Detection and quantification of sars-cov-2 wastewater treatment plants from different cities in chile, towards to a permanent sentinel surveillance.

Background: The quantification of SARS-CoV-2 in wastewater is a tool that allows determining the trend of viral circulation in a particular geographical area. Aim(s): To quantify the SARS-CoV-2 virus in 15 wastewater treatment plants in different Chilean cities to establish a comparison with the variables of: I) Active cases per 100,000 inhabitants;ii) daily positivity (novel cases);and iii) phases of the lockdown strategy. Method(s): SARS-CoV-2 was concentrated from wastewater samples. To obtain the number of virus genomes per liter, absolute quantification was performed using qRT-PCR. Result(s): Between January and June 2021, 253 samples were processed, all of which were positive for the presence of the virus. Likewise, it will be determined that the rate of active cases per 100,000 inhabitants is the variable that best fits the trends obtained with the quantification of the viral load in wastewater. Conclusion(s): The quantification of SARS- CoV-2 in wastewater as a continuous strategy is an efficient tool to determine the trend of the viral circulation in a delimited geographical area and, combined with genomic surveillance, it can constitute an ideal sentinel surveillance alert on future outbreaks.Copyright © 2022, Sociedad Chilena de Infectologia. All rights reserved.

Monitoring influenza and respiratory syncytial virus in wastewater. Beyond COVID-19.

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.

Is SARS-CoV-2 a concern in the largest wastewater treatment plant in middle east?

The surveillance of wastewater treatment plant (WWTP) as the end point of SARS-CoV-2 shed from infected people arise a speculation on transmission of this virus of concern from WWTP in epidemic period. To this end, the present study was developed to comprehensively investigate the presence of SARS-CoV-2 in raw wastewater, effluent and air inhaled by workers and employee in the largest WWTP in Tehran for one-year study period. The monthly raw wastewater, effluent and air samples of WWTP were taken and the SARS-CoV-2 RNA were detected using QIAamp Viral RNA Mini Kit and real-time RT-PCR assay. According to results, the speculation on the presence of SARS-CoV-2 was proved in WWTP by detection this virus in raw wastewater. However, no SARS-CoV-2 was found in both effluent and air of WWTP; this presents the low or no infection for workers and employee in WWTP. Furthermore, further research are needed for detection the SARS-CoV-2 in solid and biomass produced from WWTP processes due to flaks formation, followed by sedimentation in order to better understand the wastewater-based epidemiology and preventive measurement for other epidemics probably encountered in the future.

Wastewater surveillance could serve as a pandemic early warning system for COVID‐19 and beyond

Wastewater‐based surveillance can be used as an early warning system to identify COVID‐19 outbreaks because the viral load can be observed in sewage before it is clinically verified. Wastewater surveillance of SARS‐CoV‐2 can trace the transmission dynamics of infection in communities when using the scale of a wastewater diversion and treatment system. Using this early detection method can help protect human health and mitigate socio‐economic losses. It can help quantify the epidemiological data of a given population in real‐time and circumvent the need for other epidemiological indicators. There are challenges in using this technique in areas with underdeveloped sewerage infrastructure. It is especially the case in developing nations where uniform protocols for viral detection are lacking, and wastewater is heterogeneous because of environmental and operational conditions. This article explains the need for and importance of wastewater‐based surveillance for SARS‐CoV‐2. It lays out the most recent methodological approaches for detecting SARS‐CoV‐2 in municipal wastewater and outlines the main challenges associated with wastewater‐based epidemiology (WBE). The article includes a case study of surveillance work across India to demonstrate how a developing nation manages research and locational challenges. The socio‐economic, ethical, and policy dimensions of WBE for SARS‐CoV‐2 are also discussed.This article is categorized under: Engineering Water > Water, Health, and Sanitation Engineering Water > Sustainable Engineering of Water Engineering Water > Methods [ FROM AUTHOR] Copyright of WIRES Water is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full . (Copyright applies to all s.)

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 surveillance uncovers regional diversity and dynamics of SARS-CoV-2 variants across nine states in the USA.

