29 August 2023
When researchers monitor viruses circulating in a community, testing only people who are already sick doesn’t create a full picture or help public health officials work proactively. “Human testing is really, really important, but it’s inherently biased by how sick someone feels, differences in access to health care, and several other societal characteristics,” says Crystal Hepp, PhD, associate director of the Pathogen and Microbiome Division of the Translational Genomics Research Institute, known as TGen North, part of City of Hope. Her Flagstaff, Arizona, lab is working to demonstrate that the pathogens found in southwestern US wastewater sampling correlate with the illnesses that are presenting in the region’s clinics.
The Pathogen and Microbiome Division’s sequencing projects seek to better understand and characterize infectious diseases, and they often focus on environmental samples. The aim is to generate data that helps the team estimate where and when human risk will be at its highest. Public health authorities can then use the information to urge people to take precautions, prevent transmission, and, if available, get vaccinated.
The lab monitors pathogens such as West Nile virus, SARS-CoV-2, adenovirus 41, and norovirus. “Norovirus outbreaks occur each year, and rafters going through the Grand Canyon on the Colorado River have recently been hard hit,” says Hepp. “In 2022, more than 200 rafters fell ill, so we started monitoring norovirus in our area more closely.” With wastewater testing in place, her team was able to let public health officials in Northern Arizona know that norovirus season was starting earlier in 2023 than it had in the previous year, allowing for earlier mitigation planning.
TGen’s wastewater surveillance project was established in 2019, in collaboration with several city, county, and state partners throughout Arizona. And from the onset of the pandemic, they were organized and ready to design protocols that could identify increases of SARS-CoV-2 in a region’s wastewater approximately 10 days prior to an increase in cases there.
Studying influenza
Another respiratory virus, influenza A, was the subject of a 2022 study TGen conducted in collaboration with Northern Arizona University (NAU), where Hepp also has an appointment of associate professor in the School of Informatics, Computing, and Cyber Systems. For their research, the team was able to access both wastewater and clinical samples that were known to be positive for influenza A. Daryn Erickson, lead author on the manuscript in progress and a member of Hepp’s group, wanted to know what subtype of influenza A was circulating.
They prepped several high-viral-load samples for whole-genome sequencing with the Illumina Respiratory Pathogen Infectious Disease/Antimicrobial Resistance (ID/AMR) Panel, or RPIP. The panel targets over 280 respiratory pathogens, including influenza, SARS-CoV-2, respiratory syncytial virus, bacteria, fungi, and more than 2000 antimicrobial resistance markers. “The really nice thing about the RPIP kit is how broad the target list is and that it can pull out different subtypes of influenza A from a single sample,” says Erickson. “We knew the samples were positive for influenza A. We just didn’t know anything past that.”
RPIP employs a bait-capture approach, which uses probes to capture specific library molecules to take to the sequencer. It allows for greater mismatches, which helps identify pathogens that are evolving. It can also handle more probes, or targets, as compared to a PCR-based approach. After the team ran the samples on their Illumina MiSeq System, the Illumina Explify analysis pipeline processed the data and determined from the whole genome of the virus that it was the H3N2 subtype of influenza A.
Next, they used this full genome output to design a tiled amplicon scheme for H3N2—a panel of 43 primer pairs. Amplicon sequencing was a quicker and more feasible way to amplify the rest of the samples—obtained both from wastewater and from clinics—and confirm they were all the H3N2 subtype.
A rapidly evolving flu
TGen was also able to use the amplicon panel to a test an H3N2 positive sample collected in 2012, but the panel failed to amplify the genome. “The influenza A virus mutates quickly, and the primers that we had designed were very targeted for more contemporary influenza A genomes,” Erickson says.
“What we realized is that [the] RPIP [workflow] allowed for detection of more divergent subtypes of influenza while the tiled amplicon sequencing protocol was more specific,” explains Hepp. In the future, they will likely use amplicon sequencing to characterize similar influenza targets and RPIP for the initial characterization of new strains.
Erickson created a phylogenetic tree to put the influenza A H3N2 sequences into a larger context and look at how they relate genetically to other H3N2 genomes from across the US over multiple years. Different clusters were identified for these samples, one of which seems to be an outbreak of H3N2 over a short period of time in Northern Arizona. However, the team points out that the analysis is limited, as some states have more data than others. “Moving forward, we plan to perform a broader comparison of Arizona versus other US and global genomes,” Erickson says. “This will allow for a more complete understanding of transmission dynamics as they relate to community characteristics like vaccination rates and travel within the state and beyond.”
Surveillance for all viruses
Through August 2023, the NAU and TGen collaborative team are wrapping up an ongoing project in which they've performed pooled COVID-19 testing on school-age children and residents of long-term-care facilities every other week. In August of 2022, they began testing for other respiratory viruses, enabling them to inform the schools and their communities what threats are circulating at a given time. The team has tried some of these samples with the RPIP workflow, particularly when they wanted to identify additional respiratory pathogens.
In August of 2020, COVID-19 cases in the US temporarily dropped, but the number of people presenting at clinics with COVID-like symptoms remained high. Hepp happened to hear medical staff discussing this conundrum during one of her own doctor’s appointments, and she mentioned that her lab was also seeing a very low SARS-CoV-2 viral load in the wastewater, but they were running a pan-adenovirus assay and saw extremely high adenovirus viral loads. Because some adenoviruses can produce COVID-like symptoms, that information was useful and circulated quickly through the ranks to different decision makers. “The concern was that maybe COVID tests were failing, because a lot of people had symptoms” Hepp says. “However, several pathogens can cause COVID-like illness, so testing for pathogens beyond the one target of interest can enhance overall situational awareness.
“We were running the pan-adenovirus assay because there are several human viruses within the family that can cause seasonal respiratory or gastrointestinal symptoms. It turns out that the assay likely detected what was causing mild COVID-like symptoms in the absence of COVID-positive tests. However, we also realize that a bit of luck resulted in the assay being incorporated into routine surveillance—we had the primers and probes in-house for a previous one-off question we wanted to get at. One nice thing about RPIP is that it integrates a large number of assays, which can take some of the guesswork out of figuring out which assays should be included for wastewater testing.”
Looking ahead, Hepp’s team wants to build on the knowledge and data they’ve collected over the last few years so they can catalog the many respiratory viruses that circulated during the pandemic. Their vision is to enhance awareness of the breadth of viruses that can infect humans, by providing additional surveillance tools and bioinformatic pipelines that can be integrated into ongoing community and health care practices. With that vision, the team is interested in developing solutions that can be equitably distributed and applied across agencies, institutions, and borders.
To learn more about wastewater surveillance, click here.
