RESUMEN
During April-July 2022, outbreaks of severe acute hepatitis of unknown etiology (SAHUE) were reported in 35 countries. Five percent of cases required liver transplantation, and 22 patients died. Viral metagenomic studies of clinical samples from SAHUE cases showed a correlation with human adenovirus F type 41 (HAdV-F41) and adeno-associated virus type 2 (AAV2). To explore the association between those DNA viruses and SAHUE in children in Ireland, we quantified HAdV-F41 and AAV2 in samples collected from a wastewater treatment plant serving 40% of Ireland's population. We noted a high correlation between HAdV-F41 and AAV2 circulation in the community and SAHUE clinical cases. Next-generation sequencing of the adenovirus hexon in wastewater demonstrated HAdV-F41 was the predominant HAdV type circulating. Our environmental analysis showed increased HAdV-F41 and AAV2 prevalence in the community during the SAHUE outbreak. Our findings highlight how wastewater sampling could aid in surveillance for respiratory adenovirus species.
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Infecciones por Adenovirus Humanos , Adenovirus Humanos , Hepatitis , Infecciones del Sistema Respiratorio , Humanos , Niño , Aguas Residuales , Irlanda/epidemiología , Adenovirus Humanos/genética , Hepatitis/epidemiología , Brotes de Enfermedades , Enfermedad Aguda , Infecciones por Adenovirus Humanos/epidemiología , Filogenia , Infecciones del Sistema Respiratorio/epidemiologíaRESUMEN
Natural mineral waters contain indigenous bacteria characteristic of each spring source. Once bottled, these communities change over time until the water is consumed. Bottle material is believed to play a major role in the succession of these populations, but very few studies to date have evaluated the effect of this material on bacterial communities. In this study, we examined the microbial community structure of three natural mineral waters over 3 months after bottling in glass and polyethylene terephthalate (PET) bottles. To this end, we used culture-dependent (heterotrophic plate count) and culture-independent methods (16S rRNA massive gene sequencing, denaturing gradient gel electrophoresis (DGGE) and fluorescent microscopy with vital dyes). Total and viable cell counts increased by around 1-2 log10 units between 1 and 2 weeks after bottling and then remained constant over 3 months for all waters regardless of the bottle material. DGGE fingerprints and 16S rRNA massive sequencing analysis both indicated that different communities were established in the waters two weeks after bottling in the different bottle materials. In conclusion, no differences in total, viable and culturable bacteria counts were observed between mineral waters bottled with PET or glass during shelf life storage. Nevertheless, in spite of changes in the communities, each water brand and material presented a distinct microbial community structure clearly distinguishable from the others, which could be interesting for traceability purposes.
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Bacterias/aislamiento & purificación , Agua Potable/microbiología , Almacenamiento de Alimentos , Aguas Minerales/microbiología , Microbiología del Agua , Bacterias/clasificación , Recuento de Colonia Microbiana , Variación Genética , Vidrio , Secuenciación de Nucleótidos de Alto Rendimiento , Metagenómica , Tereftalatos Polietilenos , Reacción en Cadena de la Polimerasa , ARN Ribosómico 16S/genéticaRESUMEN
The aim of this work was to assess the suitability of matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) for routine heterotrophic monitoring in a drinking water treatment plant. Water samples were collected from raw surface water and after different treatments during two campaigns over a 1-year period. Heterotrophic bacteria were studied and isolates were identified by MALDI-TOF MS. Moreover, the diversity index and the coefficient of population similarity were also calculated using biochemical fingerprinting of the populations studied. MALDI-TOF MS enabled us to characterize and detect changes in the bacterial community composition throughout the water treatment plant. Raw water showed a large and diverse population which was slightly modified after initial treatment steps (sand filtration and ultrafiltration). Reverse osmosis had a significant impact on the microbial diversity, while the final chlorination step produced a shift in the composition of the bacterial community. Although MALDI-TOF MS could not identify all the isolates since the available MALDI-TOF MS database does not cover all the bacterial diversity in water, this technique could be used to monitor bacterial changes in drinking water treatment plants by creating a specific protein profile database for tracking purposes.
