Wastewater-based epidemiology: A Brazilian SARS-COV-2 surveillance experience.

Since 2020, developed countries have rapidly shared both publicly and academically relevant wastewater surveillance information. Data on SARS-CoV-2 circulation is pivotal for guiding public health policies and improving the COVID-19 pandemic response. Conversely, low- and middle-income countries, such as Latin America and the Caribbean, showed timid activities in the Wastewater-Based Epidemiology (WBE) context. In these countries, isolated groups perform viral wastewater monitoring, and the data are unevenly shared or accessible to health agencies and the scientific community. This manuscript aims to highlight the relevance of a multiparty effort involving research, public health, and governmental agencies to support usage of WBE methodology to its full potential during the COVID-19 pandemic as part of a joint One Health surveillance approach. Thus, in this study, we explored the results obtained from wastewater surveillance in different regions of Brazil as a part of the COVID-19 Wastewater Monitoring Network ANA (National Water Agency), MCTI (Ministry of Science, Technology, and Innovations) and MS (Ministry of Health). Over the epidemiological weeks of 2021 and early 2022, viral RNA concentrations in wastewater followed epidemiological trends and variations. The highest viral loads in wastewater samples were detected during the second Brazilian wave of COVID-19. Corroborating international reports, our experience demonstrated usefulness of the WBE approach in viral surveillance. Wastewater surveillance allows hotspot identification, and therefore, early public health interventions. In addition, this methodology allows tracking of asymptomatic and oligosymptomatic individuals, who are generally underreported, especially in emerging countries with limited clinical testing capacity. Therefore, WBE undoubtedly contributes to improving public health responses in the context of this pandemic, as well as other sanitary emergencies.

Sampling strategies for wastewater surveillance: Evaluating the variability of SARS-COV-2 RNA concentration in composite and grab samples.

The shedding of SARS-CoV-2 RNA titers by infected individuals, even asymptomatic and oligosymptomatic ones, allows the use of wastewater monitoring to track the COVID-19 spread in a community. This approach is interesting especially for emerging countries with limited clinical testing capabilities. However, there are still important methodological aspects that need validation so that wastewater monitoring data become more representative and useful for public health. This study evaluated the between-day and within-day variability of SARS-CoV-2 RNA concentrations in 24-hour composite and grab samples from three different sampling points, including two wastewater treatment plants (WTTP) and a sewer manhole. In the between-day evaluation (17 weeks of monitoring), a good agreement between the SARS-CoV-2 RNA concentration of each sampling method was observed. There were no significant differences between the mean concentrations of the grab and composite samples (p-value > 0.05), considering N1 and N2 gene assays. The strong relationship between composite and grab samples was proven by correlation coefficients: Pearson's r of 0.83 and Spearman's rho of 0.78 (p-value < 0.05). In within-day evaluation, 24-hour cycles were analyzed and low variability in hourly viral concentrations was observed for three sampling points. The coefficient of variation (CV) values ranged from 3.0% to 11.5%. Overall, 24-hour profiles showed that viral RNA concentrations had less variability and greater agreement with the mean values between 8 a.m. and 10 a.m, the recommended time for grab sampling. Therefore, this study provides important information on wastewater sampling techniques for COVID-19 surveillance. Wastewater monitoring information will only be useful to public health and decision-makers if we ensure data quality through best practices.

First case of SARS-CoV-2 RNA detection in municipal solid waste leachate from Brazil.

This work presents the first case of SARS-CoV-2 RNA detection in leachate collected from a transfer station in the city of São Paulo, Brazil. After calibration of the viral detection method already used for wastewater samples with a pilot leachate sample and virus fragments in laboratory, twelve polyethylene glycol concentrated leachates samples were tested by RT-qPCR. The results confirmed the presence of N1 gene in 9 of the 12 analyzed samples between epidemiological weeks 33 and 38 of the year 2021 (08/15/2021 to 09/19/2021). The occurrence of the N2 gene was only observed in 5 of the 12 samples. The concentration values for N1 and N2 genes varied between 3.1 and 4.6 log10.GC·L-1, which are values close to those measured in sanitary wastewater. This method showed to be a promising procedure to verify the presence of viral RNA in municipal solid waste leachate, being especially useful where there is no treatment system and sanitation infrastructure, which makes the conventional wastewater surveillance unfeasible.

