Environmental genomic surveillance of SARS-CoV-2 in wastewater in Rio de Janeiro, Brazil

The coronavirus disease 2019 (COVID-19) caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) was identified for the first time in Wuhan, China at the end of 2019 and on 11 March 2020, the World Health Organization declared COVID-19 a pandemic (la Rosa et al. 2020). SARS-CoV-2 is an enveloped, positive-stranded RNA virus, with a spiky crown-shaped surface and belongs to the Coronaviridae family; other members of the family such as Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-1) emerged in China in 2002, and Middle East Respiratory Syndrome (MERS-CoV) emerged in Saudi Arabia in 2012 (Algaissi et al. 2020; Magro et al. 2020; Mordecai & Hewson 2020; Girón-Navarro et al. 2021).

While primarily infecting lung cells, SARS-CoV-2 can infect the gastrointestinal tract as well (Kitajima et al. 2020). Alongside the usual symptoms of COVID-19, including fever, dry cough, dyspnea and loss of smell, some studies indicate that patients may also display symptoms associated with the gastrointestinal tract (Li et al. 2020). Detection of viral RNA in the stool of both symptomatic and asymptomatic individuals implies that SARS-CoV-2 can be excreted in stool and other bodily secretions, and consequently transported to wastewater treatment plants (WWTPs) or indirectly into water resources (Kitajima et al. 2020; Xiao et al. 2020). The SARS-CoV-2 RNA has been detected in wastewater samples in several countries, including Italy, Japan, Netherlands, Australia, Spain, United States and Brazil (Ahmed et al. 2020; la Rosa et al. 2020; Medema et al. 2020; Randazzo et al. 2020; Sherchan et al. 2020; Fongaro et al. 2021). Increased circulation of the virus in the population enhances the viral load in sewage systems, as well as the risk of transmission of pathogens from humans to the aquatic environment (Achak et al. 2021; Black et al. 2021).

In the context of the COVID-19 pandemic, wastewater-based epidemiology (WBE) is being applied for the detection of SARS-CoV-2 discharged into sewage via feces (Kitajima et al. 2020). The WBE has also been employed in previous cases to screen for infectious diseases, monitor illicit drug use and address other health concerns (Zuccato et al. 2008; Farkas et al. 2020). WBE provides a non-invasive and cost-effective way to track the spread of disease and has become increasingly important in the context of the ongoing COVID-19 pandemic (Cervantes-Avilés et al. 2021). It allows detection of SARS-CoV-2 in wastewater samples before they are clinically screened and can be used to estimate the dissemination virus in each region (Wu et al. 2020). SARS-CoV-2 was detected as early as 27 November 2019, 56 days in advance of the first confirmed COVID-19 case in the Americas (in the USA) (Fongaro et al. 2021). Medema et al. (2020) observed the presence of SARS-Coronavirus-2 RNA in sewage in the early stage of the epidemic in the Netherlands. In the same way, SARS-CoV-2 has been repeatedly detected in human sewage since January 2020 in Barcelona, Spain (Chavarria-Miró et al. 2021).

Several studies have shown that SARS-CoV-2 RNA can persist in wastewater for several days, even after patients have recovered from COVID-19 (Wu et al. 2020; Robinson et al. 2022). This means that WWTPs may serve as a source of ongoing exposure to the virus, especially in communities with high rates of COVID-19 transmission (Gholipour et al. 2021; Girón-Navarro et al. 2021). With the onset of the pandemic and the emergence of SARS-CoV-2 variants, the scientific community began to track and monitor genomic alterations present in the virus through Genomic Surveillance (Cyranoski 2021). Whole-genome sequencing followed by bioinformatic analysis are essential to understand the factors affecting SARS-CoV-2 occurrence, including genetic mutations and new variants description (Lauring & Hodcroft 2021).

This study applied wastewater surveillance to investigate the presence of SARS-CoV-2 from hospital and mixed wastewater effluents between January and December 2021. For this purpose, we evaluate the genetic diversity of SARS-CoV-2 in raw and treated sewage from two WWTPs. Sewage samples concentrated by PEG8000 were analyzed by RT-qPCR using primers toward genes E and N1. Although we were not able to assemble whole genomes in our samples, interpretation in data analysis software resulted in possible variants of concern (VOCs) such as Delta, Gamma, Omicron and Mu, according to characteristic mutations. The data obtained in the present study suggest that genomic surveillance could be considered an innovative approach capable of revealing SARS-CoV-2 variants in wastewater samples.

