key: cord-0899741-p7nsdimi
authors: Masachessi, Gisela; Castro, Gonzalo; Cachi, Ariana Mariela; Marinzalda, María de los Ángeles; Liendo, Matías; Pisano, María Belén; Sicilia, Paola; Ibarra, Gustavo; Rojas, Ricardo Manuel; López, Laura; Barbás, Gabriela; Cardozo, Diego; Ré, Viviana Elisabeth; Nates, Silvia Viviana
title: Wastewater based epidemiology as a silent sentinel of the trend of SARS-CoV-2 circulation in the community in central Argentina
date: 2022-05-04
journal: Water Res
DOI: 10.1016/j.watres.2022.118541
sha: 39f271f5ed0d675b3b55fdc2cf0356085eb9a80c
doc_id: 899741
cord_uid: p7nsdimi

Monitoring wastewater for the traces of viruses allows effective surveillance of entire communities, including symptomatic and asymptomatic infected individuals, providing information on whether a specific pathogen is circulating in a population. In the context of the COVID-19 pandemic, 261 wastewater samples from six communities of the province of Córdoba, Argentina were analyzed. From mid-May 2020 to the end of August 2021, raw sewage samples were collected from the central network pipe that enters into the Wastewater Treatment Plants (WWTP) in Córdoba city and five communities in the Punilla Valley. SARS-CoV-2 was concentrated by using the polyethylene glycol-6000 precipitation method. Viral genomes were extracted from concentrated samples, and N- and E-SARS-CoV-2 genes were detected by using real time RT-PCR. Wastewater samples that resulted positive for SARS-CoV-2 genome detection were subjected to viral variants of concern (VOCs) identification by real time RT-PCR. Overall, just by using the identification of the N gene or E gene, the rates of viral genome detection were 43.4% (86/198) and 51.5% (102/198) respectively, and by using both methodologies (positivity criterion: detection of N and / or E gene), the detection rate was 71.2% (141/198). Thereby, the optimal strategy to study the SARS-CoV-2 genome in wastewater would be the use of the combined detection of both genes. Detection of SARS-CoV-2 variants in wastewater reflected their circulation in the community, showing no VOCs detection in the first COVID-19 wave and their co-circulation with Gamma, Alpha and Delta VOCs during 2021. Therefore, SARS-CoV-2 Wastewater Based Epidemiology (WBE) described the introduction, permanence and/or the co-circulation of viral variants in the community. In geographical areas with a stable population, SARS-CoV-2 WBE could be used as an early warning sign of new COVID-19 cases, whereas in localities with a low number of inhabitants and high tourist influx, WBE may only be useful to reflect the circulation of the virus in the community. Overall, the monitoring of SARS-CoV-2 in wastewater can become a silent sentinel of the trend of viral circulation in the community, providing supplementary information for clinical surveillance to support public health measures.

• WBE can be used as a silent sentinel of SARS-CoV-2 circulation in a community.

• In stable populations, WBE can be an early warning tool of the increase in the number of COVID-19 cases.

• In small and touristic localities, WBE can only reflect the viral circulation.

• WBE is a useful tool to monitor the dynamics of circulation of SARS-CoV-2

VOCs.

• WBE of SARS-CoV-2 provides supplementary information for clinical surveillance.

Wastewater based epidemiology as a silent sentinel of the trend of SARS-CoV-2 circulation in the community in central Argentina (Su S, 2020 ) (Megyeri K, 2021) , (Ramachandran P, 2020) , (Redd WD, 2020) , (Calitri, 2021) .