Asunto(s)
Bacterias/aislamiento & purificación , Agua Potable/microbiología , Monitoreo del Ambiente/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Purificación del Agua/normasRESUMEN
Reverse osmosis membrane filtration technology (RO) is used to treat drinking water. After RO treatment, bacterial growth is still observed in water. However, it is not clear whether those microorganisms belong to species that can pose a health risk, such as Pseudomonas spp. The goal of this study is to characterize the bacterial isolates from a medium that is selective for Pseudomonas and Aeromonas which were present in the water fraction before and after the RO. To this end, isolates were recovered over two years and were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. They were then biochemically phenotyped and the population similarity indexes were calculated. The isolates were analysed for their capacity to form biofilms in vitro and antimicrobial susceptibility. There were significant differences between the microbial populations in water before and after RO. Furthermore, the structures of the populations analysed at the same sampling point were similar in different sampling campaigns. Some of the isolates had the capacity to form a biofilm and showed resistance to different antibiotics. A successful level filtration via RO and subsequent recolonization of the membrane with different species from those in the feed water was found. Pseudomonas aeruginosa was not recovered from among the isolates. This study increases the knowledge on the microorganisms present in water after RO treatment, with focus in one of the genus causing problems in RO systems associated with human health risk, Pseudomonas.
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Pseudomonas/fisiología , Microbiología del Agua , Purificación del Agua/métodos , Biopelículas , Filtración , Humanos , Membranas Artificiales , ÓsmosisRESUMEN
Antibiotic resistance genes (ARGs) have been extensively observed in bacterial DNA, and more recently, in phage particles from various water sources and food items. The pivotal role played by ARG transmission in the proliferation of antibiotic resistance and emergence of new resistant strains calls for a thorough understanding of the underlying mechanisms. The aim of this study was to assess the suitability of the prototypical p-crAssphage, a proposed indicator of human fecal contamination, and the recently isolated crAssBcn phages, both belonging to the Crassvirales group, as potential indicators of ARGs. These crAss-like phages were evaluated alongside specific ARGs (blaTEM, blaCTX-M-1, blaCTX-M-9, blaVIM, blaOXA-48, qnrA, qnrS, tetW and sul1) within the total DNA and phage DNA fractions in water and food samples containing different levels of fecal pollution. In samples with high fecal load (>103 CFU/g or ml of E. coli or somatic coliphages), such as wastewater and sludge, positive correlations were found between both types of crAss-like phages and ARGs in both DNA fractions. The strongest correlation was observed between sul1 and crAssBcn phages (rho = 0.90) in sludge samples, followed by blaCTX-M-9 and p-crAssphage (rho = 0.86) in sewage samples, both in the phage DNA fraction. The use of crAssphage and crAssBcn as indicators of ARGs, considered to be emerging environmental contaminants of anthropogenic origin, is supported by their close association with the human gut. Monitoring ARGs can help to mitigate their dissemination and prevent the emergence of new resistant bacterial strains, thus safeguarding public health.
RESUMEN
Microorganisms, including potential pathogens, can colonise plastic surfaces in aquatic environments. This study investigates the colonisation of plastic pellets by Escherichia coli (E. coli) as a proxy for faecal pathogens in aquatic environments. Plastic pellets from a polluted beach were placed in seawater aquaria spiked with E. coli. Diverse bacteria, primarily from the Proteobacteria phylum, rapidly colonised the pellets within 24 h, with notable species known for plastic or hydrocarbon degradation. Over 26 days, biofilms formed on the plastic surfaces, reaching bacterial populations of up to 6.8·105 gene copies (gc) of the 16S rRNA mm-2. E. coli, was detected in the pellets for up to 7 days using culture methods, exhibiting varying attachment densities regardless of source or environmental factors. The study highlights plastic biofilms as reservoirs for E. coli, contributing to the survival and persistence of faecal bacteria in aquatic systems. These findings deepen our understanding of the risks associated with plastic pollution in marine settings, offering insights into the behaviour of faecal indicators and their implications for water quality assessments, while providing valuable information on potential pathogen dissemination within plastic-associated microbial communities.
