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CDC Wastewater Surveillance Shifts to Public Health Labs With Planned Digital PCR Pathogen Panels


NEW YORK – During the COVID-19 pandemic, PCR, digital PCR, and sequencing technologies have each demonstrated the ability to detect SARS-CoV-2 virus shed into sewage systems and determine the presence of variants.

Now, the US Centers for Disease Control and Prevention's National Wastewater Surveillance System (NWSS) will begin a planned expansion beyond COVID-19, using a multipathogen digital PCR panel for use on platforms from Bio-Rad Laboratories and Qiagen. The agency will also support sequencing-based SARS-CoV-2 variant monitoring.

Wastewater-based epidemiology provides up to two weeks of lead time on clinical case surges and has become more useful with the popularization of at-home testing and increasing prevalence of variants causing asymptomatic or mild infection.

Initially, the NWSS program relied on data generated by contract and academic labs, which it made available in a public dashboard.

But, as previously reported, the division has always intended for wastewater surveillance to ultimately become integrated into public health labs.

While a RT-qPCR approach has the advantage of being the most quickly and broadly applied for the least cost, developers of digital PCR systems have been advocating for their technologies in this space, touting its inherent inhibition resistance, ability to detect low concentrations of nucleic acids, and quantitation without the need to generate a standard curve.

Until now, the NWSS program has standardized incoming results from a host of different workflows and technologies in order to generate wastewater surveillance data.

At separate presentations at the Clinical Virology Symposium and at the Association of Public Health Laboratories annual meeting in May, NWSS director Amy Kirby disclosed that the program will support digital PCR as its primary test platform as it transitions into public health lab sites.

"It is the most sensitive approach to wastewater surveillance and it is very robust to the inhibitors present in wastewater," she said. "We will be supporting assays for both the Qiagen QIAcuity system and the Bio-Rad QX systems."

At APHL, Kirby also said NWSS will institute a next-generation sequencing-based SARS-CoV-2 variant tracking approach, but the program has not shared specific details about the workflows it plans to employ.

The NWSS digital PCR panel

The NWSS program will collaborate with stakeholders to develop a 30-target panel of assays for digital PCR-based wastewater surveillance.

The initial targets designated for core, routine wastewater surveillance include SARS-CoV-2, influenza, and high-consequence antimicrobial resistance genes such as those conferring resistance to antimicrobials like colistin, carbapenemases, and beta lactamases.

The panel will also target Candida auris, and foodborne infections like salmonella, Shiga toxin-producing Escherichia coli, norovirus, and Campylobacter.

Targets for occasional or emergency use will likely include pathogens that cause outbreaks of waterborne illnesses, emerging diseases, and biosecurity concerns. And, the panel can be adapted to provide insights into special targets of concern, such as EV-D68, Kirby said in her CVS presentation.

The initial panel will be finalized in the next two months, Kirby said, with pilot testing through 2023 and a rollout in 2024. This will also lead to a new public-facing dashboard to present the data.

The NWSS program currently supports testing in 37 states, four major cities, and two territories. It aggregates data from more than 900 wastewater collection sites, representing the sampling of more than 110 million people, or a third of the US population.

NWSS is also supplementing this with 500 additional sites under a commercial contract with LuminUltra, in part to better support vulnerable communities, Kirby said. A send-out service is lower impact, she said, and if communities discover how useful the data is, they may be convinced to implement testing in their local public health systems.

The program is expected to expand to all 50 states, with an upper wastewater surveillance limit of 80 percent of the US population, as the remainder is not on sewer systems.

Gary Nealey, VP of Qiagen's US business, said in an interview that wastewater-based epidemiology is a powerful tool. It has the ability to detect a single case in a sample from 2 million people, Nealey said, and provide regular monitoring of almost 80 percent of the population. Furthermore, "it is cost effective, and it doesn't put a burden or strain on the healthcare system," he said.

The QIAcuity provides fully automated testing with minimal hands-on time and results in about two hours, Nealey said. It is also a small-footprint, plate-based system that can be scaled to run up to eight 96-well plates.

Qiagen claims that 70 percent of US states now have at least one public or privately owned laboratory that can monitor wastewater with QIAcuity, in part due to a previous contract with the US Department of Health and Human Services to supply public health labs, Nealey said.

At the JP Morgan Healthcare Conference in January, Qiagen CEO Thierry Bernard also specified that the firm secured business at 40 out of 52 US public health labs. Going forward, wastewater is expected to be a significant part of the overall QIAcuity business. 

Bio-Rad, meanwhile, has also been targeting the wastewater testing space for its QX systems since the early days of the COVID-19 pandemic. Last year, the firm estimated the market opportunity to be approximately $200 million over the next five years.

Simon May, Bio-Rad’s executive VP and president of the life science group, noted in an email that Droplet Digital PCR has been endorsed by the CDC NWSS group since early in the pandemic, and, "their guidelines for disease and outbreak surveillance testing note that digital PCR is a more reliable detection method for wastewater as compared to qPCR."

