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. 2026 Mar 6;18(1):12. doi: 10.1007/s12560-026-09687-1

Assessing Urban Ditches for Tracking SARS-CoV-2 in Low Sanitation Areas in Southeastern Brazil

Regina Keller 1, Gizely Azevedo Costa 1,✉, Paola de Avelar Carpinetti 1, Ricardo Franci Gonçalves 1, Mariane Vedovatti Monfardini Sagrillo 1, Rodrigo Nunes Oss 1, Danielsan de Jesus Dias 1, Isabella Balarini de Azevedo Chaiben 1, Laila de Oliveira Vaz Oliveira 1, Rodrigo Pratte 1, Alice Barros Câmara 1, Sérvio Túlio Alves Cassini 1, Daniel Rigo 1, Diogo Costa Buarque 1, Liliana Cruz Spano 2, Sandra Ventorin Von Zeidler 2, Marize Pereira Miagostovich 3
PMCID: PMC12966239  PMID: 41790378

Abstract

Several studies investigated the presence of SARS-CoV-2 in water samples and raw urban wastewater collected from urban sewage treatment plants (STPs) during the COVID-19 pandemic and correlated viral load and epidemiological data. However, limited information is available regarding the potential of urban ditches as sampling sites for epidemiological surveillance. This study aimed to detect SARS-CoV-2 RNA in urban ditches and evaluate its correlation with epidemiological data. A total of 168 samples were collected from January 2021 to February 2022 in three urban ditches and two STPs in the metropolitan region of Vitória, Espírito Santo (ES). Physicochemical and microbiological water quality parameters were analyzed, and SARS-CoV-2 RNA was detected using a TaqMan RT-qPCR system. SARS-CoV-2 was detected in 145/168 of the samples, with 87.2% (75/86) found in the urban ditches and 85.3% (70/82) in the STPs. The viral load in the three urban ditches ranged from 1.59 × 104 to 2.56 × 104 genome copies/litre (GC/L) while in the two STPs, it ranged from 7.79 × 103 to 1.26 × 104 GC/L. A moderate to strong positive correlation was observed between viral load and confirmed COVID-19 cases, particularly for samples collected from urban ditches (ditch 1, r = 0.58; ditch 2, r = 0.78; ditch 3, r = 0.86). The results indicate greater correlation of SARS-CoV-2 RNA concentration in ditches of low sanitation areas as compared with STPs, showing that ditches could be an important and equivalent sampling point for viral epidemiological studies.

Keywords: COVID-19, Environmental virology, Epidemiological monitoring, SARS-CoV-2, Urban ditches, Wastewater surveillance

Introduction

The SARS-CoV-2 pandemic has posed complex problems to the public health sector, challenging our understanding of the dynamics of viral spread and persistence in various environments (Bhattarai et al., 2021; Carraturo et al., 2020; Giacobbo et al., 2021; Kumar et al., 2022; Yang et al., 2022). Although initial attention was directed toward contact surfaces and clinical settings, aquatic environments soon became a growing concern. Several studies investigated the presence of SARS-CoV-2 in different water bodies, including surface waters, groundwaters, wastewaters, recreational marine waters, and, more recently, offshore marine waters (Ahmed et al., 2020; Augusto et al., 2022; Bonanno Ferraro et al., 2022; De Freitas Bueno et al., 2022; Hata et al., 2021; La Rosa et al., 2020, 2024; Maidana-Kulesza et al., 2021; Mazumder et al., 2022; Medema et al., 2020; Mota et al., 2021; Prado et al., 2021; Sherchan et al., 2020).

However, limited research has been conducted in urban ditches. Urban ditches are natural or artificial waterways for stormwater runoff and flood control. These water bodies are commonly subject to the discharge of urban and industrial wastewater contaminated with chemical pollutants and pathogenic microorganisms (Giuseppe et al., 2022; Razzolini et al., 2021; Rimoldi et al., 2020). In regions without adequate sanitation infrastructure, such as many cities in Latin America, urban ditches may receive untreated wastewater discharged from residences not connected to the sewer system, posing a risk to nearby communities, especially during periods of floods (Giuseppe et al., 2022; Prado et al., 2023). Problems related to urban wastewater collection and treatment can contribute to the spread of viruses in aquatic environments, contaminating local water bodies. The microbiology of urban ditches is not well understood, possibly due to challenges related to sample collection, since many ditches pass through difficult-to-access areas and cover both surface and subsurface sections. The discovery that SARS-CoV-2 is excreted in the feces of symptomatic and asymptomatic individuals, along with its detection in water and urban wastewater samples, has led to the adoption of additional surveillance measures in line with a wastewater-based epidemiology approach (Barcellos et al., 2023; De Freitas Bueno et al., 2022; Masachessi et al., 2022; Monteiro et al., 2022; Sangsanont et al., 2023; Sims & Kasprzyk-Hordern, 2020). Studies showed a strong correlation between virus concentration in urban wastewater and the incidence and prevalence of the disease in the general population (Ai et al., 2021; Barrios et al., 2021; Joseph-Duran et al., 2022; Kitamura et al., 2021; Medema et al., 2020). Urban wastewater surveillance for SARS-CoV-2 may be a valuable complementary approach in COVID-19 surveillance and diagnostic strategies. In addition to helping address critical problems related to underreporting (Robotto et al., 2022), this method offers more effective indicators of infection at the community level and can deliver faster warnings of disease outbreaks than traditional human surveillance methods based on testing and tracking infected individuals (Solo-Gabriele et al., 2023).

