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      Detection of thermotolerant coliforms and SARS-CoV-2 RNA in sewage and recreational waters in the Ecuadorian coast: A call for improving water quality regulation

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          Abstract

          Wastewater surveillance represents an alternative approach to regulating contamination and the early detection of infectious agents and outbreaks of diseases of public health importance. This study evaluated domestic wastewater effects on recreational waters in estuarine and seawater bodies in Guayas and Santa Elena provinces in Ecuador, South America. Fecal indicator bacteria (thermotolerant coliforms) served as key indicators for evaluation. Physical, chemical, and microbiological quality markers following the Ecuadorian environmental quality standard and the discharge of effluents to the water resource were analyzed. Samples were collected from 44 coastal sites and 2 oxidation lagoons during the dry and rainy seasons of 2020 and 2021, respectively. SARS-CoV-2 RNA was detected in samples with higher E. coli concentrations using reverse transcription quantitative PCR to detect the genes N and ORF1ab. All samples analyzed for SARS-CoV-2 showed Ct ˂ 40 for at least one gene. Four samples showed at least 20 genome copies of gene N per reaction. These were at an artisanal fishing port, an estuarine area (Palmar), a recreational bay, and an oxidation lagoon. A moderate correlation was found between SARS-CoV-2 RNA, thermotolerant coliform and E. coli (p-value ≤ 0.0037), and a strong and positive correlation between thermotolerant coliform and E. coli. (p-value ≤ 0.00001), highlighting the utility of these established parameters as a proxy of the virus. Significant differences were found in the concentrations of thermotolerant coliforms between seasons (p-value = 0.016) and sites (p-value = 0.005). The highest levels of coliforms were found in the dry season (63000 MPN/100 mL) in Anconcito and during the rainy season (14000 MPN/100 mL) at Esterillo in Playas County. It is recommended that the decentralized autonomous governments of the surveyed provinces in Ecuador implement urgent corrective actions and establish medium-term mechanisms to minimize a potential contamination route. Additional parameters must be included in the monitoring, such as Enterococcus and intestinal parasites, due to their public health implications. In the oxidation lagoons, maintenance actions must be carried out, including the dissolution of sediments, an increase in water retention times, and in situ treatment of the sludge, to improve the system’s performance.

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          The Presence of SARS-CoV-2 RNA in Feces of COVID-19 Patients

          In December 2019, coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged in Wuhan, China, and has spread globally. However, the transmission route of SARS-CoV-2 has not been fully understood. In this study, we aimed to investigate SARS-CoV-2 shedding in the excreta of COVID-19 patients. Electronical medical records, including demographics, clinical characteristics, laboratory and radiological findings of enrolled patients were extracted and analyzed. Pharyngeal swab, stool, and urine specimens were collected and tested for SARS-CoV-2 RNA by real-time reverse transcription polymerase chain reaction. Viral shedding at multiple time points in specimens was recorded, and its correlation analyzed with clinical manifestations and the severity of illness. A total of 42 laboratory-confirmed patients were enrolled, 8 (19.05%) of whom had gastrointestinal symptoms. A total of 28 (66.67%) patients tested positive for SARS-CoV-2 RNA in stool specimens, and this was not associated with the presence of gastrointestinal symptoms and the severity of illness. Among them, 18 (64.29%) patients remained positive for viral RNA in the feces after the pharyngeal swabs turned negative. The duration of viral shedding from the feces after negative conversion in pharyngeal swabs was 7 (6-10) days, regardless of COVID-19 severity. The demographics, clinical characteristics, laboratory and radiologic findings did not differ between patients who tested positive and negative for SARS-CoV-2 RNA in the feces. Viral RNA was not detectable in urine specimens from 10 patients. Our results demonstrated the presence of SARS-CoV-2 RNA in the feces of COVID-19 patients and suggested the possibility of SARS-CoV-2 transmission via the fecal-oral route.
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            SARS-CoV-2 in wastewater: State of the knowledge and research needs

