COVID-19 Wastewater Testing: Overview and Product Roundup

Wastewater testing is one the many tools public health officials have used to track the spread of COVID-19, and has the advantages of covering specific geographic areas and providing data for communities where clinical testing is unavailable or underutilized. Widespread wastewater surveillance has been used as a strategy to better get ahead of outbreaks and epidemics since the 1990s, and has previously been used to monitor poliovirus infections and opioid use.¹ In response to the COVID-19 pandemic, federal public health agencies including the Centers for Disease Control and Prevention (CDC), Department of Health and Human Services (HHS) and National Institutes of Health (NIH) have helped organize wastewater testing programs across the country, forming the National Sewage Surveillance Interagency Leadership Committee to offer resources and funding for implementation and research. 

Local communities, ranging from large cities to small towns to college campuses, have used wastewater surveillance to gauge the impact of the virus on their population, and data reported to the National Wastewater Surveillance System can be analyzed by CDC officials to further guide the response of public health agencies. Researchers have worked to develop more efficient methods for testing wastewater and implementing surveillance programs,² and have used wastewater detection to analyze and map virus trends³ and track the appearance of virus variants.⁴ Millions of dollars in government grants have been dedicated to research focused on COVID-19 wastewater epidemiology. 

Wastewater surveillance programs begin with developing a sampling strategy that fits the public health needs of a community. This includes determining the best locations to collect from, how often to sample, which sample type to collect and which method of sampling will be used. Sampling may take place at wastewater treatment plants covering multiple populations, or sewersheds, served by different collection systems in one area, such as a large city. An effective wastewater surveillance program requires at least three samplings to detect trends over time, with the time between consecutive samples determining the minimum length of time over which a trend may be detected, according to the CDC. Either untreated wastewater or primary sludge may be used for testing, with the latter potentially offering high virus concentrations but being not as well researched as testing of fully untreated wastewater. The two main collection methods are grab and composite sample collection, with the latter allowing for automated collection of multiple samples over a 24 hour time period for a more representative pool. 

After collection, wastewater samples are homogenized and large solids may be removed through filtration or centrifugation. Samples are then concentrated through ultrafiltration, ultracentrifugation or similar approaches, and RNA is extracted and purified. Direct extraction methods without pre-concentration of the sample are also available. RNA measurement is performed either through RT-qPCR or RT-ddPCR. The N1, N2 and E genes of SARS-CoV-2 are reported to be sensitive and specific targets for quantifying viral RNA in wastewater, according to the CDC. Matrix recovery controls are used to assess the amount of virus lost during sample processing, and human fecal normalization controls account for differences in human waste input and wastewater dilution over time. Data can then be analyzed to calculate durational and directional trends in SARS-CoV-2 concentrations. Although wastewater testing can not provide an estimated number of COVID-19 cases among a population, the data it provides can gauge the virus’ prevalence over time and can be a leading indicator for forecasting infection dynamics, making it a valuable tool for early detection and mitigation of outbreaks. 

Entities engaged in wastewater surveillance include not only government agencies but also academic institutions and private laboratories. The University of Arizona was one of the first universities to use wastewater testing at its dormitories to catch virus cases early and prevent a larger outbreak through additional clinical antigen and PCR tests.⁵ Many other universities are using this strategy to help protect students and faculty as classes get back in session, and both academic and private laboratories have been contracted by government agencies at local and federal levels to aid in surveillance efforts across the country. Additionally, many manufacturers of laboratory products and equipment have introduced new solutions and published resources specifically for COVID-19 wastewater testing efforts. Here is a roundup of some of the COVID-19 products and resources available to aid laboratories in their wastewater monitoring efforts. 

Products for COVID-19 Wastewater Testing

• Promega’s SARS-CoV-2 RT-qPCR Kit for Wastewater contains a combination of primer/probe sets as well as internal process and amplification controls recommended by the CDC for detection and quantification of SARS-CoV-2 viral RNA in wastewater. Promega scientists also published a direct capture workflow for wastewater surveillance in Science of the Total Environment, which forms the basis of the company’s new Maxwell RSC Enviro Total Nucleic Acid Kit for automated methods and Wizard Enviro Total Nucleic Acid Kit for manual methods. 

• GT Molecular and QIAGEN collaborated to develop the GT-Digital SARS-CoV-2 Wastewater Surveillance Assay For QIAcuity, a molecular reagent kit containing CDC-compliant primers, probes and controls for SARS-CoV-2 wastewater surveillance testing. The assay is designed for use with QIAGEN’s QIAcuity Digital PCR System and each kit can be used for 200 tests. QIAGEN’s recommended workflow for COVID-19 wastewater testing can be found on the company’s website.

• LuminUltra developed the GeneCount SARS-CoV-2 Wastewater Test Kit for direct extraction of SARS-CoV-2 RNA using magnetic binding bead technology. The kit is designed for use with the portable GeneCount qPCR device and was developed in collaboration with Hach to include Hach’s AS950 Portable Sampler for a complete on-site testing system. 

• Bio-Rad’s PREvalence ddPCR SARS-CoV-2 Wastewater Quantification Kit is based on CDC and WHO primers for detection of the N2 and E genes of SARS-CoV-2 through droplet digital PCR. The kit includes a matrix recovery control and can also be used in conjunction with a PREvalence ddPCR crAssphageFecal Indicator Assay or PREvalence ddPCR PMMoV Fecal Indicator Assay for COVID-19 wastewater testing. Each Wastewater Quantification Kit can be used for 200 reactions and each Fecal Indicator Assay can be used for 100 reactions.

• Combinati offers a SARS-CoV-2 Wastewater Surveillance 4-plex Kit designed for use on its Absolute Q Digital PCR platform. The kit includes a process control and human fecal control and quantifies the N1 and N2 gene of SARS-CoV-2. 

• IDEXX’s Water SARS-CoV-2 RT-PCR Test targets the N1 and N2 regions of SARS-CoV-2 and is validated for use on common RT-qPCR instruments. The test kit includes an RNA master mix, a positive control and PCR grade water, and one kit can be used for 100 tests. Workflow recommendations and compatible controls and instruments can be found on the IDEXX website.

References

1. Larsen, D.A., Wigginton, K.R. Tracking COVID-19 with wastewater. Nat Biotechnol 38, 1151–1153 (2020). https://doi.org/10.1038/s41587-020-0690-1

2. Keshaviah A, Hu XC, Henry M. 2021. Developing a flexible national wastewater surveillance system for COVID-19 and beyond. Environ Health Perspect129(4):45002, PMID: 33877858, 10.1289/EHP8572.

3. Raul Gonzalez, Kyle Curtis, Aaron Bivins, Kyle Bibby, Mark H. Weir, Kathleen Yetka, Hannah Thompson, David Keeling, Jamie Mitchell, Dana Gonzalez, COVID-19 surveillance in Southeastern Virginia using wastewater-based epidemiology, Water Research, Volume 186, 2020, 116296, ISSN 0043-1354, https://doi.org/10.1016/j.watres.2020.116296.

4. "A new way to detect the SARS-CoV-2 Alpha variant in wastewater," Singapore-MIT Alliance for Research and Technology, MIT News. https://news.mit.edu/2021/new-way-to-detect-sars-cov-2-alpha-variant-in-wastewater-0728

5. "Wastewater testing at UArizona stops coronavirus spread; garners national attention," Water & Energy Sustainable Technology Center, University of Arizona. https://west.arizona.edu/news/2020/08/wastewater-testing-uarizona-stops-coronavirus-spread-garners-national-attention