Identifying trends in SARS-CoV-2 RNA in wastewater to infer changing COVID-19 incidence: Effect of sampling frequency

SARS-CoV-2 RNA concentrations in wastewater solids and liquids are correlated with reported incident COVID-19 cases. Reporting of incident COVID-19 cases has changed dramatically with the availability of at-home antigen tests. Wastewater monitoring therefore represents an objective tool for continued monitoring of COVID-19 occurrence. One important use case for wastewater data is identifying when there are sustained changes or trends in SARS-CoV-2 RNA concentrations. Such information can be used to inform public health messaging, testing, and vaccine resources. However, there is limited research on best approaches for identifying trends in wastewater monitoring data. To fill this knowledge gap, we applied three trend analysis methods (relative strength index (RSI), percent change (PC), Mann-Kendall (MK) trend test) to daily measurements of SARS-CoV-2 RNA in wastewater solids from a wastewater treatment plant to characterize trends. Because daily measurements are not common for wastewater monitoring programs, we also conducted a downsampling analysis to determine the minimum sampling frequency necessary to capture the trends identified using the "gold standard" daily data. The PC and MK trend test appear to perform similarly and better than the RSI in terms of early warning signaling for increasing and decreasing trends, so we only considered the PC and MK trend test methods in the downsampling analysis. Using an acceptable sensitivity and specificity cutoff of 0.5, we found that a minimum of 4 samples/week and 5 samples/week is necessary to detect trends identified by daily sampling using the PC and MK trend test method, respectively. If a higher sensitivity and specificity is needed, then more samples per week would be needed. Public health officials can adopt these trend analysis approaches and sampling frequency recommendations to wastewater monitoring programs aimed at providing information on how incident COVID-19 cases are changing in the contributing communities.

Wastewater surveillance of human influenza, metapneumovirus, parainfluenza, respiratory syncytial virus (RSV), rhinovirus, and seasonal coronaviruses during the COVID-19 pandemic

BackgroundRespiratory disease is a major cause of morbidity and mortality; however, current surveillance for circulating respiratory viruses is passive and biased. Seasonal circulation of respiratory viruses changed dramatically during the COVID-19 pandemic. More active methods for understanding respiratory disease dynamics are needed to better inform public health response and to guide clinical decision making. Wastewater-based epidemiology has been used to understand COVID-19, influenza A, and RSV infection rates at a community level, but has not been used to investigate other respiratory viruses. MethodsWe measured concentrations of influenza A and B, RSV A and B, human parainfluenza (1-4), rhinovirus, seasonal human coronaviruses, and human metapneumovirus RNA in wastewater solids three times per week for 17 months spanning the COVID-19 pandemic at a wastewater treatment plant in California, USA. Novel probe-based assays were developed and validated for non-influenza viral targets. We compared viral concentrations to positivity rates for viral infections from clinical specimens submitted to sentinel laboratories. FindingsWe detected RNA from all target viruses in wastewater solids. Human rhinovirus and seasonal coronaviruses were found at highest concentrations. Concentrations of viruses correlated significantly and positively with positivity rates of associated viral diseases from sentinel laboratories. Measurements from wastewater indicated limited circulation of RSV A and influenza B, and human coronavirus OC43 dominated the seasonal human coronavirus infections while human parainfluenza 1 and 4A dominated among parainfluenza infections. InterpretationWastewater-based epidemiology can be used to obtain information on circulation of respiratory viruses at a community level without the need to test many individuals because a single sample of wastewater represents the entire contributing community. Results from wastewater can be available within 24 hours of sample collection, allowing real time information to inform public health response, clinical decision making, and individual behavior modifications.

Regional replacement of SARS-CoV-2 variant BA.1 with BA.2 as observed through wastewater surveillance

An understanding of circulating SARS-CoV-2 variants can inform pandemic response, vaccine development, disease epidemiology, and use of monoclonal antibody treatments. We developed custom assays targeting characteristic mutations in SARS-CoV-2 variants Omicron BA.1 and BA.2 and confirmed their sensitivity and specificity in silico and in vitro. We then applied these assays to daily wastewater solids samples from eight publicly owned treatment works in the greater Bay Area of California, USA, over four months to obtain a spatially and temporally intensive data set. We documented regional replacement of BA.1 with BA.2 in agreement with, and ahead of, clinical sequencing data. This study highlights the utility of wastewater surveillance for real time tracking of SARS-CoV-2 variant circulation. SynopsisWastewater surveillance was used to document regional emergence of SARS-CoV-2 variant Omicron BA.2 ahead of clinical surveillance. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/22274160v2_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@10e4406org.highwire.dtl.DTLVardef@1941dd9org.highwire.dtl.DTLVardef@133b438org.highwire.dtl.DTLVardef@17ce1c4_HPS_FORMAT_FIGEXP M_FIG C_FIG