Wastewater-based epidemiology (WBE) is a non-invasive and cost-effective approach for monitoring the spread of a pathogen within a community. WBE has been adopted as one of the methods to monitor the spread and population dynamics of the SARS-CoV-2 virus, but significant challenges remain in the bioinformatic analysis of WBE-derived data. Here, we have developed a new distance metric, CoVdist, and an associated analysis tool that facilitates the application of ordination analysis to WBE data and the identification of viral population changes based on nucleotide variants. We applied these new approaches to a large-scale dataset from 18 cities in nine states of the USA using wastewater collected from July 2021 to June 2022. We found that the trends in the shift between the Delta and Omicron SARS-CoV-2 lineages were largely consistent with what was seen in clinical data, but that wastewater analysis offered the added benefit of revealing significant differences in viral population dynamics at the state, city, and even neighborhood scales. We also were able to observe the early spread of variants of concern and the presence of recombinant lineages during the transitions between variants, both of which are challenging to analyze based on clinically-derived viral genomes. The methods outlined here will be beneficial for future applications of WBE to monitor SARS-CoV-2, particularly as clinical monitoring becomes less prevalent. Additionally, these approaches are generalizable, allowing them to be applied for the monitoring and analysis of future viral outbreaks.

Wastewater Levels of Respiratory Syncytial Virus Associated with Influenza-like Illness Rates in Children-A Case Study in Larissa, Greece (October 2022-January 2023).

The emergence of the COVID-19 pandemic has led to significant progress in the field of wastewater-based surveillance (WBS) of respiratory pathogens and highlighted its potential for a wider application in public health surveillance. This study aimed to evaluate whether monitoring of respiratory syncytial virus (RSV) in wastewater can provide a comprehensive picture of disease transmission at the community level. The study was conducted in Larissa (Central Greece) between October 2022 and January 2023. Forty-six wastewater samples were collected from the inlet of the wastewater treatment plant of Larissa and analyzed with a real-time reverse transcription polymerase chain reaction (RT-PCR) based method. RSV and SARS-CoV-2 wastewater viral loads (genome copies/100,000 inhabitants) were analyzed against sentinel surveillance data on influenza-like illness (ILI) to identify potential associations. Univariate linear regression analysis revealed that RSV wastewater viral load (lagged by one week) and ILI notification rates in children up to 14 years old were strongly associated (std. Beta: 0.73 (95% CI: 0.31-1.14), p = 0.002, R2 = 0.308). A weaker association was found between SARS-CoV-2 viral load and ILI rates in the 15+ age group (std. Beta: 0.56 (95% CI: 0.06-1.05), p = 0.032, R2 = 0.527). The results support the incorporation of RSV monitoring into existing wastewater-based surveillance systems.

Impact of COVID- 19 pandemic on antidepressants consumptions by wastewater analysis in Turkey.

The COVID-19 pandemic has been a major challenge worldwide, forcing countries to take restrictive measures beyond conventional methods in their fight against the spread of the disease. Followingly, many studies have been conducted on the effects of these measures on mental health. Wastewater-based epidemiology (WBE) was used in this study to monitor and estimate changes in antidepressant use under normal conditions (2019) and COVID-19 pandemic conditions (2020). Likewise, this study utilized wastewater-based epidemiology (WBE) to monitor and assess changing trends from the pre-pandemic period (2019) to COVID-19 pandemic conditions in antidepressant use (2020). Wastewater samples were collected from 11 cities in Turkey throughout six sampling periods covering the pre-pandemic and during-pandemic periods (June 2019-December 2020). Then, samples were analyzed via LC-MS/MS method. As a result, we observed that venlafaxine was the drug with the highest concentration (mean ± SD: 103.6 ± 112.1 mg/1000p/day). Moreover, city number 6 presented the highest venlafaxine use and the most dramatic increase during the pandemic period. Finally, this study revealed the potential of WBE to estimate the changing trends in mental health during the ongoing pandemic.

Wastewater analysis and drug consumption: useful assessment tool.

