Next-generation sequencing for surveillance of antimicrobial resistance and pathogenicity in municipal wastewater treatment plants.

The World Health Organization (WHO) ranks antimicrobial resistance (AMR) and various pathogens among the top 10 health threats. It is estimated that by 2050, the number of human deaths due to AMR will reach 10 million annually. On the other hand, several infectious outbreaks such as SARS, H1N1 influenza, Ebola, Zika fever, and COVID-19 have severely affected human populations worldwide in the last 20 years. These recent global diseases have generated the need to monitor outbreaks of pathogens and AMR to establish effective public health strategies. This review presents AMR and pathogenicity associated with wastewater treatment plants (WWTP), focusing on Next Generation Sequencing (NGS) monitoring as a complementary system to clinical surveillance. In this regard, WWTP may be monitored at three main points. First, at the inlet (raw wastewater or influent) to identify a broad spectrum of AMR and pathogens contained in the excretions of residents served by sewer networks, with a specific spatio-temporal location. Second, at the effluent, to ensure the elimination of AMR and pathogens in the treated water, considering the rising demand for safe wastewater reuse. Third, in sewage sludge or biosolids, since their beneficial use or final disposal can represent a significant risk to public health. This review is divided into two sections to address the importance and implications of AMR and pathogen surveillance in wastewater and WWTP, based on NGS. The first section presents the fundamentals of surveillance techniques applied in WWTP (metataxonomics, metagenomics, functional metagenomics, metaviromics, and metatranscriptomics). Their scope and limitations are analyzed to show how microbiological and qPCR techniques complement NGS surveillance, overcoming its limitations. The second section discusses the contribution of 36 NGS research papers on WWTP surveillance, highlighting the current situation and perspectives. In both sections, research challenges and opportunities are presented.

Beyond COVID: The Value of Wastewater Based Epidemiology (WBE) Monitoring for Municipal and Industrial Systems

The goal of this paper is to demonstrate how Wastewater Based Epidemiology (WBE) can be used after COVID-19 in both Municipal and Industrial wastewater systems to proactively monitor, manage, and avoid risks that could negatively impact the business continuity and resiliency of an organization. The history of WBE will first be reviewed to show how it has been used to maximize public health protection and social well-being while minimizing economic impacts and unintended consequences in public and private settings. The design of a WBE monitoring program for Closed, Semi-Closed, and Open Municipal and Industrial wastewater systems will be evaluated through a couple of case studies. Alignment between WBE programs and an organizations' risk management programs, sustainability goals, and ethical considerations will also be explored. Copyright © 2022 Water Environment Federation.

Wastewater monitoring of SARS-CoV-2 RNA at K-12 schools: comparison to pooled clinical testing data.

Background: Wastewater measurements of SARS-CoV-2 RNA have been extensively used to supplement clinical data on COVID-19. Most examples in the literature that describe wastewater monitoring for SARS-CoV-2 RNA use samples from wastewater treatment plants and individual buildings that serve as the primary residence of community members. However, wastewater surveillance can be an attractive supplement to clinical testing in K-12 schools where individuals only spend a portion of their time but interact with others in close proximity, increasing risk of potential transmission of disease. Methods: Wastewater samples were collected from two K-12 schools in California and divided into solid and liquid fractions to be processed for detection of SARS-CoV-2. The resulting detection rate in each wastewater fraction was compared to each other and the detection rate in pooled clinical specimens. Results: Most wastewater samples were positive for SARS-CoV-2 RNA when clinical testing was positive (75% for solid samples and 100% for liquid samples). Wastewater samples continued to test positive for SARS-CoV-2 RNA when clinical testing was negative or in absence of clinical testing (83% for both solid and liquid samples), indicating presence of infected individuals in the schools. Wastewater solids had a higher concentration of SARS-CoV-2 than wastewater liquids on an equivalent mass basis by three orders of magnitude.

Assessment of two volumetrically different concentration approaches to improve sensitivities for SARS-CoV-2 detection during wastewater monitoring.