Weekly samples were obtained from two WWTPs, between January and December 2021. The WWTP1, localized in a hospital in Rio de Janeiro city, exclusively receives effluents from hospitals and has a tertiary treatment system, with biological treatment using the Moving Bed Biofilm Reactors (MBBR) process followed by disinfection with sodium hypochlorite. Meanwhile, the WWTP2, localized in a research institution in Rio de Janeiro city, receives effluents composed of a mixture of laboratory and domestic wastewater. This plant has a biological treatment system at the secondary level with the activated sludge process, extended aeration variant and has the following units: grating, raw sewage lift, desander, aeration tank, secondary decanter, sludge recirculation lift, digester tank and drying beds. Through sampling consisting of 50 mL aliquots every 1 h, 500 mL was collected from each point (affluent and effluent). The samples were transported under refrigeration to the laboratory and processed within 24 h.

The samples were filtered using a 0.22-μm syringe filter to remove probable bacterial cells and other debris (Wu et al. 2020; Nour et al. 2021). Then, 4 g of PEG 8000 (10% w/v, Sigma) and 0.9 g of sodium chloride (NaCl) were added in an aliquot of 40 mL of the filtrate. The samples were homogenized to dissolve the solutes and then subjected to centrifugation at 15,000g for 2 h at 4 °C. After centrifugation, the supernatant was discarded and the pellet was resuspended with 1 mL of saline-phosphate buffer solution (0.1M PBS, pH 7.2) (Lu et al. 2020; Wu et al. 2020).

The extraction of RNA viral was performed using the Bio-Gene DNA/RNA extraction of Viral Kit (Bioclin) according to the protocol of the manufacturer. The RNA concentration was measured using Nanodrop (Thermo Fisher Scientific) to assess the quality of the genetic material extraction process. For the detection of SARS-CoV-2 through real-time reverse transcription polymerase chain reaction (RT-qPCR), the molecular kit SARS-CoV-2 (Biomanguinhos) was used for detection of the E gene using the primers E_Sarbeco_F (5′-ACAGGTACGTTAATAGTTAATAGCGT-3′), E_Sarbeco_R (5′-ATATTGCAGCAGTACGCACACA-3′) and probe E_Sarbeco_P1 (5′-FAM-ACACTAGCCATCCTTACTGCGCTTCG-BBQ-3′) (Corman et al. 2020). Thus, the E gene was the initial marker used to detect SARS-CoV-2 and the N gene was used to confirm that the sample would not be a false negative. Negative samples for the E gene were subjected for amplification of the N1 gene using primers 2019-nCoV_N1 (5′-GACCCCAAAATCAGCGAAAT-3′), 2019-nCoV_N1-R (5′-TCTGGTTACTGCCAGTTGAATCTG-3′) and probe 2019-nCov_N1-P (FAM-ACCCCGCAT/ZEN/TACGTTTGGTGGACC-3IABkFQ) (CDC 2020).

The positive samples for SARS-CoV-2 were sequenced on the MiSeq platform (Illumina) at the National Institute for Quality Control in Health (INCQS) using the CovidSeq kit (Illumina) as described by the manufacturer's instructions. The libraries were sequenced on a V2-type cartridge for 300 cycles according to kit protocols. The kit was used as described by the manufacturer's instructions. The libraries were sequenced on a V2-type cartridge for 300 cycles according to kit protocols. The Fastp was used to trim reads with a cutoff >30 quality Phred Score (Chen et al. 2018). The trimmed reads were assembled using SPAdes through pipeline coronaSPAdes, a special mode of rnaviralSPAdes specifically aimed for SARS-CoV-2 de novo assembly (Meleshko et al. 2022). For lineage classification, the output FASTA file from coronaSPAdes was submitted to NextClade (https://clades.nextstrain.org) and Pangolin (Phylogenetic Assignment of Lineages of Named Global Outbreaks) (https://github.com/cov-lineages/pangolin) (Cleemput et al. 2020; Rambaut et al. 2020). The presence of characteristic mutations in the VOCs and variants of interest (VOIs) obtained in our study were confirmed on the website (https://outbreak.info/) in accordance with the WHO recommendation (WHO 2022).

The sequence sets were deposited in GenBank under BioProject PRJNA950421.

Out of 152 wastewater samples, 76 were from WWTP1 (38 from the affluent and 38 from the effluent) and 76 from WWTP2 (38 from the affluent and 38 from the effluent). In WWTP1, 47.4% (36/76) of the samples presented the E gene (22 in the affluent and 14 in the effluent). The negative samples for the E gene (n = 40) were evaluated for the presence of the N1 gene, where 11.6% (13/40) of the WWTP1 samples presented the N1 gene (5 in the affluent and 8 in the effluent). In WWTP2, only 5.3% (4/76) of the samples presented the SARS-CoV-2 E gene. In contrast, none of the samples presented the N1 gene. Weekly samples from WWTP1 were grouped according to the month of collection to carry out genomic sequencing (Table 1).