This is because SARS-CoV-2 has been shown to enter the cell by the Angiotensin Converting Enzyme-2 (ACE2) receptor (Hikmet, 2020) , (Zou X, 2020) which is widely expressed in many types of cells and tissues of the GI tract. Specifically, SARS-CoV-2

can infect and replicate in esophageal cells and enterocytes, leading to direct damage to the intestinal epithelium. Different studies report that individuals infected with SARS-CoV-2 can excrete the virus by feces (Tian Y, 2020) , (Wu Y, 2020) , (Wang W, 2020) . It is estimated that feces of people infected with SARS-CoV-2 contain 10 4 -10 8 RNA copies per gram up to approximately three weeks after the onset of symptoms, with a mean excretion time of 17 days, regardless of the presence or absence of the GI and other COVID-19 symptoms (Sender R, 2021) , (Pan Y, 2020) , (Xu Z, 2021) , (Akiyama Y, 2021 ) (Yuan C, 2021) , (Guo M, 2021) . (Randazzo W, 2020) , (Ahmed W A. N., 2020) , (Medema G, 2020) , (Izquierdo-Lara R E. G., 2021), (Peccia J, 2020) , (Wurtz N, 2021) , (Sharif S, 2021) , (Haramoto E, 2020) , (Ai Y, 2021) , (Fongaro G, 2021 ) , (Chavarria-Miró G, 2021) . In this way, wastewater data could supplement current clinical measures of the spread of the virus at a community level.

It is known that SARS-CoV-2 variants with multiple amino acid changes at the spike protein are emerging in different parts of the world, raising concerns regarding their possible impact on human health and vaccine immune response efficacy against the virus (Wilton T, 2021) , (Tegally H, 2021) , (Faria NR, 2021) . The World Health Organization (WHO) has classified SARS-CoV-2 variants into variants of concern (VOC) and variants of interest (VOI), according to the virus's features given by the mutations (transmissibility, virulence, clinical disease presentation, response to the vaccine) (WHO, https://www.who.int/activities/tracking-SARS-CoV-2-variants/tracking-SARS-CoV-2-variants., 2021). In this sense, environmental surveillance of SARS-CoV-2 could constitute a useful tool for viral surveillance in a community, providing an early warning of the introduction of variants into the population (Wilton T, 2021) .

In the present study, the wastewater samples of six communities in the province of 

Cordoba city is the capital of the province of Córdoba, located in the central region of Argentina (31°25′00″S 64°11′00″O) and has 1,317,298 inhabitants with a population, density of 2308 habitants/km 2 (INDEC, 2010) ( Fig. 1 ). The sewerage system covers 50% of the population and no industrial wastewater is treated in this facility. The main wastewater treatment plant (WWTP) is named "Bajo Grande" and receives approximately the discharge of 50% of the sewage network coverage.

Punilla Valley is a quasi-urbanized valley in the metropolitan area, which is located in the center-northwest of the province of Córdoba (31°02′S 64°30′O). At present, it is one of the main tourist centers of Argentina ( Fig. 1) . In this region, there are a lot of lakes-reservoirs, such as San Roque dam, on whose shores is located the city of Villa Carlos Paz, the largest city in Punilla Valley. According to the most recent National Census, Villa Carlos Paz has a population of 62,423 inhabitants and 37% of the population is connected to the sewage network. In summer, this city triples its population due to the large influx of tourists. La Falda additionally resides in this valley and maintains a population of 16,335 inhabitants. There are also smaller towns, that stand out as tourist centers, as Villa Giardino (6,702 inhabitants), Huerta Grande (5,925 inhabitants), and

Valle Hermoso (6,187 inhabitants). La Falda has a sewage effluent plant that treats its own effluents and wastewater from Villa Giardino and Huerta Grande towns. In total, the sewage network coverage of these three localities is 51%. Valle Hermoso, whose main economic activity is tourism due to its spas and rivers with crystal clear waters, has a sewer network coverage of 20%.

From mid-May 2020 to the end of August 2021, raw sewage samples were collected from central network pipes from four wastewater treatment plants ( 

SARS-CoV-2 was concentrated in sewage specimens by using the polyethylene glycol-6000 (PEG-6000) precipitation method, according to the WHO guideline for environmental surveillance of poliovirus circulation (WHO, Guidelines for environmental surveillance of poliovirus circulation, 2003) using the method described previously by Lewis and Metcalf (1988) . Briefly, sewage was centrifuged at 4750 x g for 20 min at 4 °C. Supernatant (S1) was maintained at 4 °C to be used later, and the sediment was 

Each concentrated sample (1 mL) was subjected to RNA automated extraction using (Orf1ab) genes. In this study, only results derived from N gene detection were analyzed since it has a higher analytical sensitivity for detection than the Orf1ab gene (i.e., from the total samples that resulted positive for N gene detection (n=88), only 54.5% (n=48) were also positive for ORF 1ab gene. None of the samples resulted negative for N gene and positive for Orf1ab gene detection).