Wastewater monitoring for severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), the virus responsible for the global coronavirus disease 2019 (COVID-19) pandemic, has highlighted the need for methodologies capable of assessing viral prevalence during periods of low population infection. To address this need, two volumetrically different, methodologically similar concentration approaches were compared for their abilities to detect viral nucleic acid and infectious SARS-CoV-2 signal from primary influent samples. For Method 1, 2 L of SARS-CoV-2 seeded wastewater was evaluated using a dead-end hollow fiber ultrafilter (D-HFUF) for primary concentration, followed by the CP Select™ for secondary concentration. For Method 2, 100 mL of SARS-CoV-2 seeded wastewater was evaluated using the CP Select™ procedure. Following D-HFUF concentration (Method 1), significantly lower levels of infectious SARS-CoV-2 were lost (P value range: 0.0398-0.0027) compared to viral gene copy (GC) levels detected by the US Centers for Disease Control (CDC) N1 and N2 reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) assays. Subsamples at different steps in the concentration process were also taken to better characterize the losses of SARS-CoV-2 during the concentration process. During the centrifugation step (prior to CP Select™ concentration), significantly higher losses (P value range: 0.0003 to <0.0001) occurred for SARS-CoV-2 GC levels compared to infectious virus for Method 1, while between the methods, significantly higher infectious viral losses were observed for Method 2 (P = 0.0002). When analyzing overall recovery of endogenous SARS-CoV-2 in wastewater samples, application of Method 1 improved assay sensitivities (P = <0.0001) compared with Method 2; this was especially evident during periods of lower COVID-19 case rates within the sewershed. This study describes a method which can successfully concentrate infectious SARS-CoV-2 and viral RNA from wastewater. Moreover, we demonstrated that large volume wastewater concentration provides additional sensitivity needed to improve SARS-CoV-2 detection, especially during low levels of community disease prevalence.

SARS-CoV-2 Testing of Aircraft Wastewater Shows That Mandatory Tests and Vaccination Pass before Boarding Did Not Prevent Massive Importation of Omicron Variant into Europe.

BACKGROUND: Most new SARS-CoV-2 epidemics in France occurred following the importation from abroad of emerging viral variants. Currently, the risk of new variants being imported is controlled based on a negative screening test (PCR or antigenic) and proof of up-to-date vaccine status, such as the International Air Transport Association travel pass. METHODS: The wastewater from two planes arriving in Marseille (France) from Addis Ababa (Ethiopia) in December 2021 was tested by RT-PCR to detect SARS-CoV2 and screen for variants. These tests were carried out between landing and customs clearance and were then sequenced by MiSeq Illumina. Antigenic tests and sequencing by NovaSeq were carried out on respiratory samples collected from the 56 passengers on the second flight. RESULTS: SARS-CoV-2 RNA suspected of being from the Omicron BA.1 variant was detected in the aircraft's wastewater. SARS-CoV2 RNA was detected in 11 [20%) passengers and the Omicron BA.1 variant was identified. CONCLUSION: Our work shows the efficiency of aircraft wastewater testing to detect SARS-CoV-2 cases among travellers and to identify the viral genotype. It also highlights the low efficacy of the current control strategy for flights entering France from outside Europe, which combines a requirement to produce a vaccine pass and proof of a negative test before boarding.

Maintaining a social license to operate for wastewater-based monitoring: The case of managing infectious disease and the COVID-19 pandemic.

Wastewater monitoring as a public health tool is well-established and the SARS-CoV-2 (COVID-19) pandemic has seen its widespread uptake. Given the significant potential of wastewater monitoring as a public health surveillance and decision support tool, it is important to understand what measures are required to allow the long-term benefits of wastewater monitoring to be fully realized, including how to establish and/or maintain public support. The potential for positive SARS-CoV-2 detections to trigger enforced, community-wide public health interventions (e.g., lockdowns and other impacts on civil liberties) further emphasises the need to better understand the role of public engagement in successful wastewater-based monitoring programs. This paper systematically reviews the processes of building and maintaining the social license to operate wastewater monitoring. We specifically explore the relationship between different stakeholder communities and highlight the information and actions that are required to establish a social license to operate and then prevent its loss. The paper adds to the literature on social license to operate by extending its application to new domains and offers a dynamic model of social license to help guide the agenda for researcher and practitioner communities.

