Combining community wastewater genomic surveillance with state clinical surveillance: A framework for SARS-CoV-2 public health practice

Study objective: To garner a framework for combining community wastewater surveillance with state clinical surveillance that influence confirmation of SARS-CoV-2 variants within the community, and recommend how the flow of such research evidence could be expanded and employed for public health response. Design, setting, and participants: This work involved analyzing wastewater samples collected weekly from 17 geographically resolved locations in Louisville/Jefferson County, Kentucky from February 10 to November 29, 2021. Genomic surveillance and RT-qPCR platforms were used as screening to identify SARS-CoV-2 in wastewater, and state clinical surveillance was used for confirmation. Main results: The results demonstrate increased epidemiological value of combining community wastewater genomic surveillance and RT-qPCR with conventional case auditing methods. The spatial scale and temporal frequency of wastewater sampling provides promising sensitivity and specificity to be useful to gain public health screening insights about community emergence, seeding, and spread. Conclusions: Better national surveillance systems are needed for future pathogens and variants, and wastewater-based genomic surveillance represents opportune coupling. This paper presents current evidence that complementary wastewater and clinical testing is enhanced cost-effectively when linked; making a strong case for a joint public health framework. The findings suggest significant potential for rapid progress to be made in extending this work to consider pathogens of interest as a whole within wastewater, which could be examined in either a targeted fashion as we currently do with SARS-CoV-2 or in terms of a global monitoring of all pathogens found, and developing evidence based public health practice to best support community health

Detection of human, porcine and canine picornaviruses in municipal sewage sludge using pan-enterovirus amplicon-based long-read Illumina sequencing

We describe the successful detection of human, porcine and canine picornaviruses (CanPV) in sewage sludge (at each stage of treatment) from Louisville, Kentucky, USA, using Pan-enterovirus amplicon-based long-read Illumina sequencing. Based on publicly available sequence data in GenBank, this is the first detection of CanPV in the USA and the first detection globally using wastewater-based epidemiology. Our findings also suggest there might be clusters of endemic porcine enterovirus (which have been shown capable of causing systemic infection in porcine) circulation in the USA that have not been sampled for around two decades. Our findings highlight the value of WBE coupled with amplicon based long-read Illumina sequencing for virus surveillance and demonstrates this approach can provide an avenue that supports a “One Health” model to virus surveillance. Finally, we describe a new CanPV assay targeting the capsid protein gene region that can be used globally, especially in resource limited settings for its detection and molecular epidemiology

The rapid assessment of aggregated wastewater samples for genomic surveillance of sars-cov-2 on a city-wide scale

Throughout the course of the ongoing SARS-CoV-2 pandemic there has been a need for approaches that enable rapid monitoring of public health using an unbiased and minimally invasive means. A major way this has been accomplished is through the regular assessment of wastewater samples by qRT-PCR to detect the prevalence of viral nucleic acid with respect to time and location. Further expansion of SARS-CoV-2 wastewater monitoring efforts to include the detection of variants of interest/concern through next-generation sequencing has enhanced the understanding of the SARS-CoV-2 outbreak. In this report, we detail the results of a collaborative effort between public health and metropolitan wastewater management authorities and the University of Louisville to monitor the SARS-CoV-2 pandemic through the monitoring of aggregate wastewater samples over a period of 28 weeks. Through the use of next-generation sequencing approaches the polymorphism signatures of Variants of Concern / Interest were evaluated to determine the likelihood of their prevalence within the community on the basis of their relative dominance within sequence datasets. Our data indicate that wastewater monitoring of water quality treatment centers and smaller neighbor-hood-scale catchment areas is a viable means by which the prevalence and genetic variation of SARS-CoV-2 within a metropolitan community of approximately one million individuals may be monitored, as our efforts detected the introduction and emergence of variants of concern in the city of Louisville. Importantly, these efforts confirm that regional emergence and spread of variants of interest/concern may be detected as readily in aggregate wastewater samples as compared to the individual wastewater sheds. Furthermore, the information gained from these efforts enabled targeted public health efforts including increased outreach to at-risk communities and the deployment of mobile or community-focused vaccination campaigns