Considering the above, this study aimed to identify the presence of SARS-CoV-2 in three urban ditches and two urban sewage treatment plants (STPs) in three municipalities in the metropolitan region of Vitória, in Espírito Santo State, Brazil. Additionally, the correlation between the concentration of the virus in these environments and COVID-19 epidemiological surveillance data was analyzed to understand the role of these water bodies as tools for epidemiological surveillance in vulnerable areas with poor sanitation infrastructure. By understanding the complex dynamics of SARS-CoV-2 in drainage waters and urban wastewater in vulnerable regions (areas with limited urban infrastructure, low income, and education levels), this study is expected to provide important information for the design of public health policies, sanitation plans, and specific interventions targeting marginalized populations.

Materials and Methods

Description of Sampling Sites

The study was conducted in Vitória, Vila Velha, and Serra, three municipalities located in the metropolitan region of Vitória in Espírito Santo State, Southeastern Brazil. Water samples were collected from three urban ditches and two urban sewage treatment plants STPs (Fig. 1).

Fig. 1.

Fig. 1

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Location map showing sampling points along three urban ditches (Ditches 1–3) and at two urban sewage treatment plants (STP 1 and STP 2) in Vitória, Vila Velha, and Serra, Espírito Santo State, Brazil

Urban ditches 1 and 2 are located in the municipality of Vila Velha, while ditch 3 is located in the municipality of Vitória, Espírito Santo (ES) state. These ditches were selected based on the ease of sample collection and their relevance as urban wastewater discharge points for nearby residences. The water in these urban ditches is a mixture of drainage water, domestic urban wastewater, and rainwater, and it is discharged directly into the sea without prior treatment at a wastewater treatment plant. The STP 1 is located in the municipality of Vila Velha and treats urban wastewater produced by a population of 296.000 residents from 51 surrounding neighborhoods, while the STP 2 is located in the municipality of Serra and treats urban wastewater from 13.000 residents from 5 neighborhoods.

Geographic and population characteristics of the studied urban ditches are presented in Table 1.

Table 1.

Description of sampling points along urban ditches in Vila Velha and Vitória, Espírito Santo State, Brazil

Sampling point Geographic coordinates Contributing population Description
Ditch 1

20°21′00″S

40°17′24″W

 47.261 Measures about 6.1 km in length. It is currently ditchized with concrete walls. Some sections are concealed by underground pipes that run underneath the urban area.
Ditch 2

20°25′48″S

40°19′48″W

 54.145 A tributary of the Jucu River belonging to the Congo River basin. The ditch has a total drainage area of about 30.03 km2. Both the ditch and its tributaries are under the influence of tides. It is subject to the discharge of untreated urban wastewater by the local population.
Ditch 3

20°27′61″S

40°32′93″W

 7.090 It is located in the demographic complex of Greater São Pedro in Vitória, Espírito Santo State, and covers an area of about 3.6 km2 at the interface between human settlements and mangrove areas. The area was previously an open dump. Untreated urban wastewater is discharged directly into the estuarine region, without concrete structures or underground pipes.
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Table 2 shows the description of the STPs included in the study.

Table 2.