As of September 2020, N gene detection was coupled to envelope (E) detection, using the LightMix® Modular SARS and Wuhan CoV E-gene kit (TIB MOLBIOL GmbH, Berlin, Germany, distributed by Roche), to increase the SARS-CoV-2 genome detection sensitivity.

In all cases, assays were run according to the manufacturer's instructions. Real time RT-PCR reactions were carried out using a Cobas Z 480 equipment (Roche, Germany).

The cycle conditions and the temperature ramps used for each of the amplification kits used are described in tables 2 and 3. To estimate the SARS-CoV-2 recovery rate from wastewater by the PEG method, sewage subsamples from a sewage matrix previously determined as SARS-CoV-2 negative were seeded with a SARS-CoV-2 viral suspension to a final concentration of 300 PFU/mL. As negative control sewage did not seed with the SARS-CoV-2 viral dilution was tested in parallel. Samples were then concentrated and analyzed by real time -RT-PCR following the methodology described above (2.3, 2.4 and 2.5 sections).

Ct values obtained were extrapolated in the curve to obtain the corresponding viral PFU/mL. SARS-CoV-2 recovery rate was calculated based on the PFU/mL as follows: Recovery rate (%) = (viral PFU/mL recovered / viral PFU/mL seeded) × 100. The recovery rate mean value was 15.75%; with a range of 12.65% -22.85%. Table 4 .

The number of individuals excreting the virus by faecal matter at the time the wastewater sample was obtained was estimated based on the following: 1) the 35% of individuals infected with SARS-CoV-2 excrete the virus via faecal matter (Wu Y, 2020) , (Wang W, 2020) ; 2) the average SARS-CoV-2 genome excretion time is 17 days (Tian Y, 2020) ; 3) the sewage network coverage of the communities studied (official data available from the study communities) (WHO, Guidelines for environmental surveillance of poliovirus circulation, 2003); and 4) the daily report of SARS-CoV-2 cases in each studied locality studied. 

The sampling of wastewater from the main pipeline of Bajo Grande WWTP started on 11 th May 2020, when clinical cases continued to increase gradually in Córdoba city. The 

The SARS-CoV-2 detection in wastewater collected in WWTP from the localities of Valle Hermoso and La Falda (which includes sewage from La Falda, Huerta Grande and Villa Giardino) reflected a common viral pattern, which was characterized by a continued SARS-CoV-2 genome detection. This pattern reflected the circulation of the virus in the studied localities. Besides, the epidemic curves in all the studied localities were defined by peaks of increasing and decreasing incidence of clinical COVID-19

cases over time (Figures 5 and 6 ). Alpha were detected for the first time in wastewaters in co-circulation with no VOCs. In the month of August 2021, VOC Delta was registered in co-circulation with Gamma and no VOCs (Figures 4, 5 and 6 ).

The study of SARS-CoV-2 epidemiology based on wastewater from Córdoba and (2020) and indicate that SARS-CoV-2 genome shedding may occur soon after infection. Therefore, different authors highlight the utility of SARS-CoV-2 monitoring in wastewater as a non-invasive early warning tool to support health surveillance (Fongaro G, 2021 ) , (Larsen DA, 2020) , (Mousazadeh M, 2021) .

However, in La Falda, Villa Giardino, Huerta Grande and Valle Hermoso, the SARS-CoV-2 genome in sewage was continuously detected throughout the studied period, accompanying a pattern characterized by abrupt increases and decreases in the reported incidence of COVID-19 cases. This could be explained because the studied localities have a low number of inhabitants and a high influx of tourists. During summer and winter holidays and special holiday periods such as Easter and carnival, their populations can even be four times higher with tourists entering and leaving the place week after week. It is likely that infected individuals remain a few days in the locality, being registered as cases in the health system but no longer contributing to the excretion of SARS-CoV-2 in the sewage system. Therefore, WBE is a population-based study tool, which necessarily requires a stable population.

