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NIST Creating Reference Material for SARS-CoV-2 Diagnostic Assay Developers

NEW YORK – The National Institute of Standards and Technology (NIST) is developing a synthetic SARS-CoV-2 RNA reference material, with the aim of helping companies that are developing molecular diagnostics for the virus to measure the sensitivity of their assays by running tests against known quantities of viral RNA.

The agency is providing the material to labs and diagnostics developers and asking for feedback on how to improve it before disseminating more widely.

Though there are now dozens of assays that have received Emergency Use Authorization from the US Food and Drug Administration for the detection of SARS-CoV-2, the first few weeks of the pandemic saw companies rushing to develop tests for the virus as regulatory authorities scrambled to provide frameworks for approval.

The US Centers for Disease Control and Prevention provided the first test protocol at the end of January and began distributing test kits to state health departments and public health labs, but some state laboratories soon found that the CDC kits provided inconclusive results during the quality control process. The agency was forced to remanufacture one of the kit reagents as a result.

It wasn't until the end of February that the CDC had validated a new protocol for its assay. By then, the Association of Public Health Labs had asked the FDA to grant an exception so that labs could develop their own tests for the virus. The agency initially declined, but soon developed EUA guidelines to allow labs and companies to develop and manufacture SARS-CoV-2 diagnostic assays.

As dozens of companies began to develop tests, others began creating a variety of reference materials that the diagnostics manufacturers could use to calibrate the performance of the assays, particularly in cases where patient samples were too difficult to obtain or handle.

According to Peter Vallone, leader of the Applied Genetics group at NIST and head of the SARS-CoV-2 reference material development project, it became obvious that there would be a need for a reference standard in March, when companies began developing tests in large numbers.

"One of the things that we started to look into was how we could help offer some different reference materials, what would be needed, or what would it look like if NIST were to produce something," he said.

The agency began its design work in late March. What resulted was synthetic fragments of SARS-CoV-2 virus RNA that are characterized for concentration using digital PCR methods and that are intended for use in assessing limits of detection for SARS-CoV-2 assays, as well as calibrating other in-house or commercial SARS-CoV-2 controls. NIST characterized this material by multiple reverse transcription droplet digital PCR (RT-ddPCR) assays as well as qPCR methods.

"We wanted to have freely available reference material that we knew to be homogeneous so that labs could start to get the same material and compare assays against each other, and also use for things like limit of detection," said NIST Research Biologist Megan Cleveland, who did most of the lab work for the project. "It's two separate in vitro transcribed materials, in vitro transcribed RNA in a background with human Jurkat RNA. And we produced it and bottled it at NIST."

Cleveland noted that the material is research-grade testing material, which is of a lower quality than NIST's standard reference materials, but that's because the agency went through the process of designing it and manufacturing it so quickly in order to provide for an obvious need.

"It hasn't had the two to three years that we normally take for reference material development," she noted. "So, the purpose of this is to make this initial material and send it out to a bunch of labs for free, and they can offer feedback and suggestions to us on how to improve the material."

Some of the types of feedback NIST has asked for include whether the provided concentration range and volume are useful, or whether the material can stand up to multiple freeze/thaw cycles, Vallone said.

"What we're good at is that we assign a general value — so, we'll tell you that with this method, with this target, we're detecting five million copies per microliters. We're asking other labs to make a measurement on this and let us know, are they obtaining a value of five million copies per microliter or are they getting something higher or lower?" he explained. "We've got some feedback that after multiple freeze/thaw cycles, the concentration seems to go down, which is probably pretty normal for an RNA material. So, that's useful information for us moving forward to either find a better storage solution or provide better guidance that this is really good for one freeze/thaw, or you might want to aliquot, things like that."

Importantly, the team is also looking to figure out whether the material has any kind of bias toward a specific type of testing platform. If there is any kind of test that gives a very low value or a very high value outside the expected range, then Vallone, Cleveland, and their colleagues can look into those biases to see what's behind them.

Technically, Cleveland said, the material should work with any test that targets the regions of the virus that are in the viral fragments included in the reference kit. The NIST reference material comprises two separate 4,000 base-pair fragments that cover different regions of the SARS-CoV-2 genome.

Vallone noted that the material is not composed of packaged virus, but rather that it's a vial of naked RNA. "If you have testing methods that perform an extraction — when you collect, say, a saliva sample, that might sit in a storage media that goes to extraction, then that goes through the detection and that might be qPCR or digital PCR — this material is really intended to be put into the process at the point of the digital or qPCR. You wouldn't put this material in a storage media; you wouldn't put this through an extraction process. It's really for testing the analytical capabilities of the assay."

Importantly, the reference material plays to NIST's strength as an arbiter of measurements standards, "making the measurements, not necessarily producing liters and buckets of the material," Vallone said. "It's really in the high level of characterization that goes into it. And if we can help disseminate that measurement technique or that material to make other measurements better, that's our goal."

Indeed, said LGC SeraCare Life Sciences CSO Russell Garlick, analytical characterization of standards is a critical component of standardizing diagnostics around the world, and NIST is central to that "because they do the measurements and they do it precisely, accurately, and reproducibly."

SeraCare is one of the many companies that manufactures a reference kit for SARS-CoV-2 diagnostics. Even though the company's kit works in a different way than the agency's, SeraCare relies on NIST to help calibrate its own materials, he added.

"What we provide is to help validate the whole process of sample extraction or isolation to reporting results," Garlick said. "And so consequently, what we do is we basically make a recombinant version of the SARS-CoV-2 sequence and construct it in a way where it ends up being an encapsulated virus that is not infectious but represents what is being detected by the PCR assays. And it's more complicated than what NIST has done. But what NIST does beautifully is assessment, statistical analysis via quantitation of these materials that nobody else does."

In other words, the company thinks about what the laboratory needs to validate the whole testing process from beginning to end, whereas NIST asks whether a test can detect a virus and quantify it.

"By NIST making the materials free of charge and available, it's easy to start down that path of proving concordance between the assays," he added. "I also think what these materials will help on is the specificity of the assays. Are they detecting only the SARS-CoV-2 virus?"

Now, the NIST team is waiting for feedback from interested stakeholders before finalizing the material's design. Vallone has extended the deadline for feedback to the end of September — interested parties can still get a free vial of material if they agree to send back their comments and criticisms.

The agency is also performing its own internal studies to understand the material's stability and bias over time, he said. A major design issue could cause a six-month setback or longer. But assuming that there aren't any major problems, such as a strong bias due to a design flaw, NIST could have vials of material ready to ship in a few months.