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SARS-CoV-2 Mutations Present Unexpected Opportunities, Challenges for MDx Developers


NEW YORK – A new, more contagious variant of the SARS-CoV-2 virus is currently being tracked in an unexpected way: through failure of components of RT-qPCR assays.

On a call with test developers on Wednesday, the US Food and Drug Administration addressed the issue, which primarily involves the dropout of the SARS-CoV-2 S-gene target. Certain assays from Thermo Fisher Scientific appear to be the most widely used tests experiencing S-gene dropout at this point, and the company is currently positioning its tests as a solution for variant surveillance.

Assays from Applied DNA Sciences and Helix are also experiencing S-gene dropout, while tests with S gene components from BioFire and Altona Diagnostics are not currently affected. While researchers have quickly developed RT-qPCR tests specific for the variants, the occasion has also highlighted the potential public health benefits of disclosing proprietary commercial test formulations.

Two SARS-CoV-2 variant lineages — called B.1.1.7 and B.1.351 — each contain several mutations in the S gene. Commonly known as the UK variant because it appears to have emerged there, the B.1.17 variant has a large number of changes, while the B.1.351 variant, which emerged in South Africa, has mutations overlapping some carried in B.1.1.7 in the S gene.

Both variants are thought to be more transmissible, according to the CDC, but not inherently more likely to cause severe disease in an individual. Yet, a more transmissible strain has implications for containment and testing needs, and as a larger number of people become ill, mortality may increase proportionally.

The S-gene dropouts in Thermo Fisher's Emergency Use Authorized TaqPath COVID-19 Combo Kit test in particular are being used to track the B.1.1.7 lineage of SARS-CoV-2.

The test targets the nucleocapsid gene, or N gene, a gene called Orf1ab, and the S gene. The S-gene target happens to overlap a deletion at position 69/70 that is carried by the B.1.1.7 variant, so a dropout of the TaqPath S-gene target can effectively alert labs to the variant, with results confirmed using sequencing.

In a news release on Friday, FDA also alerted the public that false negative results can occur with any molecular test for the detection of SARS-CoV-2 if a mutation occurs in the part of the virus’s genome assessed by that test.
"Three currently authorized molecular tests, Mesa Biotech Accula, TaqPath COVID-19 Combo Kit, and Linea COVID-19 Assay Kit, may be impacted by genetic variants of SARS-CoV-2, but the impact does not appear to be significant," the agency said.
The Mesa Biotech Accula test targets the N gene of SARS-CoV-2 and the instructions for use of the test, which were updated on Thursday, state: "Analytical studies evaluating the SARS-CoV-2 N gene 28881 GGG->AAC mutation have shown a potential reduced sensitivity may occur when viral concentrations are at or near the limit of detection of the assay."

FDA talks mutants

On a town hall call with developers Wednesday, Timothy Stenzel, director of the Office of In Vitro Diagnostics and Radiological Health, said it is important to understand the SARS-CoV-2 variants that are circulating, noting that a handful of UK variants have already been confirmed in the US, and highlighting that the South African variant is overlapping, but also different.

"Both of these variants appear to be of higher infectivity, due to the viral mutations which enhance binding to the receptor ... [and] some of them may also have increased amounts of virus," which could in turn increase transmissibility, Stenzel said.

The FDA has an interest in sequencing-based tests, Stenzel commented, and has authorized a kit from Illumina to sequence the entire genome of SARS-CoV-2. "That's been a great step forward," he said, adding, "We authorized it a long time ago, before we really knew how important new mutations would be."

Illumina announced on Wednesday that is collaborating with Helix and the US Centers for Disease Control and Prevention to surveil for the new variants. The Helix COVID-19 Test is also designed to detect the SARS-CoV-2 N gene, ORF1ab, and S genes, with the S-gene target overlapping B.1.1.7 variant regions.

Stenzel said the FDA has requested that developers check known variant sequences in databases and "explain any challenges with their primer and probe sets" at least twice during the EUA submission process.

In addition, Stenzel reiterated that the FDA now has its own bioinformatics capabilities. "We have the list of all EUA primers and probes for molecular assays, and we have been interrogating the sequence databases to see if there are any mutations that could impact test performance," he said.

The FDA has analyzed the UK and South African variants, and previously for any variant that was seen in database sequences at a rate of 5 percent or more, Stenzel said.