Partial sequencing of the SARS-CoV-2 genomes resulted in the identification of VOCs and VOIs throughout the collection period. It was possible to identify the Mu, Delta, Gamma and Omicron variants in the affluents and effluents of WWTP1 (Table 2); on the contrary, no variants were observed in WWTP2. The table with all suggested variants is available in Supplementary Table S2.

Table 2

Suggested variants of concern after analysis of sequencing of SARS-CoV-2 of WWTP1

Genes:Mutations marked in red represent the characteristic mutations of variants deposited in databases (https://outbreak.info/).

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WBE could become an extremely crucial tool in the management of public health during and after the COVID-19 pandemic, capable of obtaining epidemiological information and signaling risk factors for the environment and public health (Cervantes-Avilés et al. 2021). This requires the application of effective methods in the concentration and detection of SARS-CoV-2 in wastewater (Ahmed et al. 2020). The advancement of molecular approaches, such as RT-qPCR, allowed obtaining information on the presence of viruses in different environments, including wastewater (Haramoto et al. 2018). It is important to note that many assays were aimed at clinical samples and, therefore, may have limitations when performed in environments containing inhibitors, in addition to the presence of other viruses (Ahmed et al. 2022).

Although there is no consensus on the best targets, the detection of SARS-CoV-2 has been carried out using the N, E, ORF, M and S genes (CDC 2020; Corman et al. 2020; Vogels et al. 2020). In the present study, the SARS-CoV-2 was detected by E and N1 genes in wastewater samples. Meanwhile, in Spain's sewage, the assay suggested by CDC/USA using the detection of the N1 gene outperformed the assay toward M gene by Charité Institute (Kim et al. 2020; Pérez-Cataluña et al. 2021). On the other hand, another study by Corman et al. (2020) reported that analyses using the E gene were superior to analyses using the N gene.

In the present study, it was possible to detect SARS-CoV-2 in WWTP1 during all analyzed months of 2021. This plant exclusively receives effluents from hospitals and performs tertiary treatment with MBBR followed by disinfection with sodium hypochlorite. Although MBBR treatment is considered an efficient procedure in reducing SARS-CoV-2 RNA, it does not guarantee the complete removal of viral RNA fragments (Kostrytsia et al. 2022). The WWTP2, which presented SARS-CoV-2 only in January 2021, receives mixed sewage and secondary treatment, where there is sludge formation, which can lead to low RNA concentration in the liquid part and high concentration in the solid part. Westhaus et al. (2021) revealed that the RNA of SARS-CoV-2 was detected in the sludge, while the aqueous phase was negative, suggesting a possible adhesion of viral particles to the sedimented solids. It is important to emphasize that virus detection is correlated with the prevalence of the number of cases of COVID-19 infection in each location (Medema et al. 2020). These aspects associated with the lockdown in 2021 probably contributed to the sharp reduction in SARS-CoV-2 detection in WWTP2 samples. Furthermore, although viruses can remain in the aquatic environment by adhering to suspended solids in the environment, factors such as temperature, pH change and the presence of other microorganisms can also affect viral detection (Rollemberg et al. 2020).

Viral genetics and evolution have been the main topics investigated since the first published Sars-CoV-2 genome, which boosted the investigation of the viral genome through sequencing (Zhu et al. 2020). The data generated in this study, based on the genomic sequencing of WWTP1 samples, resulted in the recovery of partial genomes, which enabled the identification of characteristic mutations of different SARS-CoV-2 variants, based on database analyses (Table 2). It is necessary to consider that the SARS-CoV-2 present in wastewater may be inserted into a complex set of genomic RNA of circulating variants in each community. This RNA may be present in an intact or fragmented viral capsid in the environment and may make the recovery of genomes unfeasible (Wurtzer et al. 2020; Robinson et al. 2022). On the other hand, a clinical sample may contain numerous RNA copies of a given SARS-CoV-2 variant.

