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Singapore-MIT Alliance Shows Benefits of Wastewater SARS-CoV-2 Variant Test

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NEW YORK – A team of researchers at the Singapore-Massachusetts Institute of Technology Alliance for Research and Technology has shown its SARS-CoV-2 variant detection assay for wastewater agrees with clinical trends. The SMART assay to detect the Alpha variant of the virus is now being used by testing lab Biobot Analytics, and SMART expects to have a Delta variant assay publicly available within the week.

Wastewater testing for pathogens, also called wastewater-based epidemiology, or WBE, has been increasingly utilized during the SARS-CoV-2 pandemic. Raw sewage samples yield RNA fragments shed by a multitude of individual infections, so it is impossible to reconstruct any individual virus' genome and determine exactly the strain present. Nevertheless, WBE can be used to track the spread of known viral variants by genotyping samples for characteristic SNPs and mutations.

The SMART assay detects three mutations of the SARS-CoV-2 genome — HV69/70del, Y144del, and A570D — that are hallmarks of the Alpha variant, formerly known as B.1.1.7. Preliminary studies of the assay were published as a pre-print earlier this year and it was formally described this month in Environmental Science and Technology Letters.

In the full analysis, the SMART team looked at remnant wastewater samples that were collected as part of Biobot's ongoing surveillance activities in the US, comparing the variant assay to local clinical trends.

Specifically, the team looked at a period in the fall of last year when no Alpha variant was known to be circulating, and compared it to a period in January of this year, when Alpha was first emerging, and in February and March, when Alpha was circulating in most US states. They found that the variant SNPs were first detectable in samples from locations that had the first clinical cases, in January, and that the fraction increased more than threefold across all sites between January and March.

Prevalence of variants in the community is typically tracked through sequencing of clinical samples. 

But Lee Wei Lin, a research scientist at SMART — MIT's research enterprise in Singapore — noted in an email that using WBE to track variants has major advantages.

"Clinical genomic surveillance is expensive, involves specialized infrastructure, and requires sequencing a significant fraction of the cases for accurate representation," Lee said. "This is extremely challenging to achieve, especially for developing countries which unfortunately are the hardest hit in this pandemic," she said.

In essence, the SMART test is a standard PCR-based genotyping assay. But, unlike other clinical variant genotyping tests, it is designed to be quantitative so it can give information on the abundance of the variants, Lee said.

The method generates a direct variant abundance readout at the amplification stage using RT-qPCR, and from sample to data takes only an average of a couple of hours, she said.

The team enhanced the assay by designing and testing a library of primers targeting mutations characteristic of the variant of concern and assessing these primer sets for sensitivity, while incorporating synthetic mismatches to improve assay specificity.

"The final candidates reported in our work were similar in sensitivity and quantitatively matched viral titers obtained with US CDC N1/N2 SARS-CoV-2 assays, an important aspect other assays were not able to achieve or have not reported," Lee also said.

The three targets help increase the confidence in detection of the Alpha variant. Lee noted that while the HV69/70del and Y144del mutations are also common in the Eta variant, the A570D is almost exclusive to the Alpha variant.

Other variant detection assays for wastewater have also recently been described in the scientific literature.

A primer extension PCR strategy targeting a signature mutation in the virus's N gene was published last week in the pre-print server MedRxiv by researchers in Ottawa, Canada. The method quantifies the proportion of Alpha and non-Alpha alleles in wastewater, "without the need to employ quantitative RT-PCR standard curves," according to the publication. The group also demonstrated that the wastewater Alpha profile correlates with the local clinical counterpart but noted that WBE has the benefit of "a near real-time and facile data collection and reporting pipeline."

Another recently published method from a team in the UK uses nested PCR then uses sequences to identify SNPs, while a team at Clear Labs has publicly disclosed preliminary data on sequencing SARS-CoV-2 in wastewater samples using its automated Oxford Nanopore-based system.

Meanwhile, Netherlands researchers previously described monitoring wastewater for the N501Y mutation, a signature of the Gamma variant, using digital PCR-based techniques.

Lee said that the SMART team purposefully developed its assay to use widely available instrumentation and reagents with an RT-qPCR workflow that is already used by most SARS-CoV-2 diagnostics. In contrast, sequencing tends to have a longer turnaround time and lower sensitivity in wastewater samples since the viral titers tend to be very low, she said, while digital PCR approaches require instrumentation that not all labs have.

Biobot collab

The collaboration with Biobot came about in part because the principal investigator at SMART's antimicrobial resistance research group, Eric Alm, is also an adviser to the startup.