            The ongoing global pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been a Public Health Emergency of International Concern, which was officially declared by the World Health Organization. SARS-CoV-2 is a member of the family Coronaviridae that consists of a group of enveloped viruses with single-stranded RNA genome, some of which have been known to cause common colds. Although the major transmission routes of SARS-CoV-2 are inhalation from person-to-person and aerosol/droplet transmission, currently available evidence indicates that the viral RNA is present in wastewater, suggesting the need to better understand wastewater as potential sources of epidemiological data and human health risks. Here, we review the current knowledge related to the potential of wastewater surveillance to understand the epidemiology of COVID-19, methodologies for the detection and quantification of SARS-CoV-2 in wastewater, and information relevant for human health risk assessment of SARS-CoV-2. There has been growing evidence of gastrointestinal symptoms caused by SARS-CoV-2 infections and the presence of viral RNA not only in feces of COVID-19 patients but in wastewater. One of the major challenges in SARS-CoV-2 detection/quantification in wastewater samples is the lack of an optimized and standardized protocol. Currently available data are also limited for conducting a quantitative microbial risk assessment (QMRA) for SARS-CoV-2 exposure pathways. However, modeling-based approaches have a potential role to play in reducing the impact of the ongoing COVID-19 outbreak, and QMRA parameters obtained from previous studies on relevant respiratory viruses help to inform risk assessments of SARS-CoV-2. Our understanding on the potential role of wastewater in SARS-CoV-2 transmission is largely limited by knowledge gaps in its occurrence, persistence, and removal in wastewater. There is an urgent need for further research to establish methodologies for wastewater surveillance and understand the implications of the presence of SARS-CoV-2 in wastewater.
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              First environmental surveillance for the presence of SARS-CoV-2 RNA in wastewater and river water in Japan