Description of urban sewage treatment plants (STP) 1 and 2

Sampling point Geographic coordinates Served population Treatment method Nominal flow rate (L/s) Description
STP 1

20°38′0″S

40°33′0″W

 296.000 Activated sludge 400 Receives urban wastewater from 51 neighborhoods through a 325,000 m long collection system
STP 2

20°12′36″S

40°13′12″W

 13.000 Step-feed activated sludge 111 Receives urban wastewater from five neighborhoods through a 65,000 m long collection system
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Sample Collection

Raw urban wastewater samples were collected in the morning weekly between January 2021 and February 2022. Samples were not collected on rainy days or within five days following heavy rains. At STPs, individual samples of raw urban wastewater (1.5 L) were collected at the inlet located after the pretreatment step, which removes coarse solids and sand. Urban ditches samples (1.5 L) were collected at a depth of about 10 cm. After collection, samples were maintained under refrigeration on ice, transported to the Sanitation Laboratory at the Federal University of Espírito Santo, and processed on the same day.

Physicochemical parameters (pH, dissolved oxygen, salinity, temperature, conductivity, and turbidity) were evaluated immediately after collection by using a portable multiparameter meter (HI-9829, Hanna Instruments). Total solids and thermotolerant coliform counts (membrane filtration) were analyzed according to the Standard Methods for the Examination of Water and Wastewater (APHA et al., 2017).

SARS-CoV-2 Detection in Urban Wastewater Samples

For SARS-CoV-2 detection, urban wastewater samples were first inactivated at 60 °C for 90 min and concentrated by membrane filtration, as described by Katayama et al. (2002). After inactivation, 200 mL of sample was filtered through a negatively charged membrane (0.45 μm pore size, 90 mm diameter; Merck Millipore, Billerica, USA; catalog no. HAWP-09000) previously hydrated with sterile water. Then, the membrane was washed with 40 mL of (0.5 mM) H2SO4. After the washing solution was removed, a Falcon tube containing 10 µL of (50 mM) H2SO4 and 10 µL of 100× Tris-EDTA was connected to the filtration system and the membrane was washed with 2 mL of NaOH (1 mM). The filtrate was separated into 200 µL aliquots and stored at − 20 °C until RNA extraction.

RNA Extraction and RT-qPCR

SARS-CoV-2 RNA extraction was performed on 200 µL concentrated samples using the Bio Gene Viral DNA/RNA Extraction kit (Bioclin, Belo Horizonte, MG, Brazil), according to the manufacturer’s recommendations. Isolated RNA was eluted in 50 µL of ribonuclease-free water. SARS-CoV-2 RNA was detected using a StepOnePlus™ Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA), and the 2019-nCoV RUO Primer and Probe kit (Integrated DNA Technologies, Coralville, IA, USA), which includes primers and probes targeting the N1 and N2 regions of the SARS-CoV-2 nucleocapsid gene.

The N1 and N2 targets were analyzed in independent reactions because both probes are labeled with the same fluorophore (FAM) and therefore emit fluorescence in the same detection channel; multiplexing was not performed. An internal amplification control was not included in the same reaction due to limited sample volume, which was prioritized for SARS-CoV-2 detection. In addition, there is no clear consensus on the most appropriate endogenous gene to serve as an internal control for environmental samples, particularly when comparing samples from sewage treatment plants and urban ditches. Assay performance was monitored using positive and negative controls in each run.

Reactions were performed in a total volume of 20 µL containing 8.5 µL of nuclease-free water, 1.5 µL of Primer and Probe Mix (#10006713), 5 µL of TaqPath 1-Step RT-qPCR Master Mix (Thermo Fisher Scientific, Coralville, IA, USA), and 5 µL of RNA. Nuclease-free water was used as negative template control. Amplification was achieved by using the following program: 25 °C for 2 min, 50 °C for 15 min, 95 °C for 2 min, followed by 45 cycles of 95 °C for 15 s and 55 °C for 30 s. The data were analyzed using StepOne software v2.3 (Applied Biosystems, Foster City, CA, USA). Appropriate Ct values were observed for all positive controls, and no amplification was observed in negative controls.

SARS-CoV-2 RNA concentrations were determined using a standard curve. Each qPCR run included quadruplicates of five different concentrations (100, 101, 102, 103, and 104 copies per reaction) of the 2019-nCoV_N_Positive Control (plasmid DNA targets containing one copy each of N1 and N2), that generated a standard curve with an R2 value of 0.99 with an efficiency of 93.35% (slope = − 3.49; y intercept = 38.29). All samples were quantified in triplicate using the N1 primer set. Samples that did not show amplification with this primer set were also quantified using the N2 primer set.

The limit of detection (LoD) is defined as the smallest spike amount of a target organism in dilution that can be detected in 95% of replicates (Kralik & Ricchi, 2017). This parameter was estimated using the N1 primer set and low viral concentrations (genome copies, GC). Thus, the 2019-nCoV_N_Positive Control (Integrated DNA Technologies) was used as a spike for SARS-CoV-2 to evaluate amplification by qRT-PCR (2, 4, 8, 16, and 32 GC/reaction). A total of 15 replications were performed for each concentration. The limit of detection calculated with a 95% confidence interval was 7 GC/reaction.