Microbiome Analysis for Wastewater Surveillance during COVID-19.

Wastewater surveillance (WS), when coupled with advanced molecular techniques, offers near real-time monitoring of community-wide transmission of SARS-CoV-2 and allows assessing and mitigating COVID-19 outbreaks, by evaluating the total microbial assemblage in a community. Composite wastewater samples (24 h) were collected weekly from a manhole between December 2020 and November 2021 in Maryland, USA. RT-qPCR results showed concentrations of SARS-CoV-2 RNA recovered from wastewater samples reflected incidence of COVID-19 cases. When a drastic increase in COVID-19 was detected in February 2021, samples were selected for microbiome analysis (DNA metagenomics, RNA metatranscriptomics, and targeted SARS-CoV-2 sequencing). Targeted SARS-CoV-2 sequencing allowed for detection of important genetic mutations, such as spike: K417N, D614G, P681H, T716I, S982A, and D1118H, commonly associated with increased cell entry and reinfection. Microbiome analysis (DNA and RNA) provided important insight with respect to human health-related factors, including detection of pathogens and their virulence/antibiotic resistance genes. Specific microbial species comprising the wastewater microbiome correlated with incidence of SARS-CoV-2 RNA, suggesting potential association with SARS-CoV-2 infection. Climatic conditions, namely, temperature, were related to incidence of COVID-19 and detection of SARS-CoV-2 in wastewater, having been monitored as part of an environmental risk score assessment carried out in this study. In summary, the wastewater microbiome provides useful public health information, and hence, a valuable tool to proactively detect and characterize pathogenic agents circulating in a community. In effect, metagenomics of wastewater can serve as an early warning system for communicable diseases, by providing a larger source of information for health departments and public officials. IMPORTANCE Traditionally, testing for COVID-19 is done by detecting SARS-CoV-2 in samples collected from nasal swabs and/or saliva. However, SARS-CoV-2 can also be detected in feces of infected individuals. Therefore, wastewater samples can be used to test all individuals of a community contributing to the sewage collection system, i.e., the infrastructure, such as gravity pipes, manholes, tanks, lift stations, control structures, and force mains, that collects used water from residential and commercial sources and conveys the flow to a wastewater treatment plant. Here, we profile community wastewater collected from a manhole, detect presence of SARS-CoV-2, identify genetic mutations of SARS-CoV-2, and perform COVID-19 risk score assessment of the study area. Using metagenomics analysis, we also detect other microorganisms (bacteria, fungi, protists, and viruses) present in the samples. Results show that by analyzing all microorganisms present in wastewater, pathogens circulating in a community can provide an early warning for contagious diseases.

Passive Samplers, an Important Tool for Continuous Monitoring of the COVID-19 Pandemic.

The global pandemic caused by COVID-19 has resulted in major costs around the world, costs with dimensions in every aspect, from peoples' daily living to the global economy. As the pandemic progresses, the virus evolves, and more vaccines become available, and the 'battle against the virus' continues. As part of the battle, Wastewater-Based Epidemiology (WBE) technologies are being widely deployed in essential roles for SARS-CoV-2 detection and monitoring. While focusing on demonstrating the advantages of passive samplers as a tool in WBE, this review provides a holistic view of the current WBE applications in monitoring SARS-CoV-2 with the integration of the most up-to-date data. A novel scenario example based on a recent Nanjing (China) outbreak in July 2021 is used to illustrate the potential benefits of using passive samplers to monitor COVID-19 and to facilitate effective control of future major outbreaks. The presented contents and how the application of passive samplers indicates that this technology can be beneficial at different levels, varying from building to community to regional. Countries and regions that have the pandemic well under control or have low positive case occurrences have the potential to significantly benefit from deploying passive samplers as a measure to identify and suppress outbreaks.

