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Testing sewage (wastewater-based surveillance, or WBS) for pathogens is an increasingly important tool for monitoring the health of populations. During the COVID-19 pandemic, some residential institutions including colleges, prisons, and skilled nursing facilities used facility-level wastewater data to inform their pandemic responses. To understand how these early adopters used WBS data in decision making, we conducted in-depth, semistructured interviews with multiple decision makers at 6 residential institutions in the United States (universities, prisons, and nursing homes) encompassing a total of more than 70 000 residents and staff about interpretation, uses, and limitations of these data. We found that WBS data were used in extremely diverse ways. WBS combined with clinical surveillance informed a wide range of public health actions at residential institutions, including transmission reduction measures, public health communications, and allocation of resources. WBS also served other institutional purposes, such as maintaining relationships with external stakeholders and helping alleviate decision makers' pervasive stress. Recognizing these diverse ways of using WBS data can inform expansion of this practice among institutions as well as development of community-scale systems.

Drinking water stagnation can lead to degradation of chlorine residual, bacterial growth (including of opportunistic pathogens and nitrifiers), and metals release from plumbing materials; however, few studies have characterized building water quality and bacterial communities during the extended stagnation periods that occurred during COVID-19 pandemic-related building closures. Additionally, despite a lack of evidence-based guidance, flushing fixtures has been recommended to restore building water quality. We aimed to evaluate the impacts of reduced building occupancy (>2 months) and weekly restorative flushing on drinking water quality, bacterial communities, and the occurrence of undesirable microorganisms in three university buildings. Reduced occupancy led to diminished chloramine and elevated intact cell counts, but values remained stable after additional weeks of limited water use. Flushing temporarily improved water quality, with chlorine and cell counts remaining stable for at least 1 day but returning to levels measured prior to flushing within 1 week. Alpha diversity was lower under more stagnant conditions, and fixture identity, not flushing, was the most influential factor on bacterial community composition, suggesting a strong influence from local biofilm. Although Mycobacterium, Legionella, Pseudomonas, Nitrosomonas, and Nitrospira were detected in samples via amplicon sequencing, concentrations measured via qPCR of M. avium complex, L. pneumophila, P. aeruginosa, and ammonia-oxidizing bacteria were very low or were undetected, supporting that stagnation alone did not lead to high occurrence of undesirable microorganisms. Findings from this study contribute to our understanding of the effects of stagnation on building water microbiomes and the efficacy of flushing to improve water quality. Under the conditions of this case study, repeated flushing on a weekly timescale during low occupancy periods was not sufficient to maintain chlorine residual and prevent bacterial growth in fixtures. Building managers need to weigh the temporary water quality benefits of flushing against the labor and water resources required considering local context.

The following protocol describes the reverse transcription quantitative polymerase chain reaction (RT-qPCR) methods that we have used at the University of California, Berkeley in the Nelson Lab and COVID-WEB (covid-web.org) for downstream analysis of extracted wastewater samples for the purpose of SARS-CoV-2 surveillance. We have been successfully using this protocol paired with the Sewage, Salt, Silica, and SARS-CoV-2 (4S) method. We are including multiple assays that we have used throughout this project, although not all of them may be in active use. This method may need to be adapted based on your lab's application, available equipment, and safety requirements. It is intended for use in a BSL2+ lab with extra precautions for processing environmental specimens for SARS-CoV-2 as described in the CDC Interim Laboratory Biosafety Guidelines. Version 1 included the following assays: N1, PMMoV, BCoV, and SOC. Version 2 was amended to include the following assays: crAssphage, Bacteroides HF183 rRNA, and human 18S rRNA.

Clinical and surveillance testing for the SARS-CoV-2 virus relies overwhelmingly on RT-qPCR-based diagnostics, yet several popular assays require 2–3 separate reactions or rely on detection of a single viral target, which adds significant time, cost, and risk of false-negative results. Furthermore, multiplexed RT-qPCR tests that detect at least two SARS-CoV-2 genes in a single reaction are typically not affordable for large scale clinical surveillance or adaptable to multiple PCR machines and plate layouts. We developed a RT-qPCR assay using the Luna Probe Universal One-Step RT-qPCR master mix with publicly available primers and probes to detect SARS-CoV-2 N gene, E gene, and human RNase P (LuNER) to address these shortcomings and meet the testing demands of a university campus and the local community. This cost-effective test is compatible with BioRad or Applied Biosystems qPCR machines, in 96 and 384-well formats, with or without sample pooling, and has a detection sensitivity suitable for both clinical reporting and wastewater surveillance efforts.

The following protocol describes the reverse transcription quantitative polymerase chain reaction (RT-qPCR) methods that we have used at the University of California, Berkeley in the Nelson Lab and COVID-WEB (covid-web.org) for downstream analysis of extracted wastewater samples for the purpose of SARS-CoV-2 surveillance. We have been successfully using this protocol paired with the Sewage, Salt, Silica, and SARS-CoV-2 (4S) method. We are including multiple assays that we have used throughout this project, although not all of them may be in active use. This method may need to be adapted based on your lab's application, available equipment, and safety requirements. It is intended for use in a BSL2+ lab with extra precautions for processing environmental specimens for SARS-CoV-2 as described in the CDC Interim Laboratory Biosafety Guidelines.

This protocol describes the procedure of the "4S" (Sewage, Salt, Silica and SARS-CoV-2) method for SARS-CoV-2 RNA extraction from wastewater. Offering a highly efficient, modular and economical alternative to existing wastewater RNA purification methods, this procedure lowers the barrier to entry for SARS-CoV-2 wastewater-based epidemiology. This procedure is intended to be carried out in a BSL2+ laboratory space, with precautions when handling raw wastewater samples.

This protocol describes the procedure of the "4S" (Sewage, Salt, Silica and SARS-CoV-2) method for SARS-CoV-2 RNA extraction from wastewater. Offering a highly efficient, modular and economical alternative to existing wastewater RNA purification methods, this procedure lowers the barrier to entry for SARS-CoV-2 wastewater-based epidemiology. This procedure is intended to be carried out in a BSL2+ laboratory space, with precautions when handling raw wastewater samples.

This protocol describes the procedure of the "4S" (Sewage, Salt, Silica and SARS-CoV-2) method for SARS-CoV-2 RNA extraction from wastewater. Offering a highly efficient, modular and economical alternative to existing wastewater RNA purification methods, this procedure lowers the barrier to entry for SARS-CoV-2 wastewater-based epidemiology. This procedure is intended to be carried out in a BSL2+ laboratory space, with precautions when handling raw wastewater samples.

The following protocol describes the "4S" (Sewage, Salt, Silica and SARS-CoV-2) workflow applied to using dry silica powder as an RNA-binding matrix instead of silica spin columns. This offers an even more economical alternative, requiring only centrifugation to extract RNA from wastewater. This procedure is intended to be carried out in a BSL2+ laboratory space, with precautions when handling raw wastewater samples.

This protocol describes the procedure of the "4S" (Sewage, Salt, Silica and SARS-CoV-2) method for SARS-CoV-2 RNA extraction from wastewater. Offering a highly efficient, modular and economical alternative to existing wastewater RNA purification methods, this procedure lowers the barrier to entry for SARS-CoV-2 wastewater-based epidemiology. This procedure is intended to be carried out in a BSL2+ laboratory space, with precautions when handling raw wastewater samples.