Effect of SARS-CoV-2 digital droplet RT-PCR assay sensitivity on COVID-19 wastewater based epidemiology

We developed and implemented a framework for examining how molecular assay sensitivity for a viral RNA genome target affects its utility for wastewater-based epidemiology. We applied this framework to digital droplet RT-PCR measurements of SARS-CoV-2 and Pepper Mild Mottle Virus genes made using 10 replicate wells, and determined how using fewer wells affected assay sensitivity and its performance for wastewater-based epidemiology applications. We used a computational, downsampling approach. When percent of positive droplets was between 0.024% and 0.5% (as was the case for SARS-CoV-2 genes during the Delta surge), measurements obtained with 3 or more wells were similar to those obtained using 10. When percent of positive droplets was less than 0.024%, then 6 or more wells were needed to obtain similar results as those obtained using 10 wells. When COVID-19 incidence is low, as it was before the Delta surge and SARS-CoV-2 gene concentrations are <104 cp/g, using 6 wells will yield a detectable concentration 90% of the time. Overall, results support an adaptive approach where assay sensitivity is increased by running 6 or more wells during periods of low SARS-CoV-2 gene concentrations, and 3 or more wells during periods of high SARS-CoV-2 gene concentrations. SynopsisAdaptive approaches developed with assay sensitivity in consideration may reduce cost and increase sensitivity for wastewater-based epidemiology. Abstract Art O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=62 SRC="FIGDIR/small/22273949v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@19cebdaorg.highwire.dtl.DTLVardef@14054d0org.highwire.dtl.DTLVardef@80286eorg.highwire.dtl.DTLVardef@1de7e3a_HPS_FORMAT_FIGEXP M_FIG C_FIG

SARS-CoV-2 RNA wastewater settled solids surveillance frequency and impact on predicted COVID-19 incidence using a distributed lag model

SARS-CoV-2 RNA concentrations in wastewater settled solids correlate well with COVID-19 incidence rates (IRs). Here, we develop distributed lag models (DLMs) to estimate IRs using concentrations of SARS-CoV-2 RNA from wastewater solids and investigate the impact of sampling frequency on model performance. SARS-CoV-2 N gene and PMMoV RNA concentrations were measured daily at four wastewater treatment plants in California. Artificially reduced datasets were produced for each plant with sampling frequencies of once every 2, 3, 4, and 7 days. Sewershed-specific models that related daily N/PMMoV to IR were fit for each sampling frequency with data from mid-Nov 2020 through mid-July 2021, which included the period of time during which Delta emerged. Models were used to predict IRs during a subsequent out-of-sample time period. When sampling occurred at least once every 4 days, the in- and out-of-sample root mean square error (RMSE) changed less than 7 cases/100,000 compared to daily sampling across sewersheds. This work illustrates that real-time, daily predictions of IR are possible with small error, despite changes in circulating variants, when sampling frequency is once every 4 days or more. However, reduced sampling frequency may not serve other important wastewater surveillance use cases.

Persistence of endogenous SARS-CoV-2 and pepper mild mottle virus RNA in wastewater settled solids

Limited information is available on the decay rate of endogenous SARS-CoV-2 and pepper mild mottle virus (PMMoV) RNA in wastewater and primary settled solids, potentially limiting an understanding of how transit or holding times within wastewater infrastructure might impact RNA measurements and their relationship to community COVID-19 infections. In this study, primary settled solids samples were collected from two wastewater treatment plants in the San Francisco Bay Area. Samples were thoroughly mixed, aliquoted into subsamples, and stored at 4{degrees}C, 22{degrees}C, and 37 {degrees}C for 10 days. The concentration of SARS-CoV-2 (N1 and N2 targets) and PMMoV RNA was measured using an RT-ddPCR. Limited decay (< 1 log10 reduction) was observed in the detection of viral RNA targets at all temperature conditions, suggesting that SARS-CoV-2 and PMMoV RNA can be highly persistent in solids. First-order decay rate constants ranged from 0.011 - 0.098 day-1 for SARS-CoV-2 RNA and 0.010 - 0.091 day-1 for PMMoV RNA, depending on temperature conditions. Slower decay was observed for SARS-CoV-2 RNA in primary settled solids compared to previously reported decay in wastewater influent. Further research is needed to understand if solid content and wastewater characteristics might influence the persistence of viral RNA targets. SynopsisSARS-CoV-2 and PMMoV genomic RNA is highly stable in wastewater settled solids over 10 days at several environmentally relevant temperatures. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=72 SRC="FIGDIR/small/22268855v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@1a508forg.highwire.dtl.DTLVardef@19efc23org.highwire.dtl.DTLVardef@bb7748org.highwire.dtl.DTLVardef@194371e_HPS_FORMAT_FIGEXP M_FIG C_FIG