Abstract: Drug use in Europe is now more than ever a multifaceted issue involving a broad and ever-changing range of substances. Alarmingly, recent European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) reports show that roughly 83 million adults aged between 15 and 64 years in the European Union have used illicit substances at least once in their lifetime. Polydrug use is rife, and consumption patterns range from experimental/occasional, habitual use all the way to addiction. Traditional epidemiological methods delineating drug abuse specifics (i.e. number of users, age groups, specific characteristics and patterns of use) are questionnaire-based and therefore present the classic biases linked to the selected sample, thus risking an underesti-mation of the phenomenon. The Mario Negri IRCCS Pharmacological Research Institute has developed a new population screening method based on wastewater-based epidemiology (WBE) which makes it possible to detect drug metabolites in waste water and to estimate which and how many substances are consumed by the entire population belonging to the wastewater treatment plant being monitored. Such a technique has gained great interest at the national and international level, and may be especially valuable as a detection/monitoring tool at a time when novel psychoactive substances have come to trigger a major public health crisis, on account of their elusiveness and potential as substitutes/adulterants of traditional substances of abuse.

Wastewater-based epidemiology for the assessment of population exposure to chemicals: The need for integration with human biomonitoring for global One Health actions.

WBE has now become a complimentary tool in SARS-CoV-2 surveillance. This was preceded by the established application of WBE to assess the consumption of illicit drugs in communities. It is now timely to build on this and take the opportunity to expand WBE to enable comprehensive assessment of community exposure to chemical stressors and their mixtures. The goal of WBE is to quantify community exposure, discover exposure-outcome associations, and trigger policy, technological or societal intervention strategies with the overarching aim of exposure prevention and public health promotion. To achieve WBE's full potential, the following key aspects require further action: (1) Integration of WBE-HBM (human biomonitoring) initiatives that provide comprehensive community-individual multichemical exposure assessment. (2) Global WBE monitoring campaigns to provide much needed data on exposure in low- and middle-income countries (LMICs) and fill in the gaps in knowledge especially in the underrepresented highly urbanised as well as rural settings in LMICs. (3) Combining WBE with One Health actions to enable effective interventions. (4) Advancements in new analytical tools and methodologies for WBE progression to enable biomarker selection for exposure studies, and to provide sensitive and selective multiresidue analysis for trace multi-biomarker quantification in a complex wastewater matrix. Most of all, further developments of WBE needs to be undertaken by co-design with key stakeholder groups: government organisations, health authorities and private sector.

The dynamic relationship between COVID-19 cases and SARS-CoV-2 wastewater concentrations across time and space: Considerations for model training data sets.

During the COVID-19 pandemic, wastewater-based surveillance has been used alongside diagnostic testing to monitor infection rates. With the decline in cases reported to public health departments due to at-home testing, wastewater data may serve as the primary input for epidemiological models, but training these models is not straightforward. We explored factors affecting noise and bias in the ratio between wastewater and case data collected in 26 sewersheds in California from October 2020 to March 2022. The strength of the relationship between wastewater and case data appeared dependent on sampling frequency and population size, but was not increased by wastewater normalization to flow rate or case count normalization to testing rates. Additionally, the lead and lag times between wastewater and case data varied over time and space, and the ratio of log-transformed individual cases to wastewater concentrations changed over time. This ratio decreased between the Epsilon/Alpha and Delta variant surges of COVID-19 and increased during the Omicron BA.1 variant surge, and was also related to the diagnostic testing rate. Based on this analysis, we present a framework of scenarios describing the dynamics of the case to wastewater ratio to aid in data handling decisions for ongoing modeling efforts.

Wastewater-based epidemiology approach: The learning lessons from COVID-19 pandemic and the development of novel guidelines for future pandemics.

Wastewater-based epidemiology (WBE) provides a comprehensive real-time framework of population attitude and health status. This approach is attracting the interest of medical community and health authorities to monitor the prevalence of a virus (such as the severe acute respiratory syndrome coronavirus 2, SARS-CoV-2) among a community. Indeed, WBE is currently fine-tuning as environmental surveillance tool for coronavirus disease 2019 (COVID-19) pandemic. After a bibliometric analysis conducted to discover the research trends in WBE field, this work aimed to side-by-side compare the conventional method based on clinical testing with WBE approach. Furthermore, novel guidelines were developed to apply the WBE approach to a pandemic. The growing interest on WBE approach for COVID-19 pandemic is demonstrated by looking at the sharp increase in scientific papers published in the last years and at the ongoing studies on viral quantification methods and analytical procedures. The side-by-side comparison highlighted the ability of WBE to identify the hot-spot areas faster than the conventional approach, reducing the costs (e.g., rational use of available resources) and the gatherings at medical centers. Contrary to clinical testing, WBE has the surveillance capacity for preventing the virus resurgence, including asymptomatic contribution, and ensuring the preservation of medical staff health by avoiding the exposure to the virus infection during clinical testing. As extensively reported, the time in collecting epidemiological data is crucial for establishing the prevention and mitigation measures that are essential for curbing a pandemic. The developed guidelines can help to build a WBE system useful to control any future pandemic.