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Biopelículas , Escherichia coli , Plásticos , Agua de Mar , Biopelículas/crecimiento & desarrollo , Escherichia coli/genética , Escherichia coli/fisiología , Escherichia coli/aislamiento & purificación , Escherichia coli/crecimiento & desarrollo , Agua de Mar/microbiología , ARN Ribosómico 16S/genética , Microbiología del AguaRESUMEN
Wastewater-based surveillance constitutes a valuable methodology for the continuous monitoring of viral circulation, with the capacity to function as an early warning system. It holds particular significance in scenarios where respiratory viruses exhibit overlapping clinical presentations, as occurs with SARS-CoV-2, influenza virus (IV), and respiratory syncytial virus (RSV), and allows seasonal virus outbreaks to be distinguished from COVID-19 peaks. Furthermore, sewage sludge, given it harbors concentrated human waste from a large population, serves as a substantial reservoir for pathogen detection. To effectively integrate wastewater-based epidemiology into infectious disease surveillance, the detection methods employed in wastewater samples must be adapted to the distinct characteristics of sludge matrices. In this study, we adapted and applied protocols for the detection of IV and RSV in sewage sludge, comparing their performance with the results obtained in wastewater. To assess the efficiency of these protocols, sludge and wastewater samples were spiked with IV and RSV RNA, either free or incorporated in lentiviral particles. Samples were concentrated using the aluminum hydroxide adsorption-precipitation method before viral RNA extraction. Absolute virus quantification was carried out by RT-qPCR, including an internal control to monitor potential inhibitory factors. Recovery efficiencies for both free IV and RSV RNA were 60 % in sludge, and 75 % and 71 % respectively in wastewater, whereas the values for IV and RSV RNA in lentiviral particles were 16 % and 10 % in sludge and 21 % and 17 % in wastewater respectively. Additionally, the protocol enabled the quantification of naturally occurring IV and RSV in wastewater and sludge samples collected from two wastewater treatment plants during the winter months, thus affirming the efficacy of the employed methodologies.
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Orthomyxoviridae , Virus , Humanos , Virus Sincitiales Respiratorios , Aguas del Alcantarillado , Aguas Residuales , ARN ViralRESUMEN
Wastewater-based epidemiology (WBE) has been an important tool for population surveillance during the COVID-19 pandemic and continues to play a key role in monitoring SARS-CoV-2 infection levels following reductions in national clinical testing schemes. Studies measuring decay profiles of SARS-CoV-2 in wastewater have underscored the value of WBE, however investigations have been hampered by high biosafety requirements for SARS-CoV-2 infection studies. Therefore, surrogate viruses with lower biosafety standards have been used for SARS-CoV-2 decay studies, such as murine hepatitis virus (MHV), but few studies have directly compared decay rates of both viruses. We compared the persistence of SARS-CoV-2 and MHV in wastewater, using 50 % tissue culture infectious dose (TCID50) and reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays to assess infectious virus titre and viral gene markers, respectively. Infectious SARS-CoV-2 and MHV indicate similar endpoints, however observed early decay characteristics differed, with infectious SARS-CoV-2 decaying more rapidly than MHV. We find that MHV is an appropriate infectious virus surrogate for viable SARS-CoV-2, however inconsistencies exist in viral RNA decay parameters, indicating MHV may not be a suitable nucleic acid surrogate across certain temperature regimes. This study highlights the importance of sample preparation and the potential for decay rate overestimation in wastewater surveillance for SARS-CoV-2 and other pathogens.
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Virus de la Hepatitis Murina , ARN Viral , SARS-CoV-2 , Aguas Residuales , Aguas Residuales/virología , SARS-CoV-2/genética , Virus de la Hepatitis Murina/fisiología , COVID-19 , Animales , Estabilidad del ARNRESUMEN
The objective of this study was to comprehensively characterise the resistome, the collective set of antimicrobial resistance genes in a given environment, of two rivers, from their source to discharge into the sea, as these flow through areas of different land use. Our findings reveal significant differences in the riverine resistome composition in areas of different land uses, with increased abundance and diversity of AMR in downstream agricultural and urban locations, with the resistome in urban areas more similar to the resistome in wastewater. The changes in resistome were accompanied by changes in microbial communities, with a reduction in microbial diversity in downstream agricultural and urban affected areas, driven mostly by increased relative abundance in the phyla, Bacteroidetes and Proteobacteria. These results provide insight into how pollution associated with agricultural and urban activities affects microbial communities and influences AMR in aquatic water bodies. These results add valuable insights to form effective strategies for mitigating and preserving aquatic ecosystems. Overall, our study highlights the critical role of the environment in the development and dissemination of AMR and underscores the importance of adopting a One Health approach to address this global public health threat.