If a primer and probe set is potentially impacted, FDA protocol is currently planning to reach out to test developers first. "We ask for their assessment of the impact of these mutations on the performance of the test," Stenzel said.

Assays could be impacted by mutations, particularly if they target single genes, and several such tests have received EUA, he said. Multigene assays, he added, are less likely to be impacted by future mutations.

That said, Stenzel also noted that some technologies do not lend themselves to multi-gene targets. "We understand that completely — there is nothing wrong with a single-gene assay, however there is a little bit more pressing need to understand how variants might impact performance."

The agency has also evaluated tests internally to anticipate significant performance alterations, and plans to share important findings with the community, Stenzel said.

For the UK variant, Stenzel highlighted the Thermo Fisher's TaqPath assay as a prominent test with S-gene dropout.

The agency believes that about 5 percent of the positive TaqPath tests have an S-gene dropout, Stenzel said, but that a great majority of those are not carrying the UK variant.

"For the time being, at least, sequence verification of S-gene dropout samples is something that we would recommend," he said on the call, adding that options include using the Illumina test or reaching out to public health laboratories. He also noted that the CDC has asked public health labs to send suspect samples to the agency for sequencing.

According to the CDC's website, it is partnering with public health, reference labs, and universities, to sequence 2,500 samples per week, and released $15 million of funding for its Epidemiology and Laboratory Capacity Program in December to integrate next-generation sequencing and bioinformatics into the US public health system.

Mutations in a gene also potentially change the protein it encodes, which can impact epitope structure and antibody binding. The effect of the new S-gene variants on antigen tests is more difficult to quickly estimate, so the FDA is now coordinating with the CDC to assess EUA antigen tests.

Specifically, Stenzel said that CDC is performing viral culture to generate enough UK variant- and South African-variant containing virus.

FDA plans to contact all developers of EUA antigen tests to ask them to send kits for analysis. Ultimately, he added, developers may potentially be able to obtain variant virus through a national repository, BEI Resources.

"We are hoping that none of the antigen tests are impacted," he commented later on the call. "We'll get a good idea with the first tests we look at. Frequently it is the N-gene [protein] that is targeted by antigen tests, and that probably is a bit more conserved in some respects than the spike protein." 

Serology assays can also be impacted, and the agency intends to collect serum and plasma samples from patients infected by variants to assess serology test performance, too.

Lemonade from lemons

Genetic drift in a viral genome is expected as the genomes continuously accumulate SNPs. Assay developers typically hedge against potential problems by targeting multiple conserved genomic regions.

On the call, Stenzel emphasized that the TaqPath assay detects three different SARS-CoV-2 genes, so positives can still be detected even if one drops out.

"We don't know, as of now, that there is any significant decrease in test performance for the Thermo Fisher assay, and for the moment at least it is a very helpful assay to identify potential S-gene dropouts that could be the UK variant as we try to assess whether spread is continuing in the US."

Nathan Grubaugh, an epidemiologist at Yale University, said this is likely the first instance of a non-detection event being used for detection. "This is the first time that I am aware of a clinical diagnostic test accidently being useful for this purpose," he said.

Alex Greninger, a clinical virologist at the University of Washington, concurred, saying he was "not aware of a similar case where a test was used in a backward manner to determine a variant." 

In addition to the TaqPath test, an EUA assay called the Linea COVID-19 Assay Kit from Stony Brook, NY-based developer Applied DNA Sciences can pick up the variant, as it exclusively targets the S gene but in two separate regions.

James Hayward, chairman, president, and CEO of Applied DNA Sciences, said that because the two regions are highly conserved, one would expect them to survive SARS-CoV-2's mutation rate. But, some of the deletions in the B.1.1.7 do appear likely to affect reporting for the assay's S1 target in the case of the UK variant, he said.

In general, the S-gene dropout is "a very striking PCR tracing — when you see it, it kind of takes your breath away," he said, adding that it is very easily identified in the lab, and can be subsequently confirmed with sequencing.

Applied DNA Sciences' test performance is not affected by the dropout, he said, because the EUA instructions for use specify that a patient is positive if either the S1 or S2 target is detected. "That puts us in a very good position to have utility in screening for these variants," Hayward said.

The firm has already detected S-gene dropout-causing variants that were not the UK variant.