Demographic and Epidemiological DATA

For the three urban ditches monitored, the population whose urban wastewater load contributes to each sampling point was estimated. Using the software QGIS, version 3.38.2 ‘Grenoble’, the drainage basins were manually delimited, taking the collection points as outlets. The delimitation of each basin considered the characteristics of the terrain and local hydrography, using contour lines of the studied areas, obtained through the Integrated Geospatial Base System of Espírito Santo, GEOBASES (https://geobases.es.gov.br/). The boundaries of the basins were drawn to ensure that all surface water flowed to the determined outlet point. The population estimate was carried out based on the census sectors that are within the delimited areas, since, when using contour lines to define the basins, not all neighborhoods were completely covered by the delimitation. The census sector, defined by the Brazilian Institute of Geography and Statistics (IBGE), is the smallest territorial unit, for which information is disclosed, consisting of a continuous area and located in a specific urban or rural framework. This territorial unit allows the collection of population data with high precision, since its boundaries respect hierarchically superior territorial divisions, such as neighborhoods, towns, districts, municipalities and Federation Units (IBGE, 2017). Confirmed cases of COVID-19 (prevalent cases, confirmed by RT-qPCR tests) were collected from the official website (https://coronavirus.es.gov.br/painel-covid-19-es), with data made available by neighborhood. Given the need to estimate the population covered in each delimited basin, a correlation was made between the percentage of inhabitants within the basin and the total number of inhabitants in each neighborhood. For the points located in STPs, the population of the neighborhoods served by the plants was considered, according to the respective development plans (STP 1: Municipal Basic Sanitation Plan of Vila Velha, 2014; STP 2: Municipal Basic Sanitation Plan of Serra, 2016).

Statistical Analysis

The Pearson correlation (Figueiredo Filho; Silva Júnior, 2009), was used to assess correlations between urban wastewater viral concentrations and COVID-19 cases confirmed by the Health Secretariat of the studied municipalities. Statistical analyses were performed using GraphPad Prism version 9 (GraphPad Software Inc., San Diego, CA, USA).

Results

Detection and Quantification of SARS-CoV-2 in Urban Ditches and STPs

Of the 86 samples collected in the urban ditches, 75 (87.2%) were positive for SARS-CoV-2 RNA with a virus detection frequency of 90.2%, 78.6% and 94.1% in ditches 1, 2 and 3 respectively. In the STPs, 85% (70/82) of the samples were positive for viral RNA, 81% of the samples in STP 1 and 89.74% in STP 2 (Table 4).

Table 4.

Detection frequency and viral load of SARS-CoV-2 in water samples collected from urban ditches and urban sewage treatment plants (STPs)

Sampling point Statistics pH Temperature (°C) Turbidity (UNT) Salinity (%) Conductivity (mS/cm) Total solids (mg/L) Dissolved oxygen (mg/L) Thermotolerant coliforms
Ditch 1 (n = 41) Mean 7.05 26 129 0.57 1155.31 609.89 0.49 3.23 × 106
Median 6.97 26 105 0.46 940.00 497.00 0.29 2.73 × 106
Min–max 5.2–9.58 22–31 42–750 0.26–1.36 551–2670 298–1529 0–3 0–9.00 × 106
Ditch 2 (n = 28) Mean 6.98 25 83 1.09 1648.40 901.29 0.57 1.38 × 106
Median 6.50 25 80 0.48 975.00 467.00 0.27 1.10 × 106
Min–max 5.3–9.08 20–31 40–160 0.25–10.71 519–17,860 232–7852 0–3.82 1.00 × 105–1.07 × 107
Ditch 3 (n = 17) Mean 6.39 26 92.13 11.62 19124.24 2077.47 0.92 7.01 × 106
Median 6.50 25 75.90 8.7 19160.00 927.00 0.75 6.15 × 106
Min–max 4.9–7.2 24–29 28.2–305 0.27–29.56 563–45,670 380–13,398 0–3.4 2.60 × 106–1.27 × 107
STP 1 (n = 43) Mean 7.25 27 187 0.74 1653.51 894.67 0.83 4.80 × 106
Median 7 28 183 0.6 1263.00 635.50 0.70 4.78 × 106
Min–max 5.7–9.59 24–30 45–328 0.25–1.95 520–8287 278–5234.00 0.1–2.88 6.00 × 105–1.30 × 108
STP 2 (n = 39) Mean 7.04 27.00 175 0.42 846.03 499.15 1.24 6.98 × 106
Median 6.96 27.00 163 0.41 841.00 470.00 1.30 7.13 × 106
Min–max 5–9.1 22–31 19–480 0.31–0.60 533–1212 288–737 0.20–4.09 8.00 × 105–2.65 × 107
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Table 3.