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Agricultura , Ríos , Ríos/microbiología , Agricultura/métodos , Monitoreo del Ambiente , Microbiota/efectos de los fármacos , Microbiología del Agua , Farmacorresistencia Bacteriana/genética , Aguas Residuales/microbiología , Bacterias/genética , Bacterias/efectos de los fármacosRESUMEN
Recreational bathing waters are complex systems with diverse inputs from multiple anthropogenic and zoogenic sources of faecal contamination. Faecal contamination is a substantial threat to water quality and public health. Here we present a comprehensive strategy to estimate the contribution of faecal indicator bacteria (FIB) from different biological sources on two at-risk beaches in Dublin, Ireland. The daily FIB loading rate was determined for three sources of contamination: a sewage-impacted urban stream, dog and wild bird fouling. This comparative analysis determined that the stream contributed the highest daily levels of FIB, followed by dog fouling. Dog fouling may be a significant source of FIB, contributing approximately 20 % of E. coli under certain conditions, whereas wild bird fouling contributed a negligible proportion of FIB (<3 %). This study demonstrates that source-specific quantitative microbial source apportionment (QMSA) strategies are vital to identify primary public health risks and target interventions to mitigate faecal contamination.
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Monitoreo del Ambiente , Heces , Heces/microbiología , Monitoreo del Ambiente/métodos , Animales , Irlanda , Playas , Microbiología del Agua , Contaminación del Agua/estadística & datos numéricos , Contaminación del Agua/análisis , Perros , Aguas del Alcantarillado/microbiología , Escherichia coli/aislamiento & purificación , Calidad del Agua , Bacterias/aislamiento & purificación , Aves/microbiologíaRESUMEN
The WHO recognises antimicrobial resistance (AMR) as a global health threat. The environment can act as a reservoir, facilitating the exchange and the physical movement of resistance. Aquatic environments are at particular risk of pollution, with large rivers subject to pollution from nearby human, industrial or agricultural activities. The land uses associated with these activities can influence the type of pollution. One type of pollution and a likely contributor to AMR pollution that lowers water quality is faecal pollution. Both pose an acute health risk and could have implications for resistance circulating in communities. The effects of land use are typically studied using physiochemical parameters or in isolation of one another. However, this study aimed to investigate the impact of different land uses on riverine systems. We explored whether differences in sources of faecal contamination are reflected in AMR gene concentrations across agricultural and urban areas. Water quality from three rivers impacted by different land uses was assessed over one year by quantifying faecal indicator bacteria (FIB), microbial source tracking markers (MST) and AMR genes. In addition, a multiparametric analysis of AMR gene pollution was carried out to understand whether agricultural and urban areas are similarly impacted. Faecal indicators varied greatly, with the highest levels of FIB and the human MST marker observed in urban regions. In addition, these faecal markers correlated with AMR genes. Similarly, significant correlations between the ruminant MST marker and AMR gene levels in agriculture areas were observed. Overall, applying multiparametric analyses to include AMR gene levels, separation and clustering of sites were seen based on land use characterisation. This study suggests that differences in prescription of antimicrobials used in animal and human healthcare may influence environmental resistomes across agricultural and urban areas. In addition, public health risks due to exposure to faecal contamination and AMR genes are highlighted.
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Antibacterianos , Monitoreo del Ambiente , Animales , Humanos , Farmacorresistencia Bacteriana/genética , Calidad del Agua , Ríos , Bacterias , Heces/microbiología , Contaminación del Agua , Microbiología del AguaRESUMEN
In the first and limiting step of nitrification, ammonia (NH3) is oxidised to nitrite (NO2-) by the action of some prokaryotes, including bacteria of the Nitrosomonas genus. A potential approach to nitrification inhibition would be through the application of phages, but until now this method has been unexplored and no virulent phages that infect nitrifying bacteria have been described. In this study, we report the isolation of the first phage infecting some Nitrosomonas species. This polyvalent virulent phage (named ΦNF-1) infected Nitrosomonas europaea, Nitrosomonas communis, and Nitrosomonas nitrosa. Phage ΦNF-1 has the morphology of the Podoviridae family, a dsDNA genome of 41,596 bp and a 45.1 % GC content, with 50 predicted open reading frames. Phage ΦNF-1 was found to inhibit bacterial growth and reduce NH4+ consumption in the phage-treated cultures. The application of phages as biocontrol agents could be a useful strategy for nitrification inhibition without the restrictions associated with chemical inhibitors.