The sensitive detection of SARS-CoV-2 RNA in wastewater depends in part on the molecular-based methods employed, which remain diverse and unstandardized (Hamouda M, 2021) . Different authors have reported discrepancies among CDC N1 trials with CDC N2, CDC N3, and E_Sarbeco for wastewater samples in the emerging epidemic in different parts of the world (Medema G, 2020) , (Wu F, 2020) , (Ahmed W B.

A., 2020), (Giraud-Billoud M, 2021). In our work, commercial kits were used to detect N and E SARS-CoV-2 genes. The results obtained showed discrepancies in the detection of the SARS-CoV-2 genome using one or another, indicating that the optimal strategy to study the SARS-CoV-2 genome in wastewater would be the use of the combined detection of both genes.

One of the limitations of this work is that the genomic load was not quantified clinical surveillance, the implemented strategy resulted in a useful tool that allowed the characterization of circulation dynamics of variants in the studied localities (Castro G. M., 2021) . To our knowledge, this is the first research work that implements this point mutation detection strategy for the identification of SARS-COV-2 variants in wastewater samples. In this sense, in addition to all the advantages previously described for this strategy (cost-effective, short time-consuming, no extra equipment required or specialized personnel and massive rapid typification) (Castro G. M., 2021) , it was possible to typify samples with Cts>30, which is often difficult in traditional sequencing typing techniques (Crits-Christoph A, 2021) (Izquierdo-Lara R E. G., 2021). Additionally, this methodology allowed detection of more than one VOC simultaneously in the same sample. In Córdoba province, as in some countries around the world, like Australia (Medema G, 2020) , New Zealand (Ahmed W B. A., 2020), the Netherlands (Bhattacharya P, 2021) and parts of Brazil (Claro ICM, 2021) , environmental surveillance was adopted by the Ministry of Health as a tool to track the circulation dynamics of SARS-CoV-2 in the community.

• The monitoring of SARS-CoV-2 in wastewater can become a silent sentinel of the trend of viral circulation in the community, providing different information according to the size and dynamics of the population.

• In geographical areas with a stable population, the first detection of the SARS-CoV-2 viral genome in wastewater after a period of non-detection, is an early warning sign of the subsequent increase in COVID-19 cases.

• In localities with a low number of inhabitants (lower than 16,000 inhabitants) and

high tourist influx, in which a continuous detection of SARS-CoV-2 genome in wastewater was detected through the studied period, this tool may be useful only to reflect the circulation of the virus in the community.

• The profile of variants detected in wastewater showed that WBE of SARS-CoV-2 allows to know the dynamics of circulation of the viral variants in the studied communities.

• WBE of SARS-CoV-2 could provide supplementary information for clinical surveillance to support public health measures. 

The authors declare that they do not have competing financial interests or personal relationships that could have influenced the work reported in this paper. Cooperativa Integral Regional de Provisión de Servicios Públicos, Vivienda y Consumo Limitada (COOPI) of Villa Carlos Paz, which managed the permits to perform the sampling of wastewater as well as assuming the responsibility for each sampling; and

the Ministry of Health of the Province of Córdoba, which declared this investigation of public interest and provided financial support. We also thank the Ministry of Science, Technology and Innovation of Argentina (MinCyT) for its collaboration in coordinating institutional interactions. We express our gratitude to the entire team of the Central Laboratory of the Province for the support and responsibility in processing the detection of the SARS-CoV-2 genome in each of the stages. We also want to thank MinCyT, the National Agency for the Promotion of Research, Technological Development and

Innovation and the National Defense University for subsidizing this research work. 

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Table 2-Cycling conditions for the DisCoVery SARS-CoV-2 RT-PCR Detection Kit, carried out in a Z480 Equipment

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This work was possible thanks to the collaboration and coordination of different government agencies and institutions: The Municipality of the City of Córdoba and the