              Wastewater-based epidemiology is a powerful tool to understand the actual incidence of coronavirus disease 2019 (COVID-19) in a community because severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of COVID-19, can be shed in the feces of infected individuals regardless of their symptoms. The present study aimed to assess the presence of SARS-CoV-2 RNA in wastewater and river water in Yamanashi Prefecture, Japan, using four quantitative and two nested PCR assays. Influent and secondary-treated (before chlorination) wastewater samples and river water samples were collected five times from a wastewater treatment plant and three times from a river, respectively, between March 17 and May 7, 2020. The wastewater and river water samples (200–5000 mL) were processed by using two different methods: the electronegative membrane-vortex (EMV) method and the membrane adsorption-direct RNA extraction method. Based on the observed concentrations of indigenous pepper mild mottle virus RNA, the EMV method was found superior to the membrane adsorption-direct RNA extraction method. SARS-CoV-2 RNA was successfully detected in one of five secondary-treated wastewater samples with a concentration of 2.4 × 103 copies/L by N_Sarbeco qPCR assay following the EMV method, whereas all the influent samples were tested negative for SARS-CoV-2 RNA. This result could be attributed to higher limit of detection for influent (4.0 × 103–8.2 × 104 copies/L) with a lower filtration volume (200 mL) compared to that for secondary-treated wastewater (1.4 × 102–2.5 × 103 copies/L) with a higher filtration volume of 5000 mL. None of the river water samples tested positive for SARS-CoV-2 RNA. Comparison with the reported COVID-19 cases in Yamanashi Prefecture showed that SARS-CoV-2 RNA was detected in the secondary-treated wastewater sample when the cases peaked in the community. This is the first study reporting the detection of SARS-CoV-2 RNA in wastewater in Japan.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: Project administrationRole: SoftwareRole: SupervisionRole: ValidationRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: ResourcesRole: SupervisionRole: ValidationRole: Writing – original draftRole: Writing – review & editing
                Role: MethodologyRole: SoftwareRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: Formal analysisRole: Resources
                Role: Resources
                Role: Writing – review & editing
                Role: Formal analysisRole: ValidationRole: Writing – original draftRole: Writing – review & editing
                Role: Formal analysis
                Role: Formal analysisRole: Methodology
                Role: Investigation
                Role: Investigation
                Role: Formal analysis
                Role: Writing – original draftRole: Writing – review & editing
                Role: Funding acquisitionRole: Resources
                Role: Investigation
                Role: Writing – original draft
                Role: Writing – original draft
                Role: Formal analysisRole: MethodologyRole: Writing – original draftRole: Writing – review & editing
                Role: Formal analysis
                Role: Formal analysis
                Role: Writing – original draftRole: Writing – review & editing
                Role: MethodologyRole: ResourcesRole: SupervisionRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS One
                plos
                PLOS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                6 May 2024
                2024
                : 19
                : 5
                : e0302000
                Affiliations
                [1 ] Interinstitutional Network for the Study of Aquatic Ecosystems of Ecuador, Guayaquil, Guayas, Ecuador
                [2 ] Ambiente Sociedad & Empresa Research Group, University of Guayaquil, Guayaquil, Guayas, Ecuador
                [3 ] Faculty of Chemical Engineering, University of Guayaquil, Guayaquil, Guayas, Ecuador
                [4 ] Fundación Bioelit, Guayaquil, Guayas, Ecuador
                [5 ] National Institute for Public Health Research–INSPI- Dr. Leopoldo Izquieta Pérez, Technical Direction of Research, Development and Innovation, Guayaquil, Guayas, Ecuador
                [6 ] Faculty of Natural Sciences, Natural Resources Research Institute, University of Guayaquil, Guayaquil, Guayas, Ecuador
                [7 ] Labcestta, Guayaquil, Guayas, Ecuador
                [8 ] Facultad del Mar y Medio Ambiente, Universidad del Pacífico, Guayaquil, Guayas, Ecuador
                [9 ] Environmental Microbiology Laboratory, Institute of Biology of Marine Organisms, CONICET, Puerto Madryn, Chubut, Argentina
                [10 ] National Institute for Public Health Research–INSPI- Dr. Leopoldo Izquieta Pérez, National Reference Center for Exanthematous, Gastroenteric and Vector-borne Viruses, Guayaquil, Guayas, Ecuador
                [11 ] Dirección del Medio Ambiente, Gobierno Provincial de Santa Elena, Santa Elena, Ecuador
                [12 ] Área Nacional de Recreación Playas Villamil, Ministerio de Ambiente Agua y Transición Ecológica, Playas, Ecuador
                [13 ] Department of Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada
                Universidad San Francisco de Quito, ECUADOR
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Author information
                https://orcid.org/0000-0001-5237-9137
                https://orcid.org/0000-0003-0162-0212
                https://orcid.org/0000-0001-7890-012X
                https://orcid.org/0000-0002-5948-9889
                https://orcid.org/0000-0003-2719-5111
                Article
                PONE-D-23-43313
                10.1371/journal.pone.0302000
                11073733
                38709720
                a9224933-c166-4302-bed6-eadcf3955bf9
                © 2024 Cárdenas-Calle et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 22 December 2023
                : 26 March 2024
                Page count
                Figures: 4, Tables: 3, Pages: 18
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100011099, Deutsche Gesellschaft für Internationale Zusammenarbeit;
                Award ID: $ 8,300.00
                Award Recipient :
                Funded by: Prefecture of Santa Elena
                Award ID: $ 5,500.00
                Award Recipient :
                The research outlined in this manuscript benefited from funding received by author MC. Specifically, the study received support from La Deutsche Gesellschaft für Internationale Zusammenarbeit (GIZ) under grant number $8,300 ( https://www.giz.de/en/worldwide/28451.html). Additionally, the project received funding from the Prefecture of Santa Elena, under grant number $5,500 ( https://www.santaelena.gob.ec/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
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                Physical Sciences
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                Chemical Reactions
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                Marine and Aquatic Sciences
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