SARS-CoV-2 RNA is enriched by orders of magnitude in solid relative to liquid wastewater at publicly owned treatment works

Wastewater-based epidemiology has gained attention throughout the world for detection of SARS-CoV-2 RNA in wastewater to supplement clinical testing. Methods have been developed using both the liquid and the solid fraction of wastewater, with some studies reporting higher concentrations in solids. To investigate this relationship further, we collaborated with six other laboratories to conduct a study across five publicly owned treatment works (POTWs) where both primary solids and raw wastewater influent samples were collected and quantified for SARS-CoV-2 RNA. Solids and influent samples were processed by participating laboratories using their respective methods and retrospectively paired based on date of collection. SARS-CoV-2 RNA concentrations by mass (gene copies per gram) were higher in solids than in influent by approximately three orders of magnitude. Concentrations in matched solids and influent were positively and significantly correlated at all five POTWs. RNA concentrations in both solids and influent were correlated to COVID-19 incidence rates in the sewershed and thus representative of disease burden; the solids methods appeared to produce a comparable relationship between SARS-CoV-2 RNA concentration measurements and incidence rates across all POTWs. Solids and influent methods showed comparable sensitivity, N gene detection frequency, and calculated empirical incidence rate lower limits. Analysis of solids has the advantage of using less sample volume to achieve similar sensitivity to influent methods.

Standardized and optimized preservation, extraction and quantification techniques for detection of fecal SARS-CoV-2 RNA

COVID-19 patients shed SARS-CoV-2 viral RNA in their stool, sometimes well after they have cleared their respiratory infection. This feature of the disease may be significant for patient health, epidemiology, and diagnosis. However, to date, methods to preserve stool samples from COVID patients, and to extract and quantify viral RNA concentration have yet to be optimized. We sought to meet this urgent need by developing and benchmarking a standardized protocol for the fecal detection of SARS-CoV-2 RNA. We test three preservative conditions for their ability to yield detectable SARS-CoV-2 RNA: OMNIgene-GUT, Zymo DNA/RNA shield kit, and the most common condition, storage without any preservative. We test these in combination with three extraction kits: the QIAamp Viral RNA Mini Kit, Zymo Quick-RNA Viral Kit, and MagMAX Viral/Pathogen Kit. Finally, we also test the utility of two detection methods, ddPCR and RT-qPCR, for the robust quantification of SARS-CoV-2 viral RNA from stool. We identify that the Zymo DNA/RNA shield collection kit and the QiaAMP viral RNA mini kit yield more detectable RNA than the others, using both ddPCR and RT-qPCR assays. We also demonstrate key features of experimental design including the incorporation of appropriate controls and data analysis, and apply these techniques to effectively extract viral RNA from fecal samples acquired from COVID-19 outpatients enrolled in a clinical trial. Finally, we recommend a comprehensive methodology for future preservation, extraction and detection of RNA from SARS-CoV-2 and other coronaviruses in stool.

SARS-CoV-2 in wastewater settled solids is associated with COVID-19 cases in a large urban sewershed

Wastewater-based epidemiology (WBE) may be useful for informing public health response to viral diseases like COVID-19 caused by SARS-CoV-2. We quantified SARS-CoV-2 RNA in wastewater influent and primary settled solids in two wastewater treatment plants to inform the pre-analytical and analytical approaches, and to assess whether influent or solids harbored more viral targets. The primary settled solids samples resulted in higher SARS-CoV-2 detection frequencies than the corresponding influent samples. Likewise, SARS-CoV-2 RNA was more readily detected in solids using one-step digital droplet (dd)RT-PCR than with two-step RT-QPCR and two-step ddRT-PCR, likely owing to reduced inhibition with the one-step ddRT-PCR assay. We subsequently analyzed a longitudinal time series of 89 settled solids samples from a single plant for SARS-CoV-2 RNA as well as coronavirus recovery (bovine coronavirus) and fecal strength (pepper mild mottle virus, PMMoV) controls. SARS-CoV-2 RNA targets N1 and N2 concentrations correlate positively and significantly with COVID-19 clinical confirmed case counts in the sewershed. Together, the results demonstrate that measuring SARS-CoV-2 RNA concentrations in settled solids may be a more sensitive approach than measuring SARs-CoV-2 in influent.