Implementing wastewater surveillance for SARS-CoV-2 on a university campus: Lessons learned.

Wastewater surveillance, also known as wastewater-based epidemiology (WBE), has been successfully used to detect SARS-CoV-2 and other viruses in sewage in many locations in the United States and globally. This includes implementation of the surveillance on college and university campuses. A two-phase study was conducted during the 2020-2021 academic year to test the feasibility of a WBE system on campus and to supplement the clinical COVID-19 testing performed for the student, staff, and faculty body. The primary objective during the Fall 2020 semester was to monitor a large portion of the on-campus population and to obtain an understanding of the spreading of the SARS-CoV-2 virus. The Spring 2021 objective was focused on selected residence halls and groups of residents on campus, as this was more efficient and relevant for an effective follow-up response. Logistical problems and planning oversights initially occurred but were corrected with improved communication and experience. Many lessons were learned, including effective mapping, site planning, communication, personnel organization, and equipment management, and obtained along the way, thereby paving an opportune guide for future planning efforts. PRACTITIONER POINTS: WBE was successful in the detection of many SARS-CoV-2 variants incl. Alpha, Beta, Gamma, Delta, Lambda, Mu, and Omicron. Careful planning and contingencies were essential for a successful implementation of a SARS-CoV-2 monitoring program. A surveillance program may be important for detection and monitoring of other public health relevant targets in wastewater incl. bacteria, viruses, fungi and viruses. Diverse lessons were learned incl. effective mapping, site planning, communication, personnel organization, and equipment management, thereby providing a guide for future planning efforts.

Long-term SARS-CoV-2 surveillance in the wastewater of Stockholm: What lessons can be learned from the Swedish perspective?

Wastewater-based epidemiology (WBE) can be used to track the spread of SARS-CoV-2 in a population. This study presents the learning outcomes from over two-year long monitoring of SARS-CoV-2 in Stockholm, Sweden. The three main wastewater treatment plants in Stockholm, with a total of six inlets, were monitored from April 2020 until June 2022 (in total 600 samples). This spans five major SARS-CoV-2 waves, where WBE data provided early warning signals for each wave. Further, the measured SARS-CoV-2 content in the wastewater correlated significantly with the level of positive COVID-19 tests (r = 0.86; p << 0.0001) measured by widespread testing of the population. Moreover, as a proof-of-concept, six SARS-CoV-2 variants of concern were monitored using hpPCR assay, demonstrating that variants can be traced through wastewater monitoring. During this long-term surveillance, two sampling protocols, two RNA concentration/extraction methods, two calculation approaches, and normalization to the RNA virus Pepper mild mottle virus (PMMoV) were evaluated. In addition, a study of storage conditions was performed, demonstrating that the decay of viral RNA was significantly reduced upon the addition of glycerol to the wastewater before storage at -80 °C. Our results provide valuable information that can facilitate the incorporation of WBE as a prediction tool for possible future outbreaks of SARS-CoV-2 and preparations for future pandemics.

Model training periods impact estimation of COVID-19 incidence from wastewater viral loads.

Wastewater-based epidemiology (WBE) has been deployed broadly as an early warning tool for emerging COVID-19 outbreaks. WBE can inform targeted interventions and identify communities with high transmission, enabling quick and effective responses. As the wastewater (WW) becomes an increasingly important indicator for COVID-19 transmission, more robust methods and metrics are needed to guide public health decision-making. This research aimed to develop and implement a mathematical framework to infer incident cases of COVID-19 from SARS-CoV-2 levels measured in WW. We propose a classification scheme to assess the adequacy of model training periods based on clinical testing rates and assess the sensitivity of model predictions to training periods. A testing period is classified as adequate when the rate of change in testing is greater than the rate of change in cases. We present a Bayesian deconvolution and linear regression model to estimate COVID-19 cases from WW data. The effective reproductive number is estimated from reconstructed cases using WW. The proposed modeling framework was applied to three Northern California communities served by distinct WW treatment plants. The results showed that training periods with adequate testing are essential to provide accurate projections of COVID-19 incidence.