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Bacteriófagos , Nitrosomonas europaea , Bacteriófagos/genética , Nitrosomonas , Bacterias , Nitritos , AmoníacoRESUMEN
Crassvirales (crAss-like phages) are an abundant group of human gut-specific bacteriophages discovered in silico. The use of crAss-like phages as human fecal indicators is proposed but the isolation of only seven cultured strains of crAss-like phages to date has greatly hindered their study. Here, we report the isolation and genetic characterization of 25 new crAss-like phages (termed crAssBcn) infecting Bacteroides intestinalis, belonging to the order Crassvirales, genus Kehishuvirus and, based on their genomic variability, classified into six species. CrAssBcn phage genomes are similar to ΦCrAss001 but show genomic and aminoacidic differences when compared to other crAss-like phages of the same family. CrAssBcn phages are detected in fecal metagenomes around the world at a higher frequency than ΦCrAss001. This study increases the known crAss-like phage isolates and their abundance and heterogeneity open the question of what member of the Crassvirales group should be selected as human fecal marker.
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Bacteriófagos , Humanos , Heterogeneidad Genética , Genómica , Heces , Metagenoma/genética , Genoma Viral/genética , FilogeniaRESUMEN
Bacteriophages have been shown to play an important role in harbouring and propagating antibiotic resistance genes (ARGs). Faecal matter contains high levels of phages, suggesting that faecal contamination of water bodies may lead to increased antimicrobial resistance (AMR) levels due to increased phage loading in aquatic environments. In this study, we assessed whether faecal pollution of three rivers (Rivers Liffey, Tolka, and Dodder) was responsible for increased levels of ARGs in phage particles using established phage-faecal markers, focusing on four ARGs (blaTEM, tet(O), qnrS, and sul1). We observed all four ARGs in phage fractions in all three rivers, with ARGs more frequently observed in agricultural and urban sampling sites compared to their source. These findings highlight the role of faecal pollution in environmental AMR and the impact of agricultural and urban activities on water quality. Furthermore, our results suggest the importance of including phages as indicators when assessing environmental AMR, as they serve as significant reservoirs of resistance genes in aquatic environments. This study provides important insights into the role of faecal pollution and phages in the prevalence of AMR in the environment and the need for their inclusion in future studies to provide a comprehensive understanding of environmental AMR.
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Bacteriófagos , Bacteriófagos/genética , Genes Bacterianos , Farmacorresistencia Microbiana/genética , Heces , Antibacterianos/farmacologíaRESUMEN
The EU Bathing Water Directive (BWD) requires member states to assess bathing water quality according to the levels of faecal indicator bacteria (FIB) in designated bathing areas. However, this criterion has two significant limitations given that the BWD does not; (i) account for differences in hydrodynamic properties of bathing waters and, (ii) assumes that all faecal pathogens decay equally in aquatic environments. This study simulated sewage discharge events in three hypothetical aquatic environments characterised by different advection and dispersion parameters in the solute transport equation. Temporal changes in the downstream concentration of six faecal indicators were determined in simulations that utilised measured decay rates of each faecal indicator from a programme of controlled microcosm experiments in fresh and seawater environments. The results showed that the decay rates of faecal indicators are not a critical parameter in advection dominant water bodies, such as in fast-flowing rivers. Therefore, faecal indicator selection is less important in such systems and for these, FIB remains the most cost-effective faecal indicator to monitor the public health impacts of faecal contamination. In contrast, consideration of faecal indicator decay is important when assessing dispersion and advection/dispersion dominant systems, which would pertain to transitional (estuarine) and coastal waterbodies. Results suggest that the inclusion of viral indicators, such as crAssphage and PMMoV, could improve the reliability of water quality modelling and minimise the risk of waterborne illnesses from faecal contamination.
