Tracking coronavirus in wastewater

An aerial view of a waste water sewage treatment plant.
A wastewater treatment plant. iStock

An emerging tool in the global pandemic centers upon surveilling a community’s wastewater for traces of the coronavirus or its viral variants. One person’s trash—or flushed goods, in this case—truly is another’s research treasure.  

Early in the pandemic, University of Florida researchers began sampling wastewater from the nearby island community of Cedar Key to learn about the influence of tourism on COVID-19 infection rates. They also wanted to test the possibilities of using wastewater samples to detect introduced viral variants—a feat rife with technical challenges.

They found a connection between tourism and the presence of SARS-CoV-2 virus in wastewater, and they were able to reconstruct and identify two complete genomes representing two different viral variants.

The work, which began in May 2020, was recently published in the journal Environmental Research. It showcases the power of using wastewater surveillance to communicate changing local conditions and craft public health risk messaging to community members.

“There were some limitations in our study, but we were still able to show that there was a significant impact of tourism on the concentration of SARS-CoV-2 genetic markers in the wastewater,” said Tony Maurelli, a co-author to the study and a professor in the UF College of Public Health and Health Professions where he is the associate chair of the department of environmental and global health. “This was highly successful in Cedar Key because of the island’s participation and because we were focusing on a single wastewater treatment facility that served a small community.”

A tourist destination

The island community of Cedar Key lies roughly 55 miles southwest of UF on the Gulf Coast. Its economy relies on commercial fishing, crabbing and clams raised by aquaculture. It also relies on tourists who come to dine on fresh seafood, buy local art, use the public boat ramp, or fish from the pier.

In the pandemic’s early days, the city of Cedar Key limited people coming onto the island to residents only. With one bridge to get on and off the island key, limiting travel was fairly simple. But measuring tourism proved slightly more challenging because the bridge lacks a traffic counter, according to Maurelli, who is also a faculty member at the UF Emerging Pathogens Institute.

The study authors relied instead on visitations measured by the Cedar Key Chamber of Commerce. They also analyzed data from the Florida Department of Health to track infection rates alongside visits by tourists. This too proved problematic, Maurelli said, because the state tracks infection rates by zip code, and the zip code that Cedar Key falls within also includes parts of the mainland outside of its community.

“The limitations of the data collected by the Florida Department of Health precluded establishing a relationship between tourism in Cedar Key and cases, because cases were only tracked at the zip code level,” Maurelli said. “But by testing the wastewater, we were able to show that there was an impact from tourism, on the viral markers in wastewater,” Maurelli said.

The researchers found that for every 100 visitors to the island, the odds of detecting the coronavirus in the wastewater increased by 96%.  They also found that one of the genetically sequenced viral genomes showed the effect of out-of-state tourists bringing the virus with them.

The down and dirty

Here’s how the study worked. The team collected a single 24-hour composite sample from the community’s only wastewater treatment facility each week for a full year. Senior author Joseph Bisesi, an assistant professor in the department of environmental and global health, forged productive relationships with members of Cedar Key’s city council and helped to facilitate the sample collection.

Bisesi is also a member of the EPI and worked with Maurelli and co-author Tara Sabo-Attwood to develop the wastewater surveillance techniques. Sabo-Attwood is chair of the department of environmental and global health in the College of Public Health and Health Professions and is also a faculty member of the EPI. The team leveraged Bisesi’s earlier research detecting and measuring chemical contaminants such as heavy metals and organic contaminants from environmental samples.

“This project was my first entrée into wastewater surveillance,” Bisesi said. The team retooled his environmental sampling methods to look for viral signatures unique to SARS-CoV-2, specifically:  

  • Sequences of the SARS-CoV-2 nucleocapsid gene, indicating the presence of the virus
  • The number of genome copies of the virus to determine wastewater concentrations
  • Genomic sequences comprising the entire virus to detect specific variants

This was a somewhat tall order considering the material the samples were being derived from. When an infected patient produces a sample for analysis—either by coughing up sputum, enduring a nasal swab, or urinating in a cup—the material delivered to a laboratory for analysis is enriched with viral particles. Because so much virus is present, it is easier to run tests looking for different things.