Public awareness and support for use of wastewater for SARS-CoV-2 monitoring: A community survey in Louisville, Kentucky

The majority of sewer systems in the United States and other countries, are operated by public utilities. In the absence of any regulation, public perception of monitoring wastewater for population health biomarkers is an important consideration for a public utility commission when allocating resources for this purpose. In August 2021, we conducted a survey as part of an ongoing COVID-19 community prevalence study in Louisville/Jefferson County, KY. The survey comprised of seven questions about awareness of and privacy concerns and was sent to 32,000 households randomly distributed within the county. A total of 1,220 sampled adults participated in the probability sample, and 981 were used in analysis. A total of 2,444 adults additionally responded in the convenience sample, and 1,751 were used in analysis. The samples were weighted to produce estimates representative of all adults in the county. Public awareness of tracking COVID-19 virus in the sewers was low. Opinions about how data from this activity are shared strongly supported public disclosure of monitoring results. Responses showed more support for measuring the largest areas (\u3e30,000 to 50,000 households) typically representing population levels found in a community or regional wastewater treatment plant. Those who had a history of COVID-19 infection were more likely to support highly localized monitoring. Understanding wastewater surveillance strategies and thresholds of privacy concerns requires in-depth, comprehensive analysis of public opinion for continued success and efficacy of public health monitoring

SARS-CoV-2 RNA abundance in wastewater as a function of distinct urban sewershed size

During the COVID-19 pandemic, wastewater-based epidemiology has emerged as a promising approach for monitoring SARS-CoV-2 prevalence on a community-level. Despite much being known about the utility of making these measurements in large wastewater treatment plants, little is known about the correlation with finer geographic resolution, such as those obtained through sewershed sub-area catchments. This study aims to identify community wastewater surveillance characteristics between sewershed areas that affect the strength of the association of SARS-CoV-2 RNA detection in a metropolitan area. For this, wastewater from 17 sewershed areas were sampled in Louisville/Jefferson County, Kentucky (USA), from August 2020 to April 2021 (N = 727), which covered approximately 97% of the county\u27s households. Solids were collected from the treatment plants from November 2020 to December 2020 (N = 42). Our results indicate that the sewersheds differ in SARS-CoV-2 trends; however, high pairwise correlation spatial trends were not observed, and the mean SARS-CoV-2 RNA concentrations of smaller upstream community sewershed areas did not differ from their respective treatment centers. Solid samples could only be collected at treatment plants, therefore not allowing us to evaluate SARS-CoV-2 abundance as a function of the sewershed scale. The population size sensitivity of SARS-CoV-2 concentration detection is non-linear: at low population levels the measures are either too sensitive and generate a high level of variability, or at high population levels the estimates are dampened making small changes in community infection levels more difficult to discern. Our results suggest selecting sampling sites that include a wide population range. This study and its findings may inform other system-wide strategies for sampling wastewater for estimating non-SARS-CoV-2 targets

The Detection of Periodic Reemergence Events of SARS-CoV-2 Delta Strain in Communities Dominated by Omicron

Despite entering an endemic phase, SARS-CoV-2 remains a significant burden to public health across the global community. Wastewater sampling has consistently proven utility to understanding SARS-CoV-2 prevalence trends and genetic variation as it represents a less biased assessment of the corresponding communities. Here, we report that ongoing monitoring of SARS-CoV-2 genetic variation in samples obtained from the wastewatersheds of the city of Louisville in Jefferson county Kentucky has revealed the periodic reemergence of the Delta strain in the presence of the presumed dominant Omicron strain. Unlike previous SARS-CoV-2 waves/emergence events, the Delta reemergence events were geographically restricted in the community and failed to spread into other areas as determined by wastewater analyses. Moreover, the reemergence of the Delta strain did not correlate with vaccination rates as communities with lower relative vaccination have been, to date, not affected. Importantly, Delta reemergence events correlate with increased public health burdens, as indicated by increased daily case rates and mortality relative to non-Delta wastewatershed communities. While the underlying reasons for the reemergence of the Delta variant remain unclear, these data reaffirm the ongoing importance of wastewater genomic analyses towards understanding SARS-CoV-2 as it enters the endemic phase