Detection frequency and viral load of SARS-CoV-2 in water samples collected from urban ditches and urban sewage treatment plants (STP) in Vitória, Vila Velha and Serra, Espírito Santo State, Brazil

Sampling point Positive samples Total samples Detection frequency (%) Viral load (genome copies/L)
Ditch 1 37 41 90.24 1.59 × 104
Ditch 2 22 28 78.57 1.83 × 104
Ditch 3 16 17 94.12 2.56 × 104
STP 1 35 43 81.40 7.79 × 103
STP 2 35 39 89.74 1.26 × 104
Total 145 168 86.31
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Evaluation of Viral Load and Correlation with Confirmed COVID-19 Cases

The number of COVID-19 cases and the viral concentration measured in the three urban ditches over the studied epidemiological weeks are presented in Fig. 2. When an increase in the SARS-CoV-2 concentration in the water samples from the urban ditches was observed, an increase in the number of reported cases was shown, throughout most of the study period.

Fig. 2.

Fig. 2

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Temporal variation in confirmed COVID-19 cases and SARS-CoV-2 RNA loads in water samples collected from three urban ditches in Espírito Santo State, Brazil, between January 2021 and February 2022. A Ditch 1, B Ditch 2, C Ditch 3. All SARS-CoV-2 concentrations shown represent detected values; no non-detects are included

Figure 3 shows the number of COVID-19 cases and the viral load of urban wastewater samples collected from the two STPs.

Fig. 3.

Fig. 3

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Temporal variation of confirmed COVID-19 cases in the population and SARS-CoV-2 RNA concentration in water samples collected from two-urban sewage treatment plants (STP) in Espírito Santo State, Brazil, between January 2021 and February 2022. A Urban sewage treatment plant (STP 1) and B STP 2. All SARS-CoV-2 concentrations shown represent detected values

Individually, the urban ditches presented their respective positive correlations (ditch 1, r = 0.58; ditch 2, r = 0.78; ditch 3, r = 0.86) and the STPs (STP 1, r = 0.45; STP 2, r = 0.74. In this study, a greater correlation was observed between the viral load and the number of confirmed cases of COVID-19 for the joint analysis of the urban ditches (r = 0.87) when compared with the STPs (r = 0.75).

Figure 4 depicts the correlation between RNA concentrations of SARS-CoV-2 in urban wastewater from urban ditches and STPs and the number of confirmed COVID-19 cases.

Fig. 4.

Fig. 4

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Relation between confirmed COVID-19 cases numbers and SARS-CoV-2 RNA concentrations in wastewater samples from A three urban ditches and B two urban sewage treatment plants in Espírito Santo State, Brazil. All SARS-CoV-2 concentrations shown represent detected values

The viral concentrations measured at all collection points reflect the dynamics of clinical data, with increases and reductions in viral load prior to variations in COVID-19 cases. The strong correlation (r = 0.88) confirms the effectiveness of pathogen surveillance by wastewater-based epidemiology (Fig. 5).

Fig. 5.

Fig. 5

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Historical timeline of SARS-CoV-2 RNA concentration with confirmed COVID-19 cases numbers over the monitoring period in water samples collected from three urban ditches and two urban sewage treatment plants

Physicochemical and Microbiological Characteristics of Urban Ditches and STPs

Although not completely covered by related parameters of the main Brazilian Legislation for water quality represented by CONAMA 430 (2011), the assessed ditches environment could be considered as an urban sewage drainage based on the observed values of thermotolerant coliform and dissolved oxygen (DO) reinforced by the characteristic grey coloration and aggressive odour as a sensorial parameter of ditches samples. Turbidity, salinity, and conductivity values differed slightly from those of STP samples, given that the urban ditches are open and influenced by rainfall and water tides. The concentration of thermotolerant coliforms in urban discharge ditches was similar to that of wastewater from STPs, confirming the constant dumping of urban wastewater in urban ditches (Table 5).

Table 5.

