Since the discovery that people infected with COVID-19 often shed the virus in their feces, scientists around the world have scrambled to spot signs of the virus in the stuff that we flush.
However, detecting tiny virus particles amid the wastewater that flows through our sewage pipes — which includes not only toilet water, but sink water, shower water and everything else that goes down a drain — is no easy feat.
A team of scientists at the University of California, Berkeley, has spent months refining and optimizing a rapid and low-cost new technique to test wastewater for SARS-CoV-2, the virus that causes COVID-19. This month, the team has launched a new high-throughput pop-up lab — a temporary 1200-square-foot lab, rapidly set up in an empty research space in Hildebrand Hall — that, in conjunction with UC Berkeley and wastewater utilities and public health agencies from throughout the Bay Area, will monitor the region’s wastewater for the virus.
“From the very beginning of the pandemic, it was clear that there were major limitations to the ability to test every individual in a population frequently enough to find out whether they were infected or not,” said Kara Nelson, professor of civil and environmental engineering at UC Berkeley, who leads the team. “Wastewater naturally pools the waste from hundreds to even millions of people in a single sample, so if you can collect a representative sample of wastewater and analyze it, you can gain a tremendous amount of information that you likely couldn’t gain through testing people individually.”
Monitoring wastewater from a city, neighborhood or building is an efficient way to keep track of whether COVID-19 is spreading there. It also has the potential to provide an early warning for a potential outbreak, as evidence suggests that the virus can start to shed in an infected individual’s feces even before the person starts to experience symptoms.
Working with municipalities around the Bay Area, the team has identified sewersheds to monitor, each an area of land where all the sewers flow to a single pipe or station where samples can be collected. The sewersheds they are currently monitoring represent the waste of a few thousand to several hundred thousand people, Nelson said.
Through its COVID-WEB (COVID wastewater epidemiology for the Bay Area) project, the lab can currently process approximately 30 samples a week for 11 agencies, and the team plans to scale up to as many as 200 samples a week by the end of the year to meet the growing demand from regional public health agencies.
“One of the huge bottlenecks in wastewater testing has just been testing capacity,” Nelson said. “This pop-up lab is the first high-throughput lab in the Bay Area that has the capacity to bring in a large number of samples and provide results quickly to public health officials.”
Like finding a virus in a haystack
In theory, the SARS-CoV-2 virus can be spotted in wastewater using the same types of PCR-based assays that doctors currently use to diagnose COVID-19 in humans. These tests work by first transcribing viral RNA into DNA and then using the polymerase chain reaction, or PCR, technique to make billions of copies of the viral DNA so that they can be quantified using fluorescent molecular markers.
However, detecting the virus in wastewater samples is decidedly more troublesome than finding in it a nasal swab, Nelson points out.
For one, wastewater contains chemicals, such as bleach, that can break down the SARS-CoV-2 virus as it travels through the sewer system. Wastewater also contains a plethora of other viruses that each have their own RNA molecules, making it harder to isolate the SARS-CoV-2 RNA in a sample. Finally, each individual infected person can excrete different amounts of SARS-CoV-2 virus particles in their feces, and the wastewater itself is also variable in composition.
Luckily, Nelson and her team have ample experience analyzing and understanding the contents of wastewater. Before the COVID-19 pandemic, the team was primarily concerned with detecting and removing pathogens from water sources, including wastewater, in order to make it safe to drink. Many of the techniques and processes that the group uses in that work also apply to COVID-19 wastewater testing.
“Nelson’s lab is the primary research group on campus that studies treatment processes for pathogens in wastewater and drinking water, and so we already had a lot of preexisting relationships with the wastewater utilities,” said Rose Kantor, a postdoctoral scholar in Nelson’s lab and a member of the COVID-WEB team. “So, when the pandemic hit, we were able to pretty quickly start getting samples from them and testing out different methods for detecting SARS-CoV-2.”
Most methods start by concentrating the particles of the virus, then extracting the RNA for detection. Working with Oscar Whitney, a graduate student in Robert Tjian’s lab in UC Berkeley’s Department of Molecular and Cell Biology, the team created a new technique that takes the opposite approach: Their technique first uses table salt to lyse, or slice open, the outer envelope of the virus, causing it to spill all of its RNA into the sample. The salt stabilizes the RNA, which is then concentrated in the lab.
