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.

Interpretation of temporal and spatial trends of SARS-CoV-2 RNA in San Francisco Bay Area wastewater

Wastewater surveillance for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA can be integrated with COVID-19 case data to inform timely pandemic response. However, more research is needed to apply and develop systematic methods to interpret the true SARS-CoV-2 signal from noise introduced in wastewater samples (e.g., from sewer conditions, sampling and extraction methods, etc.). In this study, raw wastewater was collected weekly from five sewersheds and one residential facility, and wastewater SARS-CoV-2 concentrations were compared to geocoded COVID-19 clinical testing data. SARS-CoV-2 was reliably detected (95% positivity) in frozen wastewater samples when reported daily new COVID-19 cases were 2.4 or more per 100,000 people. To adjust for variation in sample fecal content, crAssphage, pepper mild mottle virus, Bacteroides ribosomal RNA (rRNA), and human 18S rRNA were evaluated as normalization biomarkers, and crAssphage displayed the least spatial and temporal variability. Both unnormalized SARS-CoV-2 RNA signal and signal normalized to crAssphage had positive and significant correlation with clinical testing data (Kendalls Tau-b ({tau})=0.43 and 0.38, respectively). Locational dependencies and the date associated with testing data impacted the lead time of wastewater for clinical trends, and no lead time was observed when the sample collection date (versus the result date) was used for both wastewater and clinical testing data. This study supports that trends in wastewater surveillance data reflect trends in COVID-19 disease occurrence and presents approaches that could be applied to make wastewater signal more interpretable and comparable across studies.

Sewage, Salt, Silica and SARS-CoV-2 (4S): An economical kit-free method for direct capture of SARS-CoV-2 RNA from wastewater.

Wastewater-based epidemiology is an emerging tool to monitor COVID-19 infection levels by measuring the concentration of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in wastewater. There remains a need to improve wastewater RNA extraction methods sensitivity, speed, and reduce reliance on often expensive commercial reagents to make wastewater-based epidemiology more accessible. We present a kit-free wastewater RNA extraction method, titled "Sewage, Salt, Silica and SARS-CoV-2" (4S), that employs the abundant and affordable reagents sodium chloride (NaCl), ethanol and silica RNA capture matrices to recover 6-fold more SARS-CoV-2 RNA from wastewater than an existing ultrafiltration-based method. The 4S method concurrently recovered pepper mild mottle virus (PMMoV) and human 18S ribosomal subunit rRNA, both suitable as fecal concentration controls. The SARS-CoV-2 RNA concentrations measured in three sewersheds corresponded to the relative prevalence of COVID-19 infection determined via clinical testing. Lastly, controlled experiments indicate that the 4S method prevented RNA degradation during storage of wastewater samples, was compatible with heat pasteurization, and could be performed in approximately 3 hours. Overall, the 4S method is promising for effective, economical, and accessible wastewater-based epidemiology for SARS-CoV-2, providing another tool to fight the global pandemic. SYNOPSISThe 4S method for measuring SARS-CoV-2 in wastewater is promising for effective, economical, and accessible wastewater-based epidemiology. ABSTRACT ART O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=70 SRC="FIGDIR/small/20242131v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@192784eorg.highwire.dtl.DTLVardef@11879e7org.highwire.dtl.DTLVardef@1eb315borg.highwire.dtl.DTLVardef@1f560cf_HPS_FORMAT_FIGEXP M_FIG C_FIG