In the present study, it was possible to detect suggestive mutations of the Delta, Gamma and Omicron VOCs. The Gamma (P.1) variant was detected in genomes of clinical samples from December 2020 to August 2021 (Fiocruz 2022). However, this variant was only detected in WWTP1 in March and June, a period in which there was an increase in the number of clinical genomes deposited in the Fiocruz Genomic Network (FGN) (Fiocruz 2022). The Delta (AY.99.2) variant was revealed in August 2021 (Table 2), where an increase in clinical genomes deposited from the FGN was also observed (Fiocruz 2022). Lamarca et al. (2022) revealed that over 98% of clinical genome sequences from the state of Rio de Janeiro originated from a single introductory event of the AY.99 lineage, which diverged into AY.99.1 and AY.99.2 lineages between May and June 2021. Both strains spread across the state from this point onwards, with AY.99.2 being dominant. Another Delta variant, AY.32 detected in wastewater, in August, presented the mutation ORF1a:V2930L, described as a characteristic mutation by the WHO (WHO 2022). In addition to the Delta variant AY.32, other variants Delta, Mu, B.1618, B.1469, XD (recombinant variant Delta + BA.1) and XBA (Table 2) also showed characteristic mutations. Another significant finding was the detection of the Mu (B.1.621) variant containing characteristic mutations described by the WHO (S:E484K, S:N501Y) in the WWTP1 entry, in May 2021. In June 2021, a Mu variant, containing the same mutations, recovered from a clinical sample and had its genome deposited in the GISAID database. It is important to point out that the WWTP1 sample comes from the effluent of the same hospital where the clinical variant B.1.621 was found. These data allow us to suggest that this variant was present in wastewater one month before it was detected in the clinical sample. As in the present study, Karthikeyan et al. (2022) detected the B.1.621 variant through genomic surveillance of wastewater approximately 4 weeks before its first detection through clinical genomic surveillance. It was possible to detect mutations suggestive of the Omicron variant in May 2021 in our wastewater samples, the variant in question was only found in clinical samples in Brazil in December 2021 (Fiocruz 2022).

The continued evolution of SARS-CoV-2 and the emergence of VOCs such as Omicron VOC highlight the importance of maintaining an active vigilance for the emergence of unexpected new variants (Callaway 2021; Martin et al. 2021). The fact that the origins and initial spread of VOCs Alpha and Omicron have not been observed justifies efforts to detect and monitor new variants (Herold et al. 2021). However, as stated earlier, genome-wide sequencing of SARS-CoV-2 isolated from wastewater often suffers from low sequencing depth of coverage in epidemiologically relevant areas of the genome, such as the Spike Receptor Binding Domain (RBD) (Fontenele et al. 2021; Swift et al. 2021). Furthermore, as wastewater may contain a mixture of viral lineages and whole-genome sequencing depends on sequencing small genome fragments, computational strategies to identify variants with linked mutations often fail to identify lineages present at low concentrations (Baaijens et al. 2022). To address these issues, researchers have developed a ‘targeted’ sequencing approach that amplifies and sequences the RBD region of the Spike protein of the SARS-CoV-2 genome as a single fragment (Gregory et al. 2021; Smyth et al. 2022). The RBD region is relevant sequencing coverage. A recent study described a set of unknown lineages from various locations in the United States. While wastewater has its own lineages, the study provided evidence that some lineages could have shared the same common ancestor (Gregory et al. 2021). It is important to emphasize that in the present study, unknown lineages not reported in environmental and/or clinical samples in global databases were also revealed, despite some difficulties and limitations in the annotation and interpretation of SARS-CoV-2 genomes. Another study, also using this same approach, demonstrated that the frequencies of variants in wastewater closely followed VOC frequency estimates from clinical sampling in the same areas (Gregory et al. 2021; Smyth et al. 2022). However, in some locations, the presence of unknown strains not seen in clinical specimens anywhere in the world has been demonstrated. Several of these strains contained amino acid substitutions rarely reported in global databases such as GISAID (N460K, Q493K, Q498Y and N501S) (Smyth et al. 2022). Changes in circulating SARS-CoV-2 VOCs may require changes in public health responses to the COVID-19 pandemic, as they have the potential to evade vaccines and pharmaceutical interventions and may be more transmissive than other SARS-CoV variants. As such, it is essential to track and prevent its spread in susceptible communities (Wurtz et al. 2021).

The data obtained in this study reinforce the idea that genomic surveillance of Sars-CoV-2, through WBE, can be considered a promising and innovative approach capable of providing important information about the persistence of the virus in environmental samples, in addition to enabling the investigation of the emergence and circulation of variants even before their revelation in patients infected with COVID-19. The WBE has been applied to the surveillance of SARS-CoV-2 and its variants, including the monitoring of outbreaks and the assessment of the effectiveness of public health interventions (Ahmed et al. 2022).

• The detection of a possible B.1.621 (Mu) variant one month before the same variant was recovered from a clinical sample demonstrates the importance of this tool in investigating the emergence and circulation of variants even before their disclosure in patients infected with COVID-19.

• The sequencing of wastewater for SARS-CoV-2 surveillance resulted in small fragments that did not allow assembly of complete SARS-CoV-2 genomes, considering low viral loads, highly fragmented RNA and PCR inhibitors in complex environmental samples. However, database analyses revealed genetic mutations indicative of possible VOCs and VOIs.

• The data obtained in this study reinforce the idea that genomic surveillance of SARS-CoV-2, through WBE, could become an important tool in public health management during and after the COVID-19 pandemic, capable of obtaining epidemiological information and signaling possible risk factors for the environment and public health.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.