Descriptive statistics of physicochemical and microbiological parameters of water samples collected from urban ditches and urban sewage treatment plants (STPs)

Sampling point Statistics pH Temperature (°C) Turbidity (UNT) Salinity (%) Conductivity (mS/cm) Total solids (mg/L) Dissolved oxygen (mg/L) Thermotolerant coliforms
Ditch 1 (n = 41) Mean 7.05 26 129 0.57 1155.31 609.89 0.49 3.23 × 106
Median 6.97 26 105 0.46 940.00 497.00 0.29 2.73 × 106
Min–max 5.2–9.58 22–31 42–750 0.26–1.36 551–2670 298–1529 0–3 0–9.00 × 106
Ditch 2 (n = 28) Mean 6.98 25 83 1.09 1648.40 901.29 0.57 1.38 × 106
Median 6.50 25 80 0.48 975.00 467.00 0.27 1.10 × 106
Min–max 5.3–9.08 20–31 40–160 0.25–10.71 519–17,860 232–7852 0–3.82 1.00 × 105–1.07 × 107
Ditch 3 (n = 17) Mean 6.39 26 92.13 11.62 19124.24 2077.47 0.92 7.01 × 106
Median 6.50 25 75.90 8.7 19160.00 927.00 0.75 6.15 × 106
Min–max 4.9–7.2 24–29 28.2–305 0.27–29.56 563–45,670 380–13,398 0–3.4 2.60 × 106–1.27 × 107
STP 1 (n = 43) Mean 7.25 27 187 0.74 1653.51 894.67 0.83 4.80 × 106
Median 7 28 183 0.6 1263.00 635.50 0.70 4.78 × 106
Min–max 5.7–9.59 24–30 45–328 0.25–1.95 520–8287 278–5234.00 0.1–2.88 6.00 × 105–1.30 × 108
STP 2 (n = 39) Mean 7.04 27.00 175 0.42 846.03 499.15 1.24 6.98 × 106
Median 6.96 27.00 163 0.41 841.00 470.00 1.30 7.13 × 106
Min–max 5–9.1 22–31 19–480 0.31–0.60 533–1212 288–737 0.20–4.09 8.00 × 105–2.65 × 107
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Discussion

This research highlighted the presence of SARS-CoV-2 RNA in samples from three urban ditches and two urban wastewater treatment plants located in the municipalities of Serra, Vitória, and Vila Velha, in the metropolitan region of Vitória, in southeastern Brazil. The detection of SARS-CoV-2 viral RNA coincided with the epidemiological data collected during the surveillance period and agrees with previous studies, which demonstrated a SARS-CoV-2 positivity rate between 50% and 100% in raw urban wastewater (Acosta et al., 2022; Ahmed et al., 2020; Barbosa et al., 2022; La Rosa et al., 2020; Randazzo et al., 2020; Prado et al., 2021).

The low level of sanitation in the regions where the three ditches monitored in this study circulate was evidenced by the physical-chemical and microbiological characteristics of the waters collected during the study period, which are similar to those recorded in the analyses of raw urban wastewater flowing into the STPs. In addition, thermotolerant coliforms were detected throughout the surveillance period in concentrations typical of raw urban wastewater, above 106 MPN/100mL, demonstrating the constant discharge of fecal material into these waters. Urban ditches, whether natural or constructed, open or partially covered, in galleries, such as those analyzed in our research, are designed to prevent flooding, inundation and waterlogging that occur frequently in places where living and sanitation conditions are precarious. They can be found in several cities around the world and, despite being inadequate, are natural routes for the diffuse drainage of waste and contaminants produced by the population of cities that do not have an urban wastewater collection and treatment system (Santana & Cunha, 2019).

Another important parameter is the high concentration of solids present in the waters of the ditches such as that found in canal 3, which represents more than double the concentration of solids present in the raw urban wastewater of the STPs. SARS-CoV-2 has an affinity for the biosolids present in these types of samples (Balboa et al., 2021). The presence of solids in urban surface canals is a significant environmental concern, as these solids or sediments can contain a variety of pollutants, including pathogenic microorganisms and toxic chemicals (Arora et al., 2021). Viruses can associate with solid particles present in these environments and persist on these surfaces for varying periods depending on factors such as temperature, humidity, and exposure to sunlight (Câmara et al., 2023; Carraturo et al., 2020).