Padronização de método de concentração e extração de ácidos nucleicos em amostras de esgoto sanitário: uma ferramenta de baixo custo para ser utilizada na vigilância epidemiológica de SARS-CoV-2 / Standardization of the method of concentration and extraction of nucleic acids in wastewater samples: a low-cost tool to be used in epidemiological surveillance of SARS-CoV-2

RESUMO A vigilância da qualidade dos esgotos sanitários pode representar uma ferramenta complementar para monitoramento de doenças infecciosas e prevenção de surtos epidêmicos, especialmente quando a capacidade para testes clínicos é limitada. Dessa maneira, o presente estudo descreve o detalhamento técnico de um método de baixo custo para a concentração e extração de ácidos nucleicos de amostras de esgoto sanitário como etapa prévia para a detecção de vírus e outros agentes patogênicos. Para validar a metodologia proposta, após as etapas de concentração e extração, analisaram-se a presença do ácido ribonucleico do SARS-CoV-2 (COVID-19) nas amostras, por meio de reação em cadeia da polimerase em tempo real. O ácido ribonucleico do vírus foi detectado em 80% das amostras de esgoto sanitário analisadas, comprovando o êxito do procedimento metodológico adotado. A detecção precoce de um patógeno associado ao trabalho de equipes multidisciplinares possibilita a prática da vigilância epidemiológica, que auxilia na tomada de decisões na Saúde Única — união indissociável entre a saúde animal, humana e ambiental.

ABSTRACT Sewage quality surveillance can represent a complementary tool for monitoring infectious diseases and preventing epidemic outbreaks, especially when the capacity for clinical testing is limited. Thus, the present study describes the technical details of a low-cost method for concentrating and extracting nucleic acids from sewage samples, as a preliminary step for the detection of viruses and other pathogens. To validate the proposed methodology, after the concentration and extraction steps, the presence of the SARS coronavirus-2 (COVID-19) in the samples was analyzed using real-time polymerase chain reaction. The virus' ribonucleic acid was detected in 80% of the sewage samples analyzed, proving the success of the methodological procedure adopted. The early detection of a pathogen associated with the work of multidisciplinary teams allows the practice of epidemiological surveillance, which assists in making decisions about One Health — an inseparable union between animal, human, and environmental health.

Long-term monitoring of SARS-COV-2 RNA in wastewater in Brazil: A more responsive and economical approach.

SARS-CoV-2, the novel Coronavirus, was first detected in Wuhan, China, in December 2019, and has since spread rapidly, causing millions of deaths worldwide. As in most countries of the world, in Brazil, the consequences of the COVID-19 pandemic have been catastrophic. Several studies have reported the fecal shedding of SARS-CoV-2 RNA titers from infected symptomatic and asymptomatic individuals. Therefore, the quantification of SARS-CoV-2 in wastewater can be used to track the virus spread in a population. In this study, samples of untreated wastewater were collected for 44 weeks at five sampling sites in the ABC Region (São Paulo, Brazil), in order to evaluate the SARS-CoV-2 occurrence in the sewerage system. SARS-CoV-2 RNA titers were detected throughout the period, and the concentration ranged from 2.7 to 7.7 log10 genome copies.L-1, with peaks in the last weeks of monitoring. Furthermore, we observed a positive correlation between the viral load in wastewater and the epidemiological/clinical data, with the former preceding the latter by approximately two weeks. The COVID-19 prevalence for each sampling site was estimated via Monte-Carlo simulation using the wastewater viral load. The mean predicted prevalence ranged 0.05 to 0.38%, slightly higher than reported (0.016 ± 0.005%) in the ABC Region for the same period. These results highlight the viability of the wastewater surveillance for COVID-19 infection monitoring in the largest urban agglomeration in South America. This approach can be especially useful for health agencies and public decision-makers in predicting SARS-CoV-2 outbreaks, as well as in local tracing of infection clusters.