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Hidrodinámica , Salud Pública , Calidad del Agua , Bacterias , Playas , Monitoreo del Ambiente , Heces/microbiología , Reproducibilidad de los Resultados , Microbiología del AguaRESUMEN
Wastewater-based surveillance can be a valuable tool to monitor viral circulation and serve as an early warning system. For respiratory viruses that share similar clinical symptoms, namely SARS-CoV-2, influenza, and respiratory syncytial virus (RSV), identification in wastewater may allow differentiation between seasonal outbreaks and COVID-19 peaks. In this study, to monitor these viruses as well as standard indicators of fecal contamination, a weekly sampling campaign was carried out for 15 months (from September 2021 to November 2022) in two wastewater treatment plants that serve the entire population of Barcelona (Spain). Samples were concentrated by the aluminum hydroxide adsorption-precipitation method and then analyzed by RNA extraction and RT-qPCR. All samples were positive for SARS-CoV-2, while the positivity rates for influenza virus and RSV were significantly lower (10.65 % for influenza A (IAV), 0.82 % for influenza B (IBV), 37.70 % for RSV-A and 34.43 % for RSV-B). Gene copy concentrations of SARS-CoV-2 were often approximately 1 to 2 logarithmic units higher compared to the other respiratory viruses. Clear peaks of IAV H3:N2 in February and March 2022 and RSV in winter 2021 were observed, which matched the chronological incidence of infections recorded in the Catalan Government clinical database. In conclusion, the data obtained from wastewater surveillance provided new information on the abundance of respiratory viruses in the Barcelona area and correlated favorably with clinical data.
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COVID-19 , Gripe Humana , Infecciones por Virus Sincitial Respiratorio , Virus , Humanos , Gripe Humana/epidemiología , Virus Sincitiales Respiratorios/genética , Aguas Residuales , COVID-19/epidemiología , SARS-CoV-2 , Monitoreo Epidemiológico Basado en Aguas Residuales , Infecciones por Virus Sincitial Respiratorio/epidemiologíaRESUMEN
SARS-CoV-2 RNA quantification in wastewater is an important tool for monitoring the prevalence of COVID-19 disease on a community scale which complements case-based surveillance systems. As novel variants of concern (VOCs) emerge there is also a need to identify the primary circulating variants in a community, accomplished to date by sequencing clinical samples. Quantifying variants in wastewater offers a cost-effective means to augment these sequencing efforts. In this study, SARS-CoV-2 N1 RNA concentrations and daily loadings were determined and compared to case-based data collected as part of a national surveillance programme to determine the validity of wastewater surveillance to monitor infection spread in the greater Dublin area. Further, sequencing of clinical samples was conducted to determine the primary SARS-CoV-2 lineages circulating in Dublin. Finally, digital PCR was employed to determine whether SARS-CoV-2 VOCs, Alpha and Delta, were quantifiable from wastewater. No lead or lag time was observed between SARS-CoV-2 wastewater and case-based data and SARS-CoV-2 trends in Dublin wastewater significantly correlated with the notification of confirmed cases through case-based surveillance preceding collection with a 5-day average. This demonstrates that viral RNA in Dublin's wastewater mirrors the spread of infection in the community. Clinical sequence data demonstrated that increased COVID-19 cases during Ireland's third wave coincided with the introduction of the Alpha variant, while the fourth wave coincided with increased prevalence of the Delta variant. Interestingly, the Alpha variant was detected in Dublin wastewater prior to the first genome being sequenced from clinical samples, while the Delta variant was identified at the same time in clinical and wastewater samples. This work demonstrates the validity of wastewater surveillance for monitoring SARS-CoV-2 infections and also highlights its effectiveness in identifying circulating variants which may prove useful when sequencing capacity is limited.