Synthetic biology enables field-deployable biosensors for water contaminants

Wastewater surveillance is a powerful tool to understand community profiling in terms of health monitoring. Tracking biomarkers such as inorganic and organic pollutants, drugs, and pathogens in wastewater gives a general idea about the lifestyle and health status of a population as well as pollutant exposure caused by various toxic chemicals. Notably, tracing pathogenic clues could help predict and prevent disease outbreaks such as the ongoing COVID-19 pandemic in communities. To this end, developing portable biosensing platforms will facilitate the on-site monitoring of water contamination without requiring complex equipment. New technological developments in synthetic biology have advanced both synthetic gene circuit-based biosensors and new in vitro detection strategies coupled with easy-to-interpret visualization methods. Here, we summarize the latest advances in synthetic biology tools and discuss how they enable the development of rapid, low-cost, ease-to-use and field-deployable biosensors for monitoring a variety of water contaminants and health-related biomarkers in the environment.

Wastewater monitoring, surveillance and epidemiology: a review of terminology for a common understanding.

Response to the COVID-19 (coronavirus disease 2019) pandemic saw an unprecedented uptake in bottom-up efforts to incorporate community wastewater testing to inform public health. While not a new strategy, various specialized scientific advancements were achieved to establish links between wastewater concentrations of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) and public health outcomes. Maximizing public health benefit requires collaboration among a broad range of disciplinary experts, each bringing their own historical context to the central goal of protecting human health. One challenge has been a lack of shared terminology. Standardized terminology would provide common ground for this rapidly growing field. Based on the review herein, we recommend categorical usage of the term 'wastewater-based epidemiology' to describe the science of relating microbes, chemicals or other analytes in wastewater to public health. We further recommend the term 'wastewater surveillance' to describe continuous monitoring of health outcomes (either microbes or chemicals) via wastewater. We suggest that 'wastewater tracking' and 'wastewater tracing' be used in more narrow ways, specifically when trying to find the source of a health risk. Finally, we suggest that the phrase 'wastewater monitoring' be abandoned, except in rare circumstances when ensuring wastewater discharge is safe from a public health perspective.

Making waves: Plausible lead time for wastewater based epidemiology as an early warning system for COVID-19.

Wastewater-based epidemiology (WBE) has emerged as a useful tool in the fight to track and contain COVID-19 spread within communities. One of the motives behind COVID-19 WBE efforts is the potential for 'early warning' of either the onset of disease in a new setting or changes in trends in communities where disease is endemic. Many initial reports of the early warning potential of WBE have relied upon retrospective sample analysis, and delays in WBE analysis and reporting should be considered when evaluating the early warning potential of WBE that enable public health action. Our purpose in this manuscript is to establish a framework to critique the potential of WBE to serve as an early warning system, with special attention to the onset of viral shedding and the differential between results reporting for WBE and clinical testing. While many uncertainties remain regarding both COVID-19 clinical presentation and technical factors influencing WBE results, our analysis suggests at most a modest lead time interval ranging from six days for clinical testing to four days for WBE during community-level wastewater surveillance where clinical testing is accessible on-demand with a rapid time to results. This potential lead time for WBE subsequently increases in settings with limited clinical testing capacity or utilization. Care should be taken when reporting 'early detection' of COVID-19 disease trends via WBE to consider underlying causes (e.g., clinical testing lag or delayed result reporting) to avoid misrepresenting WBE potential.

Wastewater-Based Epidemiology as an Early Warning System for the Spreading of SARS-CoV-2 and Its Mutations in the Population.

New methodologies based on the principle of "sewage epidemiology" have been successfully applied before in the detection of illegal drugs. The study describes the idea of early detection of a virus, e.g., SARS-CoV-2, in wastewater in order to focus on the area of virus occurrence and supplement the results obtained from clinical examination. By monitoring temporal variation in viral loads in wastewater in combination with other analysis, a virus outbreak can be detected and its spread can be suppressed early. The use of biosensors for virus detection also seems to be an interesting application. Biosensors are highly sensitive, selective, and portable and offer a way for fast analysis. This manuscript provides an overview of the current situation in the area of wastewater analysis, including genetic sequencing regarding viral detection and the technological solution of an early warning system for wastewater monitoring based on biosensors.