The discharge of urban wastewater into the canals may originate from nearby homes that are not yet connected to the urban sewerage wastewater system or from the lack of separation of rainwater from domestic effluents. These canals are, therefore, impacted by the direct discharge of urban wastewater or indirectly by seasonal floods that carry various types of waste into their course. Many of these canals, which flow into the sea, are under the influence of tides, resulting in different flow rates and residence times. The three urban ditches analyzed, which flow into the sea, are strongly influenced by the tide, substantially interfering with their daily flow. In this context, human interventions during the urbanization process of cities have significantly impacted river systems, altering their natural behavior. These changes include the loss of ditch sinuosity, changes in drainage patterns, modifications in discharge regimes, variations in flow patterns and flow velocities, increased peak discharges in tributaries, and increased sediment loads. Sediments transported and deposited downstream reduce the cross-sectional area of ​​the ditch, resulting in silting of the bottom (Botelho, 2011; Oliveira & Botelho, 2014). As a result, urban streams have become examples of intensely disturbed environments, often degraded and altered to meet the new demands of urban growth, as pointed out by Santana and Cunha (2019). All of these changes can be observed in ditches 1 and 2, which had their paths straightened by man to drain water from rainfall, but which currently receive domestic wastewater from nearby homes and discharge their water into Vitória Bay without any treatment. Ditch 3 consists predominantly of a closed conduit along most of its length and runs through neighborhoods with very poor sanitation levels. This ditch receives urban wastewater from a population of approximately 7000 people and flows directly into Vitória Bay. Due to the high influence of the tides, the samples collected in ditch 3 showed high salinity and conductivity, in addition to the concentration of solids, which was also much higher than that found in the other ditches and in the STPs.

In addition to sanitation issues, urban wastewater presents several limitations for pathogen detection because it is a complex matrix. Wastewater is a complex, often inhomogeneous mixture of substances other than fecal waste, including pharmaceuticals, personal hygiene products, rainwater, sediments, household detergents, industrial effluents, metals, and other substances. Some of these substances can completely or partially inhibit the RT-PCR amplification process, leading to a false-negative result. These substances are generally referred to as PCR inhibitors and include a heterogeneous and ill-defined group of chemicals known to inhibit PCR-based methods, including multi-ring polysaccharides (e.g., humic and fulvic acids), fats, proteins, metal ions (e.g., iron and aluminum), RNases, and others (Schrader et al., 2012). In addition to the presence of contaminants typically found in raw urban wastewater, waterways are influenced by RNA dilution effects due to mixing with other wastewater flows, stormwater infiltration, diurnal variation in discharge, and hydraulic residence time in the wastewater collection system, all of which may affect the likelihood of detecting SARS-CoV-2 RNA, particularly under low prevalence conditions (Ahmed et al., 2021).

During the surveillance period of SARS-CoV-2 RNA in ditch 1, the viral load ranged from 3.40 × 103 to 9.63 × 104 GC/L and followed the trend of registered COVID-19 cases in the respective region. However, in some periods, viral load peaks were observed in the ditch, despite a low number of registered COVID-19 cases. These results can be attributed to periods in which a significant portion of the population was vaccinated and restrictive measures were relaxed, leading to a decrease in testing of the population and concerns about the disease.

In ditch 2, on the other hand, SARS-CoV-2 RNA concentrations were below the detection limit during most of the surveillance period. In December 2021 (week 50), although the number of reported cases of COVID-19 was low, the viral load in urban wastewater samples was high (1.39 × 105 GC/L), indicating that the virus remained in circulation in the population and demonstrated to be an early warning for the exponential increase in cases that would occur in January 2022 with the arrival of the Ômicron variant in the country (SESA, 2022).

Unlike ditches 1 and 2, ditch 3 runs through a low-income region, where only 58% of the area has urban sewerage (PMSB, 2015). The waters of the canal are highly influenced by the tides, resulting in significant variations in physical-chemical parameters. Of the samples collected, 94% were positive for SARS-CoV-2, with little variation in viral load during the study period. There were only two peak periods (week 37, 2021; week 4, 2022), when increases in viral load and case reporting were recorded by the health department. This result showed that the high percentage of samples positive for SARS-CoV-2 RNA in ditch 3 (94.12%) reflected the widespread presence of the virus in the population. In the evaluation of urban wastewater inflow to STPs, the number of genomic copies determined in the samples was similar to those already recorded around the world (Ahmed et al., 2021; Amereh et al., 2022; Nasseri et al., 2021; Nagarkar et al., 2022). Only from December 2021, as was also observed in the ditches, there was a significant increase in the detection of SARS-CoV-2 RNA, reaching a peak in January 2022. This increase accompanied the third wave of COVID-19 identified in Brazil, resulting from the arrival of the Ômicron variant (Xavier et al., 2022). The variant was characterized as highly contagious, and the wave coincided with the post-holiday period, resulting in an exponential increase in the number of confirmed cases of COVID-19 in Espírito Santo in early January 2022.