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COVID-19 , SARS-CoV-2 , COVID-19/epidemiología , Humanos , Irlanda/epidemiología , ARN Viral , SARS-CoV-2/genética , Aguas Residuales/análisis , Monitoreo Epidemiológico Basado en Aguas ResidualesRESUMEN
Wastewater surveillance for pathogens using reverse transcription-polymerase chain reaction (RT-PCR) is an effective and resource-efficient tool for gathering community-level public health information, including the incidence of coronavirus disease-19 (COVID-19). Surveillance of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) in wastewater can potentially provide an early warning signal of COVID-19 infections in a community. The capacity of the world's environmental microbiology and virology laboratories for SARS-CoV-2 RNA characterization in wastewater is increasing rapidly. However, there are no standardized protocols or harmonized quality assurance and quality control (QA/QC) procedures for SARS-CoV-2 wastewater surveillance. This paper is a technical review of factors that can cause false-positive and false-negative errors in the surveillance of SARS-CoV-2 RNA in wastewater, culminating in recommended strategies that can be implemented to identify and mitigate some of these errors. Recommendations include stringent QA/QC measures, representative sampling approaches, effective virus concentration and efficient RNA extraction, PCR inhibition assessment, inclusion of sample processing controls, and considerations for RT-PCR assay selection and data interpretation. Clear data interpretation guidelines (e.g., determination of positive and negative samples) are critical, particularly when the incidence of SARS-CoV-2 in wastewater is low. Corrective and confirmatory actions must be in place for inconclusive results or results diverging from current trends (e.g., initial onset or reemergence of COVID-19 in a community). It is also prudent to perform interlaboratory comparisons to ensure results' reliability and interpretability for prospective and retrospective analyses. The strategies that are recommended in this review aim to improve SARS-CoV-2 characterization and detection for wastewater surveillance applications. A silver lining of the COVID-19 pandemic is that the efficacy of wastewater surveillance continues to be demonstrated during this global crisis. In the future, wastewater should also play an important role in the surveillance of a range of other communicable diseases.
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COVID-19 , Pandemias , Humanos , Estudios Prospectivos , ARN Viral , Reproducibilidad de los Resultados , Estudios Retrospectivos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , SARS-CoV-2 , Aguas Residuales , Monitoreo Epidemiológico Basado en Aguas ResidualesRESUMEN
The introduction of SARS-CoV-2 containing human stool and sewage into water bodies may raise public health concerns. However, assessment of public health risks by faecally contaminated water is limited by a lack of knowledge regarding the persistence of infectious SARS-CoV-2 in water. In the present study the decay rates of viable infectious SARS-CoV-2 and SARS-CoV-2 RNA were determined in river and seawater at 4 and 20°C. These decay rates were compared to S. typhimurium bacteriophage MS2 and pepper mild mottle virus (PMMoV). Persistence of viable SARS-CoV-2 was temperature dependent, remaining infectious for significantly longer periods of time in both freshwater and seawater at 4°C than at 20°C. T90 for infectious SARS-CoV-2 in river water was 2.3 days and 3.8 days at 20°C and 4°C, respectively. The T90 values were 1.1 days and 2.2 days in seawater at 20°C and 4°C, respectively. In contrast to the rapid inactivation of infectious SARS-CoV-2 in river and sea water, viral RNA was relatively stable. The RNA decay rates were increased in non-sterilised river and seawater, presumably due to the presence of microbiota. The decay rates of infectious MS2, MS2 RNA and PMMoV RNA differed significantly from the decay rate of SARS-CoV-2 RNA, suggesting that their use as surrogate markers for the persistence of SARS-CoV-2 in the environment is limited.
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COVID-19 , SARS-CoV-2 , Humanos , ARN Viral , Ríos , Aguas ResidualesRESUMEN
The detection of fecal viral pathogens in water is hampered by their great variety and complex analysis. As traditional bacterial indicators are poor viral indicators, there is a need for alternative methods, such as the use of somatic coliphages, which have been included in water safety regulations in recent years. Some researchers have also recommended the use of reference viral pathogens such as noroviruses or other enteric viruses to improve the prediction of fecal viral pollution of human origin. In this work, phages previously tested in microbial source tracking studies were compared with norovirus and adenovirus for their suitability as indicators of human fecal viruses. The phages, namely those infecting human-associated Bacteroides thetaiotaomicron strain GA17 (GA17PH) and porcine-associated Bacteroides strain PG76 (PGPH), and the human-associated crAssphage marker (crAssPH), were evaluated in sewage samples and fecal mixtures obtained from different animals in five European countries, along with norovirus GI + GII (NoV) and human adenovirus (HAdV). GA17PH had an overall sensitivity of ≥83% and the highest specificity (>88%) for human pollution source detection. crAssPH showed the highest sensitivity (100%) and specificity (100%) in northern European countries but a much lower specificity in Spain and Portugal (10 and 30%, respectively), being detected in animal wastewater samples with a high concentration of fecal indicators. The correlations between GA17PH, crAssPH, or the sum of both (BACPH) and HAdV or NoV were higher than between the two human viruses, indicating that bacteriophages are feasible indicators of human viral pathogens of fecal origin and constitute a promising, easy to use and affordable alternative to human viruses for routine water safety monitoring.