A 30-day follow-up study on the prevalence of SARS-COV-2 genetic markers in wastewater from the residence of COVID-19 patient and comparison with clinical positivity.

Wastewater based epidemiology (WBE) is an important tool to fight against COVID-19 as it provides insights into the health status of the targeted population from a small single house to a large municipality in a cost-effective, rapid, and non-invasive way. The implementation of wastewater based surveillance (WBS) could reduce the burden on the public health system, management of pandemics, help to make informed decisions, and protect public health. In this study, a house with COVID-19 patients was targeted for monitoring the prevalence of SARS-CoV-2 genetic markers in wastewater samples (WS) with clinical specimens (CS) for a period of 30 days. RT-qPCR technique was employed to target nonstructural (ORF1ab) and structural-nucleocapsid (N) protein genes of SARS-CoV-2, according to a validated experimental protocol. Physiological, environmental, and biological parameters were also measured following the American Public Health Association (APHA) standard protocols. SARS-CoV-2 viral shedding in wastewater peaked when the highest number of COVID-19 cases were clinically diagnosed. Throughout the study period, 7450 to 23,000 gene copies/1000 mL were detected, where we identified 47 % (57/120) positive samples from WS and 35 % (128/360) from CS. When the COVID-19 patient number was the lowest (2), the highest CT value (39.4; i.e., lowest copy number) was identified from WS. On the other hand, when the COVID-19 patients were the highest (6), the lowest CT value (25.2 i.e., highest copy numbers) was obtained from WS. An advance signal of increased SARS-CoV-2 viral load from the COVID-19 patient was found in WS earlier than in the CS. Using customized primer sets in a traditional PCR approach, we confirmed that all SARS-CoV-2 variants identified in both CS and WS were Delta variants (B.1.617.2). To our knowledge, this is the first follow-up study to determine a temporal relationship between COVID-19 patients and their discharge of SARS-CoV-2 RNA genetic markers in wastewater from a single house including all family members for clinical sampling from a developing country (Bangladesh), where a proper sewage system is lacking. The salient findings of the study indicate that monitoring the genetic markers of the SARS-CoV-2 virus in wastewater could identify COVID-19 cases, which reduces the burden on the public health system during COVID-19 pandemics.

Does normalization of SARS-CoV-2 concentrations by Pepper Mild Mottle Virus improve correlations and lead time between wastewater surveillance and clinical data in Alberta (Canada): comparing twelve SARS-CoV-2 normalization approaches.

Wastewater-based surveillance (WBS) data normalization is an analyte measurement correction that addresses variations resulting from dilution of fecal discharge by non-sanitary sewage, stormwater or groundwater infiltration. No consensus exists on what WBS normalization parameters result in the strongest correlations and lead time between SARS-CoV-2 WBS data and COVID-19 cases. This study compared flow, population size and biomarker normalization impacts on the correlations and lead times for ten communities in twelve sewersheds in Alberta (Canada) between September 2020 and October 2021 (n = 1024) to determine if normalization by Pepper Mild Mottle Virus (PMMoV) provides any advantages compared to other normalization parameters (e.g., flow, reported and dynamic population sizes, BOD, TSS, NH3, TP). PMMoV concentrations (GC/mL) corresponded with plant influent flows and were highest in the urban centres. SARS-CoV-2 target genes E, N1 and N2 were all negatively associated with wastewater influent pH, while PMMoV was positively associated with temperature. Pooled data analysis showed that normalization increased ρ-values by almost 0.1 and was highest for ammonia, TKN and TP followed by PMMoV. Normalization by other parameters weakened associations. None of the differences were statistically significant. Site-specific correlations showed that normalization of SARS-CoV-2 data by PMMoV only improved correlations significantly in two of the twelve systems; neither were large sewersheds or combined sewer systems. In five systems, normalization by traditional wastewater strength parameters and dynamic population estimates improved correlations. Lead time ranged between 1 and 4 days in both pooled and site-specific comparisons. We recommend that WBS researchers and health departments: a) Investigate WWTP influent properties (e.g., pH) in the WBS planning phase and use at least two parallel approaches for normalization only if shown to provide value; b) Explore normalization by wastewater strength parameters and dynamic population size estimates further; and c) Evaluate purchasing an influent flow meter in small communities to support long-term WBS efforts and WWTP management.

