Like performing one giant pooling test, assessing sewage for SARS-CoV-2 has the potential to localize hotspots of the infection. Municipalities around the world are scaling up wastewater-based epidemiology programs using standard commercially available kits and start-ups have begun offering testing and epidemiology consulting services.
Monitoring for a wisp of RNA virus in liter-sized volumes of raw sewage is no small feat, and might just take the cake in terms of PCR inhibitors and nucleic acid degradation. The field of molecular wastewater-based epidemiology (WBE) is also relatively new, so in parallel to the push to rapidly deploy SARS-CoV-2 sewage surveillance, researchers around the world are hammering out the basic protocols to best sample wastewater, extract and detect viral nucleic acids, and correlate viral loads in wastewater with human infection trends.
There is increasing consensus around the idea that wastewater monitoring is a powerful tool to help direct the resources of cash-strapped public health departments. However, in the U.S. the scale up of sewage monitoring seems to be currently left to local governments. The National Science Foundation and others have recently flushed the field with funding for basic research, but some experts suggest a coordinated federal effort is also needed to more effectively bring WBE to bear on the COVID-19 pandemic.
Although environmental surveillance of wastewater has been deployed to pinpoint and monitor local polio and hepatitis outbreaks, in general the field of WBE is thought to have kicked off with a 2001 study hypothesizing the method could be used to monitor illicit drug use in a population, according to Adam Gushgari, a civil engineer and co-founder of AquaVitas, a startup in the WBE space. The technique has been adapted in the U.S. for opioid surveillance in cities and college campuses.
Although narcotics had been the main focus of the WBE, interest in molecular pathogen detection has been growing, particularly over the past three years, Gushgari said. And now, "The field has seen a definite increase in interest because of the pandemic," he said.
But, wastewater remains "an extremely complex — if not the most complex — environmental matrix that we can use as a diagnostic tool," he said, so it takes a lot of trial and error to balance the extreme extraction required with preservation of target integrity.
In the developing research space and applied market of WBE, there are many interconnected networks of players.
AquaVitas spun out of the Biodesign Center for Environmental Health Engineering at Arizona State University and was co-founded by WBE expert and center Director Rolf Halden.
Halden and his team at ASU also support one of the most mature wastewater-related public health efforts in the U.S. in the city of Tempe, Arizona. There, they have run testing and coordinated a publicly-facing online opiate monitoring dashboard since 2018.
Now, the ASU team has scaled this project to create a SARS-CoV-2 dashboard to surveil and report on seven areas of Tempe, monitoring overall infection trends among approximately 185,000 local residents.
Both the opiate and COVID dashboards in Tempe are integrated into public health management, Halden said in an email, and his team meets monthly with the mayor's office to help identify areas of concern and direct limited resources where they are needed most.
In a recent study, Halden and his collaborator Olga Hart illuminated both the challenges and the potential economic opportunities of WBE through modeling of eight global SARS-CoV-2 hotspots. The study estimated that incorporating national sewage surveillance campaigns could potentially save billions of dollars compared to clinical diagnostic testing alone. Palm Coast, Florida city leaders considering 13 weeks of WBE monitoring at six sites estimated it would cost $47,000 and be covered by CARES Act funding.
Likewise, the author of the seminal 2001 WBE study, Christian Daughton, who recently retired from the Environmental Protection Agency, penned a review in May arguing national approaches to SARS-CoV-2 sewage surveillance could be beneficial in part because large-scale diagnostic testing of individuals faces "overwhelming challenges in providing fast surveys of large populations." In contrast, WBE could help not only in mitigation and containment of the pandemic, but also could — hopefully — minimize "domino effects, such as unnecessarily long stay-at-home policies that stress humans and economies alike," Daughton wrote.
A handful of US municipalities seem to have been persuaded already. Besides Tempe, pilot programs in Utah and Montana are currently generating coronavirus wastewater monitoring data and are sharing it with the public. And, a WBE project supported by the EPA is slated to be rolled out across the state of Ohio in the near future.
Perhaps emblematic of the parallel tracks that seem to be part of this blossoming space, EPA's researchers are continuing efforts to develop analytical methods, even as the program is being rolled out.
Melissa Sullivan in EPA's office of public affairs commented in an email that the agency's researchers are currently working on methods to measure SARS-CoV-2 virus in wastewater, as well as establishing techniques for "interpreting the data generated by these evolving analytical methods and developing models to predict community COVID infection rates."
In a related effort, Sullivan said EPA is contributing data and working to finalize a plan for a statewide wastewater monitoring program in Ohio. That program includes collaborators from the Metropolitan Sewer District of Greater Cincinnati, the Ohio Department of Health, the Ohio EPA, and the Ohio Water Resources Center at the Ohio State University.
Internationally, pre-existing wastewater surveillance programs may be more deeply rooted than in the US, so SARS-CoV-2 WBE seems to be taking off at somewhat of a faster pace, albeit with identical technical hurdles.
At the National Academies' Water Science and Technology Board virtual annual meeting in May, Gertjan Medema, a researcher at Delft University of Technology, pointed out that the Netherlands, Finland, and Germany are examples of countries that have already begun the groundwork to implement national SARS-CoV-2 surveillance programs, with dozens of sentinel cities currently being surveilled.
