Researchers develop algorithm to identify SARS-CoV-2 variants in wastewater

It may be a bit smellier than other ways to monitor COVID-19, but analyzing wastewater is a cheaper, faster and more accurate way for public health officials and researchers to detect rising cases. Bits and pieces of the SARS-CoV-2 virus are flushed down toilets and sinks by infected individuals; more copies of the virus found in sewage means more people are sick. But until now, most wastewater analysis methods brought all SARS-CoV-2 viruses together as one.

Now scientists from Scripps Research and University of California, San Diego, in collaboration with the San Diego Epidemiology and Research for COVID Health (SEARCH) alliance, have changed that. The team has reported that with just two teaspoons of raw sewage, they can accurately determine the genetic mixture of SARS-CoV-2 variants in a population and identify new variants of concern up to 14 days before traditional clinical testing. In San Diego wastewater, the group discovered the Omicron variant 11 days before it was first clinically reported.

Their algorithm, called “Freyja”, for identifying SARS-CoV-2 variants in wastewater, described today in Naturehas been rapidly adapted by many public health labs and is a boon to surveillance efforts aimed at detecting novel variants of SARS-CoV-2.

“In many places, standard clinical surveillance for new variants of care is not only slow, but also extremely cost-effective,” said Kristian Andersen, PhD, professor of Immunology and Microbiology at Scripps Research and a senior author of the new work. “But with this new tool you can take a single wastewater sample and profile the entire city.”

The project required close collaboration between hospitals, state and local governments, sequencing facilities and academic scientists, including researchers in the Andersen lab and that of UC San Diego microbiologist Rob Knight, PhD. The Knight lab deployed 131 wastewater autosamplers to collect wastewater from 343 buildings on the UCSD campus and 17 public schools in 4 San Diego school districts, and collected samples from major wastewater treatment plants in the province. In nearly a year, the group analyzed more than 20,000 wastewater samples. In the process, they developed improved methods for concentrating viral RNA in wastewater, which are now widely used by public health labs across the country and the world. Next, the Andersen lab took on the challenge of quantifying viral variants from the sequence data.

“It’s a challenge to take all these tiny bits of virus floating around in wastewater and figure out which are of different variants and what their relative abundance is,” said Scripps Research postdoctoral fellow Joshua Levy, PhD, a co-first author. of the new paper featuring Smruthi Karthikeyan of UC San Diego.

Many variants of SARS-CoV-2, including Omicron and Delta, differ by a small number of mutations. But since these changes could affect how the virus spreads or infects people, public health officials need to monitor them carefully. They’ve usually done this by sequencing patients’ virus genomes, which is a slow, costly process and has become less effective at capturing the size and diversity of COVID-19 variants as many people turn to home testing.

Levy developed a library of “barcodes” that identify SARS-CoV-2 variants based on short fragments of their RNA unique to each variant. He then coded a new calculation tool that searches through the mass of genetic information in wastewater to find these barcodes. He made the new Freyja program easy to use and free.

“If you’re in a lab that can already sequence a wastewater sample, you’re good to go — just run this code and in another 20 seconds you’re done,” he says.

When the researchers applied Freyja to their wastewater samples and compared the results with clinical data collected by SEARCH in San Diego, they found that the tool detected variants of concern, including Alpha, Delta and Omicron, in wastewater for up to 14 days before start. was reported clinically. The variant Mu (B.1.621) was found in UC San Diego wastewater on July 27, 2021; four weeks before the first clinical detection on campus. And using more recent data not included in the original study period, the team also reported that the Omicron variant could be detected at the Point Loma wastewater treatment plant — in an abundance of just over one percent of all SARS-CoV-2. -viruses in a contributing population of more than two million people-; on November 27, 2021, 11 days before clinical detection in the city.

“Wastewater contains an enormous amount of very valuable information about our health, including these viral genomes that allow us to track the course of a pandemic or epidemic,” says Karthikeyan.

“It took a lot of collaboration between public health and academic players to set up this system in San Diego, and now that we’ve demonstrated its effectiveness, we hope it inspires other places to use these tools,” added Knight. “We are also very excited about its expansion to pathogens beyond SARS-CoV-2.”

The researchers say they continue to improve the set of tools they use to analyze viruses in wastewater, but the current set of methods is already a leap from previous approaches. The same strategies can be used to monitor not only variants of SARS-CoV-2, but other human pathogens as well.

“When you rely on clinical sampling, not only do you introduce a lot of socioeconomic and geographic biases about who contributes to genomic surveillance data, but you also have the problem that asymptomatic people don’t get tested and those who only use home testing don’t contribute.” to the data pool,” says Levy. “But we don’t have those blind spots with wastewater.”

In addition to Levy, Karthikeyan, Andersen and Knight, Christine Aceves, Catelyn Anderson, Karthik Gangavarapu, Emory Hufbauer, Ezra Kurzban, Justin Lee, among others, are the authors of the study “Wastewater sequencing reveals early, cryptic SARS-CoV-2 variant transmission”. , Nathaniel Matteson, Edyth Parker, Sarah Perkins, Karthik Ramesh, Refugio Robles-Sikisaka, Madison Schwab, Emily Spencer, Shirlee Wohl, Laura Nicholson, and Mark Zeller of Scripps Research, as well as contributors to UC San Diego, Rady Children’s Institute for Genomic Medicine, Scripps Health, Sharp Healthcare, Helix, the County of San Diego Health and Human Services Agency, the California Department of Public Health, and the Centers for Disease Control and Prevention.