By taking this approach, the technique is also able to catch bits of RNA from virus particles that may have already partially disintegrated in the wastewater, making it more sensitive to detecting the original number of virus particles that entered the sewer system. It also uses very simple materials, like table salt and ethanol, which helps keep the costs low and helps bypass the supply chain issues that plagued many labs during the first few months of the pandemic.
“Back in early May, when people were just starting to explore wastewater surveillance, some of the materials were hard to obtain,” said Adrian Hinkle, a graduate student in Nelson’s lab and a member of the COVID-WEB team. “So, we tried to develop a method that required as few materials as possible and was fast, because we wanted to get the results as fast as possible, to inform public health authorities.”
The technique has an exceptionally high sensitivity, compared to other techniques that have been developed. Hinkle estimates that it is sensitive enough to detect the virus from just a handful of infected individuals in wastewater produced by a few thousand people.
It is also fast. Hinkle says the whole testing technique now takes about eight hours to complete, and the pop-up lab turns around the results for any given sample to public health officials within three days or less. They have taken extra steps to further streamline the process, like including salt in the sampling kits that they send to utilities, so that the lysis step can start before a wastewater sample even reaches the lab.
“Our technique has a high sensitivity, I think, in part, because of the salt lysis early on and because of the simplicity of the process and how we have fine-tuned the filtration and rinsing steps to maximize the percent recovery [of the virus],” Hinkle said.
‘A bit of a crapshoot’
On a smoky morning in early October, Tim Pine and Al Sanchez haul a large, grey, cylindrical plastic barrel out of a sewer hole located in UC Berkeley’s University Village.
The cylinder, which the researchers call an autosampler, had been hanging in the sewer hole for the past 24 hours. Several times an hour, a hose attached to the sampler draws up a small sample of wastewater from the sewer pipe below, depositing it into a two-liter bucket tucked safely inside the barrel.
Pine and Sanchez unhook the lid of the sampler and remove it, revealing the storage bucket and its contents. The wastewater inside looks like slightly dirty water — not quite the sludge you might imagine when you think of the word sewage.
“Every time we do this, it’s a little bit of a crapshoot, pun intended,” Pine says, pointing at a piece of toilet paper, stuck on a ladder rail in the drain — a common hazard that could easily have plugged the sampling hose and ruined that day’s collection. “Today, it looks kind of clean. We don’t usually see it this clean.”
Pine, an environmental protection specialist, and Sanchez, a senior hazardous materials technician, both work in UC Berkeley’s Office of Environment, Health and Safety. Pine has been collecting weekly samples of wastewater from University Village since mid-July and also samples from sewers that drain from UC Berkeley’s undergraduate dormitories and surrounding neighborhoods.
While Sanchez loads the day’s sample into a truck for transport back to the pop-up lab on campus, Pine describes the autosampler in more detail. Before COVID-19, he says, these devices were often used for environmental regulation. For instance, an autosampler, like this one, could be used to sample the water downstream of a factory suspected of illegally dumping pollutants into the sewer system. But the COVID-19 pandemic has given autosamplers a new use.
The COVID-WEB team has partnered with a number of Bay Area utilities, including the San Francisco Public Utilities Commission (SFPUC), to form a regional working group that helps coordinate when, where and how to gather wastewater samples for COVID-19 testing.
“We have a great field monitoring team who usually sample wastewater to ensure permit compliance for industrial users, and they’ve pivoted quite deftly to using their equipment to pull composite samples from different locations in the city for COVID-19 surveillance,” said SFPUC General Manager Harlan L. Kelly Jr. “Depending on where they sample, they can get a comprehensive picture of the virus on a large scale, or we can use their skills to zoom in down to the building level.”
The regional working group also includes local public health agencies, which advise the team on how wastewater testing can best help the overall effort to slow the spread of COVID-19.