"Biobot Analytics was looking for convenient methods that would enable them to quantify and track variants of concern via wastewater surveillance while in SMART we had the technology already developed but were seeking an avenue to apply it," Lee said.

There are no financial arrangements in the collaboration, but rather it is more of a mutually beneficial partnership.

The results have been published "open-source and open-access, with detailed steps and know-how that would enable anyone interested, such as Biobot, to be able to adapt this method to tracking the Alpha variant and other variants," Lee emphasized.

Newsha Ghaeli, president and cofounder of Biobot, noted in an email that the lab was selected last month by the US Department of Health and Human Services to launch a 12-week national wastewater based disease monitoring program. As part of the program, Biobot is detecting the presence of new variants of COVID-19 by sequencing viral RNA from samples, and Ghaeli said the sequencing data will be uploaded to the National Center for Biotechnology Information data repository, NCBI SRA, by HHS.

"Our testing services now cover approximately 30 percent of the US — we have sampling locations in all US states, eight native communities, and two US territories," she said, adding that from these sources, Biobot has been testing upwards of 700 samples per week for SARS-CoV-2.

The WBE approach has its roots in monitoring wastewater for clues about community incidence of substance use, with a goal of better targeting public health outreach. As such, Biobot also has research and pilot studies ongoing for its high-risk substances testing product, Ghaeli said.

Similarly, Adam Gushgari, CEO of Scottsdale, Arizona-based WBE lab AquaVitas, said that he has seen a shift back into the chemical and mass-spec analysis arenas. Work early in the pandemic showing drug abuse trends related to lockdown has caught some interest, he said, suggesting, "the field is about to take off in many directions."

Meanwhile, AquaVitas is also supplying wastewater variant testing, as is at least one other commercial lab, Fort Collins, Colorado-based GT Molecular.

AquaVitas offers multiple variant assays using both RT-qPCR and sequencing, Gushgari said.

"We're able to detect all of the significant SARS-CoV-2 variants of concern, and are constantly reviewing advances in the field to include new variants as they are identified," he added.

Christopher McKee, CEO of GT Molecular, said his firm offers a full wastewater testing service that starts at $295 and includes digital PCR testing, shipping both ways, and data delivery via a double-encrypted web platform.

GT Molecular can test for SARS-CoV-2 and all variants of concern, including Alpha, Beta, and Gamma, as well as the newly dominant and highly transmissible Delta variant.

The lab also offers wastewater testing for influenza A/B, hepatitis A, and opioids, and it sells its assay as RT-qPCR and digital PCR kits, with 200 reactions and quantitative controls in each kit, McKee said.

In addition to its testing efforts, Biobot Analytics has launched a public-facing data visualization, called the Nationwide Wastewater Monitoring Network, that compares the lab's data to local clinical COVID-19 case numbers.

Delta, and beyond

The Delta variant is more transmissible, so understanding where it is present and prevalent could be useful to public health.

The SMART team's Delta variant test has already been developed, Lee said, and will be published on MedRxiv within the next week.

Lee noted that this assay can benefit the WBE community because "any laboratory, institution, or agency will have open access to the steps required for implementation and conducting wastewater surveillance for the Delta variant using commercially available RT-qPCR protocols."

A team in Israel also recently published a RT-qPCR assay that detects both Gamma and Delta variants in wastewater.

Biobot expects to adopt the SMART Delta variant test as soon as it is available.

"We believe the assay will have a huge public health impact," Ghaeli said.

And, in the future, with a wastewater monitoring infrastructure soon to be in place in the US and across the world, "we can be constantly on the lookout for new outbreaks of disease and potential public health threats ... so we are better prepared to protect our communities and save lives in the next global health crisis," she said.

Indeed, although the WBE method can't reconstruct individual viromes, it can theoretically be used to surveil for unusual new mutations and potentially provide some lead time to public health organizations.

For example, as described this week in a MedRxiv pre-print, a group in New York City has detected increasing frequencies of novel SARS-CoV-2 lineages in wastewater that were not recognized in a GISAID database and are rarely seen in clinical samples. The authors speculate that one possible explanation might be a local non-human animal reservoir.

And, another study published last week in MedRxiv used sequencing-based methods over many months to track single-nucleotide variants of SARS-CoV-2 in the Los Angeles area. That team detected several SNVs that are markers for clinically important SARS-CoV-2 variants, along with SNVs "of unknown function, prevalence, or epidemiological consequence," the authors wrote.

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