The associations of viral load with the number of cases registered during the epidemiological weeks revealed a moderate/strong correlation between viral loads in wastewater and the incidence and prevalence of the disease in the population. Other studies evaluating STPs effluent have reported similar correlations (Acosta et al., 2022; Ahmed et al., 2020; Barrios et al., 2021; Galani et al., 2022; La Rosa et al., 2020; Masachessi et al., 2022; Prado et al., 2021; Shrestha et al., 2023). The joint analysis of surveillance data from ditches and STPs identified some of the most important moments of the COVID-19 pandemic in Brazil, such as the confirmation of the introduction of the Delta variant in August 2021 and the emergence of the Ômicron variant at the end of that same year. These events coincide directly with peaks in the concentration of SARS-CoV-2 detected in urban wastewater samples from canals and STPs. Early detection of the presence of the virus in these locations highlights the importance of environmental surveillance in urban areas and in urban wastewater treatment systems, especially where human influence, such as improper urban wastewater disposal, has a direct impact.

Conclusions

Our results demonstrate the extent of the presence of SARS-CoV-2 in wastewater especially that collected in urban ditches. The high percentage of positive samples for SARS-CoV-2 RNA detected in ditches indicates that this matrix is ​​an option that can be considered to investigate the circulation of the virus in communities lacking basic sanitation, without urban wastewater collection and treatment.

SARS-CoV-2 was detected at high frequencies in the three urban ditches monitored in this study and present a strong positive correlation between wastewater viral load and clinical cases in the populations served by the urban ditches.

Acknowledgements

We thank the Brazilian Federal Agency for Support and Evaluation of Graduate Education (CAPES) for financing the research project (grant No. 88887.505373/2020-00) through a doctoral scholarship awarded to Gizely Azevedo Costa and postdoctoral scholarships awarded to Mariane Vedovatti Monfardini Sagrillo, Alice Barros Câmara, and Laila de Oliveira Vaz Oliveira. We also thank the Federal University of Espírito Santo (UFES) for supporting this research (grant No. 10546/2020) and for awarding a scientific initiation scholarship to Isabella Balarini de Azevedo Chaiben. We are grateful to the Espírito Santo State Agency for Water Resources for authorizing the collection of urban wastewater samples at the Araçás and Manguinhos STPs. Marize Pereira Miagostovich is a Fellowship of Conselho de Desenvolvimento Científico e Tecnológico (CNPq 305737/2023-6).

Author Contributions

Regina Keller: Conceptualization; Writing - Review & Editing; Supervision; Funding acquisition; Project administration. Gizely Azevedo Costa : Conceptualization; Methodology; Investigation; Formal analysis; Writing - Original Draft; Visualization. Paola de Avelar Carpinetti: Conceptualization; Methodology; Investigation; Formal analysis; Visualization. Ricardo Franci Gonçalves: Formal analysis; Resources; Funding acquisition. Mariane Vedovatti Monfardini Sagrillo : Methodology; Writing – review & editing. Rodrigo Nunes Oss : Methodology; Writing – review & editing. Danielsan de Jesus Dias: Methodology; review & editing. Isabella Balarini de Azevedo Chaibe : Methodology; review & editing. Laila de Oliveira Vaz Oliveira : Methodology; Project administration. Alice Barros Câmara: Methodology; Formal analysis . Rodrigo Pratte: Resources. Sérvio Túlio Alves Cassini: Resources; Funding acquisition; Writing – review & editing. Daniel Rigo: Conceptualization ; Writing – review & editing. Diogo Costa Buarque: Conceptualization ; Writing – review & editing. Liliana Cruz Spano: Conceptualization; Funding acquisition; Formal analysis; Writing – original draft. Sandra Ventorin Von Zeidler: Conceptualization; Funding acquisition; Formal analysis; Writing. Marize Pereira Miagostovich: Conceptualization; Funding acquisition; Formal analysis; Writing – review & editing.

Funding

The Article Processing Charge (APC) for the publication of this research was funded by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) (ROR identifier: 00x0ma614). This study was supported by the governmental Brazilian agency Coordination for the Improvement of Higher Education (CAPES; Portuguese: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior), grant number 88887.505373/2020-00 and the Federal University of Espírito Santo (UFES; Portuguese: Universidade Federal do Espírito Santo). Gizely Azevedo Costa received a doctoral scholarship from the same development agency.

Data Availability

The data that support the findings of this study are available from the corresponding author, on special request.

Declarations

Competing interests

The authors declare no competing interests.

Ethical Approval

Not applicable.

Consent to Participate

Not applicable.

Consent to Publish

Not applicable.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author, on special request.

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