“One goal of this regional working group is to hear from public health officials how they think this wastewater data might help inform decision-making,” said Sasha Harris-Lovett, a postdoctoral fellow at the Berkeley Water Center and a member of the COVID-WEB team. “What are the gaps that this data could fill, and how could the data allow them to make more informed decisions?”
Harris-Lovett has learned that many in public health are interested in using wastewater testing to keep tabs on the SARS-CoV-2 virus in residential facilities and other dense living arrangements, where a few cases could quickly escalate to a major outbreak. This includes not just dorms and student apartments like University Village, but also places like nursing homes or prisons.
“Public health officials have also told us that there are some neighborhoods that haven’t registered very many cases, not necessarily because people aren’t sick, but because people aren’t getting tested,” Harris-Lovett said. “So, there is an interest in using wastewater monitoring to keep track of trends in neighborhoods where perhaps people aren’t able to access health care.”
Guy Nicolette, executive director and assistant vice chancellor of UC Berkeley’s University Health Services, said the wastewater testing being done near campus and at University Village will augment the school’s COVID-19 testing strategy.
“While we need to understand more, wastewater testing has great potential to be an early warning system, especially for sites where people aren’t being tested frequently, for whatever the reason,” Nicolette said. “I can also imagine that when we see significantly reduced general transmission, (wastewater testing) could serve as large pooled testing for populations, so we can direct testing capacity to highest exposure risks and be even more adaptive and responsive, rather than try to directly test every single person.”
With COVID-19 case counts in California finally reaching a plateau, wastewater testing in the Bay Area could now play a role in helping public health officials keep an eye out for a possible resurgence of the virus, said Maya Petersen, chair of the Division of Biostatistics at UC Berkeley’s School of Public Health.
“I think there’s really exciting potential for wastewater testing for the purpose of maintaining surveillance activity in a very efficient way that allows us to keep an eye on the bigger picture, and where an early surge might be happening, while at the same time really focusing our resources on persons and communities that are still bearing the brunt of the epidemic,” Petersen said.
A model for other wastewater surveillance projects
The potential of wastewater testing for COVID-19 goes beyond early detection and pooled surveillance of COVID-19. Working with Alexander Crits-Christoph, a graduate student in professor Jill Banfield’s lab in UC Berkeley’s Innovative Genomics Institute, the team has also developed a way to sequence the RNA of the individual strains of the SARS-CoV-2 virus.
Sequencing the RNA of SARS-CoV-2 helps scientists and epidemiologists monitor the virus as it changes over time and helps them track the different strains of the virus as they travel around the world. For example, by studying the genomes of SARS-CoV-2 virus samples collected on nasal swabs, epidemiologists have been able to deduce that the virus was brought to California via multiple introduction events.
“It’s harder to sequence viral genomes in wastewater because, with a nasal swab from a patient, you expect them to just have one strain of the virus. But in wastewater, a lot of different infected people are excreting virus into that single sample,” Kantor said. “To be able to look at the individual single nucleotide variants that are present in wastewater, you need to use more sophisticated bioinformatics tools that have been developed in Jill Banfield’s lab.”
In a preprint recently posted to MedRxiv, the team compares the strains of the virus found in the Bay Area’s wastewater with those found through nasal swabs of patients. In the wastewater, they found the same strains that had been identified through nasal testing, but also found additional strains that had not yet been observed in California.
Regularly sequencing the virus is also important, Kantor pointed out, because the virus is always mutating, but COVID-19 tests only work when they are programmed to detect the correct RNA sequences in the virus.
The researchers stressed that the rapid success of the new project has hinged on the close collaboration among the team members, both those from Berkeley and those who have joined the regional wastewater monitoring group. The team also shares information, via a massive Slack channel, with hundreds of researchers around the world who are also developing wastewater testing techniques, and such efficiency helps everyone progress faster.
Now that the high throughput pop-up lab is up and running, Nelson says that she and the team are eager to keep learning and sharing how to make this tool as useful as possible, by working collaboratively with their regional partners to put it into practice.
“One of our project goals is to help other regions replicate what we’re doing,” Nelson said. “We want to share information as soon as we possibly can, so that we can speed up the process for other regions that are trying to create something similar.”