Select Concentrating Pipette (InnovaPrep LLC)30. Replicates of all water samples are stored at 4°C for one week as a backup. In addition, remaining viral concentrates are frozen at -80°C and used only if downstream molecular analyzes fail (see below).
Quantification of SARS-CoV-2 genomes is performed using RT-qPCR targeting a common genetic marker (N1) and two complementary targets, N2 and IP431.32. Mean values of RT-qPCR performance parameters for the three genetic targets analyzed are provided in Supplementary Table S2. To monitor the effectiveness of the entire procedure, a viral surrogate (transmissible gastroenteritis virus (TGEV) coronavirus or bacteriophage MS2) is added to the raw water samples along with the processing control needed to calculate the absolute value of recovery per sample (see 11 and 20 for more details). Furthermore, given the different analytical procedures carried out by each laboratory, several inter-laboratory comparison tests were periodically carried out (California. every three months during the first year) using crude samples, sample concentrates and synthetic RNA replicates to ensure an acceptable level of variability in results (z-score 33.