Comparative Analysis of RNA-Extraction Approaches and Associated Influences on RT-qPCR of the SARS-CoV-2 RNA in a University Residence Hall and Quarantine Location

Wastewater-based epidemiology (WBE) provides an early warning and trend analysis approach for determining the presence of COVID-19 in a community and complements clinical testing in assessing the population level, even as viral loads fluctuate. Here, we evaluate combinations of two wastewater concentration methods (i.e., ultrafiltration and composite supernatant-solid), four pre-RNA extraction modifications, and three nucleic acid extraction kits using two different wastewater sampling locations. These consisted of a quarantine facility containing clinically confirmed COVID-19-positive inhabitants and a university residence hall. Of the combinations examined, composite supernatant-solid with pre-RNA extraction consisting of water concentration and RNA/DNA shield performed the best in terms of speed and sensitivity. Further, of the three nucleic acid extraction kits examined, the most variability was associated with the Qiagen kit. Focusing on the quarantine facility, viral concentrations measured in wastewater were generally significantly related to positive clinical cases, with the relationship dependent on method, modification, kit, target, and normalization, although results were variable-dependent on individual time points (Kendall's Tau-b (tau) = 0.17 to 0.6) or cumulatively (Kendall's Tau-b (tau) = -0.048 to 1). These observations can support laboratories establishing protocols to perform wastewater surveillance and monitoring efforts for COVID-19.

SARS-CoV-2 RNA and N Antigen Quantification via Wastewater at the Campus Level, Building Cluster Level, and Individual-Building Level

Monitoring wastewater for SARS-CoV-2 from populations smaller than those served by wastewater treatment plants may help identify small spatial areas (subsewersheds) where COVID-19 infections are present. We sampled wastewater from three nested locations with different sized populations within the same sewer network at a university campus and quantified SARS-CoV-2 RNA using reverse transcriptase droplet digital polymerase chain reaction (PCR). SARS-CoV-2 RNA concentrations and/or concentrations normalized by PMMoV were positively associated with laboratory-confirmed COVID-19 cases for both the sewershed level and the subsewershed level. We also used an antigen-based assay to detect the nucleocapsid (N) antigen from SARS-CoV-2 in wastewater samples at the sewershed level. The N antigen was regularly detected at the sewershed level, but the results were not associated with either laboratory-confirmed COVID-19 cases or SARS-CoV-2 RNA concentrations. The results of this study indicate that wastewater monitoring based on quantification of SARS-CoV-2 RNA using PCR-based methods is associated with COVID-19 cases at multiple geographic scales within the subsewershed level and can serve to aid the public health response.

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's 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.

High-Frequency, High-Throughput Quantification of SARS-CoV-2 RNA in Wastewater Settled Solids at Eight Publicly Owned Treatment Works in Northern California Shows Strong Association with COVID-19 Incidence.

A number of recent retrospective studies have demonstrated that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA concentrations in wastewater are associated with coronavirus disease 2019 (COVID-19) cases in the corresponding sewersheds. Implementing high-resolution, prospective efforts across multiple plants depends on sensitive measurements that are representative of COVID-19 cases, scalable for high-throughput analysis, and comparable across laboratories. We conducted a prospective study across eight publicly owned treatment works (POTWs). A focus on SARS-CoV-2 RNA in solids enabled us to scale up our measurements with a commercial lab partner. Samples were collected daily, and results were posted to a website within 24 h. SARS-CoV-2 RNA in daily samples correlated with the incidence of COVID-19 cases in the sewersheds; a 1 log10 increase in SARS-CoV-2 RNA in settled solids corresponds to a 0.58 log10 (4×) increase in sewershed incidence rate. SARS-CoV-2 RNA signals measured with the commercial laboratory partner were comparable across plants and comparable to measurements conducted in a university laboratory when normalized by pepper mild mottle virus (PMMoV) RNA. Results suggest that SARS-CoV-2 RNA should be detectable in settled solids for COVID-19 incidence rates of >1/100,000 (range, 0.8 to 2.3 cases per 100,000). These sensitive, representative, scalable, and comparable methods will be valuable for future efforts to scale up wastewater-based epidemiology. IMPORTANCE Access to reliable, rapid monitoring data is critical to guide response to an infectious disease outbreak. For pathogens that are shed in feces or urine, monitoring wastewater can provide a cost-effective snapshot of transmission in an entire community via a single sample. In order for a method to be useful for ongoing COVID-19 monitoring, it should be sensitive for detection of low concentrations of SARS-CoV-2, representative of incidence rates in the community, scalable to generate data quickly, and comparable across laboratories. This paper presents a method utilizing wastewater solids to meet these goals, producing measurements of SARS-CoV-2 RNA strongly associated with COVID-19 cases in the sewershed of a publicly owned treatment work. Results, provided within 24 h, can be used to detect incidence rates as low as approximately 1/100,000 cases and can be normalized for comparison across locations generating data using different methods.

Effect of storage conditions on SARS-CoV-2 RNA quantification in wastewater solids.

SARS-CoV-2 RNA in wastewater settled solids is associated with COVID-19 incidence in sewersheds and therefore, there is a strong interest in using these measurements to augment traditional disease surveillance methods. A wastewater surveillance program should provide rapid turn around for sample measurements (ideally within 24 hours), but storage of samples is necessary for a variety of reasons including biobanking. Here we investigate how storage of wastewater solids at 4 °C, -20 °C, and -80 °C affects measured concentrations of SARS-CoV-2 RNA. We find that short term (7 or 8 d) storage of raw solids at 4 °C has little effect on measured concentrations of SARS-CoV-2 RNA, whereas longer term storage at 4 °C (35-122 d) or freezing reduces measurements by 60%, on average. We show that normalizing SARS-CoV-2 RNA concentrations by concentrations of pepper mild mottle virus (PMMoV) RNA, an endogenous wastewater virus, can correct for changes during storage as storage can have a similar effect on PMMoV RNA as on SARS-CoV-2 RNA. The reductions in SARS-CoV-2 RNA in solids during freeze thaws is less than those reported for the same target in liquid influent by several authors.