Bio-Rad's ddPCR systems are already in a majority of US states in laboratories monitoring wastewater for pathogens, May said. Indeed, "for the past decade, Droplet Digital PCR has been demonstrated to be an effective and sensitive tool for low-level detection of pathogens in wastewater so communities can act quickly to prevent the spread of disease," he said.

Bio-Rad also has assays available for pathogen detection in wastewater beyond SARS-CoV-2, such as norovirus and Ebola virus, as well as other targets currently under review by the CDC NWSS team for potential inclusion into the new panel.

An expanding role

The transition of US wastewater surveillance from private and academic labs to public health labs is ongoing and likely to be completed in the next year.

For example, one of the most well-developed programs is the Ohio Wastewater Monitoring Network (OWMN). Supported in part by the US Environmental Protection Agency, the program began in July 2020 and has been monitoring 77 sites twice weekly for almost two years.

Nichole Brinkman, an EPA researcher, noted in a presentation at the APHL conference that the Ohio wastewater surveillance system, which also reports data to NWSS, consists of eight commercial, academic, and private government labs using RT-qPCR, dPCR, and sequencing to screen for SARS-CoV-2 in wastewater. Each lab has a unique processing and QC workflow, Brinkman noted. Only two currently use digital PCR, although all of them perform targeted sequencing to detect fragments of SARS-CoV-2 variants of concern using the Illumina NextSeq 2000.

Now, the OWMN program is transitioning from eight separate labs to a single lab — and a single workflow — within the Ohio Department of Health, Brinkman said, with the move expected to be completed in July of this year.

North Carolina also supports an NWSS-affiliated wastewater testing program, sampling 25 sites twice per week, with 12 additional sites being added soon, according to Virginia Guidry, branch head of occupational and environmental epidemiology at the North Carolina Department of Health and Human Services.

The samples are tested weekly using digital PCR at two University of North Carolina labs, but the program will shift to the NC State Lab of Public Health this summer, Guidry said in an APHL meeting presentation.

Overall, the wastewater surveillance has been particularly helpful in detecting community spread in fluctuating populations not always represented in local clinical testing data, including university students and tourists, she said. Developing one-to-one relationships with stakeholders at the sewage utilities has also been critical to the success of the North Carolina program, she added.

Lauryn Massic, an infectious disease fellow at the Nevada State Public Health Lab in Reno, noted that her lab is using RT-qPCR on the Thermo Fisher QuantStudio 7 and a kit from Promega for SARS-CoV-2 detection, with Ceres Nanotrap beads for viral concentration. The lab is now in the process of transitioning to Bio-Rad Droplet Digital PCR, she said in an interview.

The Nevada lab is also using the Illumina MiniSeq to perform variant monitoring from wastewater, Massic said, and presented data on the method in a poster at the APHL annual meeting. "In my opinion, sequencing could never be beat," she said. "It gives us a lot of genomic information about changes in the virus," she added, and this in turn can have public health implications. For example, the lab was able to detect the Omicron variant on Dec. 12 last year, Massic said, even though the first local clinical case didn't turn up until Dec. 20.

"I don't see sequencing going away," she said.

Researchers at the University of California, San Diego also presented a poster at the APHL annual conference on their use of the Thermo Fisher Scientific Genexus Integrated Sequencer for variant detection. The lab reported testing took one day with only 10 minutes of hands-on time using Thermo Fisher's Ion AmpliSeq SARS-CoV-2 Insight Research Assay. The UCSD lab also developed a bioinformatics tool, called Freya, that it said is easy to use and can produce lineages and abundances for mixed SARS-CoV-2 samples.

Andy Felton, VP of product management research and platforms business in the Clinical Sequencing Division at Thermo Fisher, noted in an email that while digital PCR will be useful for monitoring of the presence and abundance of pathogens within wastewater samples, sequencing is ideal for tracking variant information.

Thermo Fisher expects customers will appreciate that in-house systems like the Genexus Integrated Sequencer can provide variant data more rapidly and consistently than send-out testing, which can take several weeks and is not as comprehensive, Felton said.

Despite the growing enthusiasm for wastewater testing, some challenges remain.

Kirby noted at APHL that with the expanded panel, close collaboration with sewer utilities will continue to be important. SARS-CoV-2 in wastewater is not infectious, but that is not the case for some of the other planned targets, and utilities will likely be tasked with removal of whatever transmissible pathogens may be detected in the samples they provide public health labs.

And, the epidemiological extrapolation also needs to be improved in order to better understand what a positive result means, to allow for a more refined public health response.

In the future, the CDC also expects to provide more messaging on the ethics of wastewater testing and sample archiving, Kirby said.

"Wastewater has lots of DNA in it, including human DNA, so we need to be very clear [with the public] about what we are detecting, what we are not detecting, and how these samples may be used in the future," she said.