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

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.

Legionella pneumophila occurrence in reduced-occupancy buildings in 11 cities during the COVID-19 pandemic (preprint)

In spring 2020, reduced water demand was an unintended consequence of COVID-19 pandemic-related building closures. Concerns arose that contaminants associated with water stagnation, such as Legionella pneumophila , could become prevalent. To investigate this potential public health risk, samples from 26 reduced-occupancy buildings across 11 cities in the United States, Canada, and Switzerland were analyzed for L. pneumophila using liquid culture (Legiolert, n=258) and DNA-based methods (qPCR/ddPCR, n=138). L. pneumophila culture-positivity was largely associated with just five buildings, each of which had specific design or operational deficiencies commonly associated with L. pneumophila occurrence. Samples from free chlorine buildings had higher culture-positivity (37%) than chloramine buildings (1%), and 78% of culture-positive samples occurred when the residual was ≤0.1 mg/L Cl 2 . Although overall sample positivities using culture- and DNA-based methods were equivalent (34% vs. 35%), there was disagreement between the methods in 13% of paired samples. Few buildings reported any water management activities, and L. pneumophila concentrations in flushed samples were occasionally greater than in first-draw samples. This study provides insight into how building plumbing characteristics and management practices contribute to L. pneumophila occurrence during low water use periods and can inform targeted prevention and mitigation efforts. Synopsis Statement Legionella pneumophila occurrence was evaluated in reduced-occupancy buildings during the COVID-19 pandemic across multiple cities. Graphic for Table of Contents (TOC)

Estimating relative abundance of two SARS-CoV-2 variants through wastewater surveillance at two large metropolitan sites (preprint)

Monitoring SARS-CoV-2 variants of concern (VOCs) is critical for public health management of COVID-19. Case isolate sequencing is resource-intensive and not all isolates can be sequenced, and thus are not representative. However, since wastewater SARS-CoV-2 RNA concentrations correlate with COVID-19 case incidence in sewersheds, tracking VOCs through wastewater is appealing. We developed targeted digital RT-PCR assays to monitor abundance of select mutations in Alpha and Delta VOCs in wastewater settled solids, applied these to July 2020-August 2021 samples from two large metropolitan sewersheds, and compared results to estimates of variant abundance from case isolate sequencing. Wastewater measurements tracked closely with case isolate estimates at each site (rp= 0.82, 0.88 for Alpha and rp= 0.97 for Delta). Mutations were detected in wastewater measurements even at levels <5% of circulating virus and in samples available weeks before case isolate results. Therefore, wastewater variant monitoring should be strategically deployed to complement case isolate sequencing.

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

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.