Meanwhile, a longstanding European wastewater monitoring program called the Sewage Analysis Core group Europe, or SCORE, is adapting from a focus on drugs-of-abuse surveillance to SARS-CoV-2 WBE.
Launched in 2011, SCORE currently tracks sewage from 86 cities in 29 countries. Lian Lundy, an environmental scientist at Middlesex University London, is helping to lead two related SCORE initiatives in the EU for SARS-CoV-2. One is an overall feasibility assessment in which all samples will be analyzed in a single lab, Lundy said in an email, while the second supports the development of an international open-access database.
"So much is happening so fast," Lundy said, and the key challenge currently is that there is "no standard analytical method" for SARS-CoV-2 detection in wastewater. She noted that on average it takes approximately three years for standardization organizations within the water sciences field to approve a method. Lundy suggested, however, that it might be possible to address this challenge through use of a robust QA/QC framework.
Methods and vendors
Research teams around the world are indeed sharing their WBE protocols and sewage solutions, and some are collaborating extensively to essentially crowdsource elaborate protocol troubleshooting.
Overall, in the burgeoning body of literature on SARS-CoV-2 WBE, the methods used for PCR-based testing are essentially the standard methods used by clinical and research labs, with some of the front-end sample prep perhaps needing extra finesse.
One particular method for SARS-CoV-2 molecular detection in wastewater that seems to be a starting place for many local teams stems from pioneering work out of Australia by Warish Ahmed and his colleagues at the Commonwealth Scientific and Industrial Research Organisation.
Ahmed uses electronegative membranes to capture viruses and then extracts RNA directly from the membranes using Qiagen PowerWater or PowerMicrobiome extraction kits, he said in an email. He then uses either a Bio-Rad CFX-96 thermal cycler or digital PCR to quantify SARS-CoV-2 RNA in wastewater. For the latter, he noted that he recently ordered a Qiagen dPCR platform because he found it to be especially fast.
Ahmed also recently co-authored a paper in Environmental Science and Technology laying the groundwork for a global collaborative effort with SCORE and the Global Water Pathogen Project that hopes to empower worldwide wastewater-based epidemiology of SARS-CoV-2.
In Australia, Ahmed said he and his team are in touch with public health officials and inform them of the WBE results to help them make decisions.
In Tempe, meanwhile, Halden said his team at ASU uses Ahmed's basic method for the detection of SARS-CoV-2 in municipal wastewater, specifically using standard qPCR probes that have been published and validated by Centers for Disease Control and the World Health Organization. The ASU team has also demonstrated temperature sensitivity and in-sewer decay of SARS-CoV-2 targets, Halden said, and so they are using "proprietary methods and devices" to prevent and compensate for these challenges. Halden and his team have opted not to use digital PCR at this time, based on prior experiences with the technology, he said.
In terms of technical challenges, Ahmed noted that if the concentration of virus in wastewater is high, detection is pretty straightforward. But, "when the concentration of virus is low, it can be challenging to detect." To combat this issue, labs can use a better recovery method or perform more replicates in the PCR reactions, he said. Ahmed's team is now working on several potential refinements, such as comparing different platforms, and single time-point analysis — also called "grab sampling" — to analyze samples of wastewater pooled over extended periods of time.
According to Halden, other technical aspects will require still more protocol refinements.
"Wastewater monitoring sounds simple, but in practice there are many challenges that require careful planning," he said, including sampling, shipping, analysis, and sample archiving. The ASU team has developed techniques and technologies to overcome signal decay, for example, and has quantified the ways temperature effects virual decay and the impact of the effective size of the monitoring network. Another issue is that automated sewage samplers that are supposed to deliver 24-hour composite samples in actuality may sample only a few minutes of each hour, Halden said, resulting in signal passing through the monitoring location uncaptured and unaccounted for. "We have developed customized equipment that improves signal capture and sample representativeness by over an order of magnitude," Halden said.
Once the best possible sample is captured, most protocols for the PCR lab work appear to use methods to capture and concentrate the nucleic acids in raw wastewater, often followed by off-the-shelf extraction and reagent kits — sometimes specifically designed for wastewater samples — and standard PCR instruments.
For example, some researchers are using the aforementioned Qiagen extraction kits designed for environmental testing of soil or water, although the firm also markets the AllPrep PowerViral DNA/RNA Kit for samples high in PCR inhibitors like wastewater. And, in a preliminary study, Medema and colleagues at Delft University in the Netherlands centrifuged and filtered sewage then used magnetic extraction.
Meanwhile, other reagent manufacturers are now jumping in with products purposely designed for SARS-CoV-2 detection from wastewater.
Madison, Wisconsin-based Promega has also now developed protocols specifically for detecting SARS-CoV-2 RNA in wastewater samples, namely for viral purification using either a type of precipitation or centrifugal filtration.
Brigitta Saul, Promega's global commercialization marketing manager said there's a viral RNA purification chemistry under development, too. The kit is for wastewater samples and can be performed for single samples using manual extraction, but is also adaptable to higher-throughput automated RT-qPCR detection.
Thermo Fisher Scientific is also extrapolating an existing footprint in WBE to more purpose-designed products. Specifically, a representative said the firm's commercially available next-generation sequencing product is being used for epidemiological studies of SARS-CoV-2, and some of Thermo Fisher's customers are using their products to analyze the virus in waste water.