Can wastewater surveillance assist China to cost-effectively prevent the nationwide outbreak of COVID-19?

China has controlled the nationwide spread of COVID-19 since April 2020, but it is still facing an enormous threat of disease resurgence originating from infected international travelers. Taking the rapid transmission and the mutation of SARS-CoV-2 into consideration, the current status would be easily jeopardized if sporadic locally-transmitted individuals are not identified at an early stage. Clinical diagnosis is the gold standard for COVID-19 surveillance, but it is hard to screen presymptomatic or asymptomatic cases in those who have not exhibited symptoms. Since presymptomatic or asymptomatic individuals are infectious, it is urgent to establish a surveillance system based on other tools that can profile the entire population. Infected people including those who are symptomatic, presymptomatic, and asymptomatic shed SARS-CoV-2 RNA in feces and thereby endow wastewater-based epidemiology (WBE) with an early-warning ability for mass COVID-19 surveillance. In the context of China's "COVID-zero" strategy, this work intends to discuss the practical feasibility of WBE applications as an early warning and disease surveillance system in hopes that WBE together with clinical testing would cost-effectively restrain sporadic COVID-19 outbreaks in China.

Relationship between SARS-CoV-2 in wastewater and clinical data from five wastewater sheds.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has resulted in a global pandemic starting in 2019 with nearly 500 million confirmed cases as of April 2022. Infection with SARS-CoV-2 is accompanied by shedding of virus in stool, and its presence in wastewater samples has been documented globally. Therefore, monitoring of SARS-CoV-2 in wastewater offers a promising approach to assess the pandemic situation covering pre-symptomatic and asymptomatic cases in areas with limited clinical testing. In this study, the presence of SARS-CoV-2 RNA in wastewater from five wastewater resource recovery facilities (WRRFs), located in two adjacent counties, was investigated and compared with the number of clinical COVID-19 cases during a 2020-2021 outbreak in United States. Statistical correlation analyses of SARS-CoV-2 viral abundance in wastewater and COVID-19 daily vs weekly clinical cases was performed. While a weak correlation on a daily basis was observed, this correlation improved when weekly clinical case data were applied. The viral fecal indicator Pepper Mild Mottle Virus (PMMoV) was furthermore used to assess the effects of normalization and the impact of dilution due to infiltration in the wastewater sheds. Normalization did not improve the correlations with clinical data. However, PMMoV provided important information about infiltration and presence of industrial wastewater discharge in the wastewater sheds. This study showed the utility of WBE to assist in public health responses to COVID-19, emphasizing that routine monitoring of large WRRFs could provide sufficient information for large-scale dynamics.

Early Detection of SARS-CoV-2 Omicron BA.4 and BA.5 in German Wastewater.

Wastewater-based SARS-CoV-2 epidemiology (WBE) has been established as an important tool to support individual testing strategies. The Omicron sub-variants BA.4/BA.5 have spread globally, displacing the preceding variants. Due to the severe transmissibility and immune escape potential of BA.4/BA.5, early monitoring was required to assess and implement countermeasures in time. In this study, we monitored the prevalence of SARS-CoV-2 BA.4/BA.5 at six municipal wastewater treatment plants (WWTPs) in the Federal State of North Rhine-Westphalia (NRW, Germany) in May and June 2022. Initially, L452R-specific primers/probes originally designed for SARS-CoV-2 Delta detection were validated using inactivated authentic viruses and evaluated for their suitability for detecting BA.4/BA.5. Subsequently, the assay was used for RT-qPCR analysis of RNA purified from wastewater obtained twice a week at six WWTPs. The occurrence of L452R carrying RNA was detected in early May 2022, and the presence of BA.4/BA.5 was confirmed by variant-specific single nucleotide polymorphism PCR (SNP-PCR) targeting E484A/F486V and NGS sequencing. Finally, the mutant fractions were quantitatively monitored by digital PCR, confirming BA.4/BA.5 as the majority variant by 5 June 2022. In conclusion, the successive workflow using RT-qPCR, variant-specific SNP-PCR, and RT-dPCR demonstrates the strength of WBE as a versatile tool to rapidly monitor variants spreading independently of individual test capacities.