But with wastewater, there’s a rich mixture of organic and inorganic matter in the sample, some of which can inactivate the virus or degrade its RNA.

“It’s very hard to isolate virus from wastewater,” said research scientist John Lednicky, who is also a professor in the UF College of Public Health and Health Professions, department of environmental and global health. “It contains detergents, alcohol, bleach and other things that will inactivate it. You have to think about how it’s been treated. Even from the same place, the samples will never be exactly alike.”

Further complicating the task was the fact that environmental samples such as wastewater tend to yield low levels of virus compared to a sample taken directly from a patient. Because of this, the researchers were unable to use advanced next-generation sequencing techniques which require large amounts of viral materials. Instead, they used a more laborious technique called Sanger sequencing.

Also, whole viruses may not be found in wastewater. The virus may fall apart, or be bound to other materials, such that only genetic parts and pieces are detected in an unorganized fashion. It’s up to the researchers to painstakingly put this puzzle back together, nucleotide by nucleotide.

“The computer programs can also get confused when multiple viral lineages are present in a single sample,” said Lednicky, who is also an EPI faculty member. “And they end up creating these crypto-genomes by stitching together genetic sequences in a composite fashion that may not be true to reality.”

Sanger sequencing, on the other hand, allowed the team to reconstruct the viral genomes nucleotide by nucleotide, through methods known as tiling and gene walking. While laborious, they are precise, Lednicky said.

Tiling involves using probes to detect overlapping sequences to comprehensively cover all coding and non-coding sequences of a genome. It ensures that nothing is missed when reconstructing a complete genome. Gene walking differs by looking for unknown sequences that are adjacent to known ones. It can grab longer sequences at a time than tiling.

The researchers were able to identify sequence amplicons specific to the SARS-CoV-2 nucleocapsid protein in 15 of the 52 samples. They were also able to count the number of nucleocapsid protein sequence amplicons present in these positive samples, which was used to quantify the viral load in each sample. They then statistically correlated the number of these genome copies with tourism visitation numbers; this showed a small but positive relationship.

Using the tiling and genome walking methods, they were able to fully reconstruct the viral genomes of two genetic variants. These genomic sequences were then fed into computer programs and global databases that can quickly search for matched hits with known sequences.  One sequenced variant from October 2020 was most closely aligned to a strain identified early in the pandemic from Italy (B.1).  The other sequenced variant was most closely related to the Alpha (B.1.1.7) variant and likely represented spread to the community by an out-of-state visitor, the researchers reported.

The study also analyzed the potential role of an increasingly vaccinated public upon its findings. Detection of the SARS-CoV-2 virus in wastewater declined later in the study. This timeframe aligns with months when the vaccine was becoming more widely available in Florida and the US.

Public health messaging

One of the plusses of wastewater surveillance is the potential to feed results to communities to communicate public health risk.

The city of Cedar Key chose to communicate findings from the study in real time to its community members. The city discussed weekly data at their city commission meetings and posted findings about the presence of the virus to social media channels. Posts were linked with suggested public health actions such as maintaining physical distance from others and wearing a face mask. City leaders used the data to help inform community decisions such as when to reopen the island to visitors and other safety precautions.

“The leadership of the community was very supportive of using this information as a public health tool. They saw this as a useful tool to provide for the safety of the community,” Maurelli said. “This is a tool that is best adapted to alerting communities to when infection rates are rising so that mitigating actions can be taken.”

Other approaches using wastewater surveillance as a public health protection tool can focus on monitoring buildings or neighborhoods to detect where hot spots are bubbling up, he added.

“The real uniqueness of this study, is that this was an isolated community. And travel was really restrictive at one point, for this very small population,” Lednicky said. “And under these conditions, you can really do the modeling well because there are fewer complicating factors.”

As the pandemic grinds on, researchers’ interest is rising in ways to use wastewater to passively monitor community transmission rates. It’s not hard to envision a day in the not-so-far-off future when push notifications to your cell phone may alert you to rising COVID-19 levels. Or, better yet, confirm a lack of detection in your area for several weeks running.

By: DeLene Beeland