Now, "We expect to market our diagnostic as effective in the case of strong pressure for mutation in the S gene, and specifically to have utility in the face of B.1.1.7," Hayward said.

Applied DNA Sciences is currently using its test to surveil essential and government workers, as well as academic centers and businesses locally through a wholly owned subsidiary, Applied DNA Clinical Laboratories. The firm is also coordinating with New York State and the FDA, Hayward said, and it is developing a B.1.1.7 variant-specific test for which it expects to seek FDA EUA.  

Meanwhile, Manoj Gandhi, Thermo Fisher Scientific's senior director of medical affairs for genetic sciences, said in an email that the firm is working with FDA to help use its TaqPath test for surveillance efforts for the new variant.

In addition, "We have proactively reached out to the CDC and the Association of Public Health Laboratories in an effort to monitor the spread of the new variant ... [and we are] working with customers to employ targeted sequencing solutions to help with surveillance efforts for emerging strains," he said.

The Waltham, Massachusetts-based firm has commercialized several kits for COVID-19 diagnostic testing. The TaqPath COVID-19 Combo Kit and Combo Kit Advanced both have EUA, while the TaqPath COVID-19 CE-IVD RT-PCR kit and the TaqPath COVID-19, Flu A/B, RSV Combo Kit are CE marked. The firm also offers research-use-only kits, including the TaqCheck SARS-CoV-2 Fast PCR Assay for coronavirus surveillance testing; a multiplex kit for SARS-CoV-2, influenza A, and influenza B; and a multiplex kit for SARS-CoV-2, influenza A/B, and respiratory syncytial virus.

"Not all the Thermo Fisher tests experience the S-gene dropout," Gandhi said, as only the two EUA kits and the CE-IVD RT-PCR kit utilize the S gene as one of the targets for detection of SARS-CoV-2

"Due to the multi-target design of our assays, the S gene dropout has not been found to impact test results obtained using our products, and the probability of a mutation in the S gene impacting the accuracy of our test results is low," Ghandi said, adding that the firm calculates it is less than 0.01 percent.

Thermo Fisher Scientific also currently offers the Ion AmpliSeq SARS-CoV-2 Research Panel on its Ion Torrent next-generation sequencing platforms to enable contact tracing and epidemiological studies, "which are critical to understand viral spread," Ghandi said. 

The firm has published Sanger sequencing primers and a protocol for confirmation of the S gene deletion. 

While the TaqPath COVID-19 Combo Kit is not intended for use for the detection of the 69-70 deletion mutation in SARS-CoV-2 variant strains, per se, the European CDC recommends that an S-gene dropout can be used as a signal for the presence of that mutant for further investigation, Ghandi said, especially if sequencing capacity is limited.

"As a part of our post-market commitments, Thermo Fisher Scientific continuously monitors assay performance globally and will make adjustments accordingly, either to the product labeling, product enhancements, or new product development," he added, but noted that no adaptations to the tests or changes in use are necessary at this time.

Other multi-target assays targeting the S-gene include ones from Altona Diagnostics, BioMérieux subsidiary BioFire, and Express Gene.

Stephan Ölschläger, Altona Diagnostics' head of marketing, said that the Hamburg, Germany-based firm constantly uses bioinformatics tools to confirm that the target regions in its assays are not affected by known mutations, and it shares these analyses with customers on a regular basis. 

The firm's EUA assay, called the RealStar SARS-CoV-2 RT-PCR Kit, targets the E and S genes, but Ölschläger said the S target may be in "a more conserved region" than is targeted by other assays. Altona has not gotten any feedback from customers about S gene dropouts, especially not from the UK where the variant is more prevalent, and the firm has shipped its test to 50 countries, he said.

BioFire's EUA Respiratory 2.1 Panel test targets the virus' membrane protein gene, or M gene, and the S gene.

Ashley Warcup, a global product manager at BioFire, said the firm routinely monitors emerging SARS-CoV-2 sequence data and compares it in silico against its existing SARS-CoV-2 assays for inclusivity and reactivity. 

The RP2.1 assays "have thus far demonstrated very robust inclusivity," she said. "The S-gene mutations have not been shown to adversely impact the sequences targeted by the RP2.1 SARS-CoV-2 assays, and we do not predict false negative results for specimens containing these strains." 

Intellectual property versus public health

The S-gene dropout situation has again raised discussion about disclosure of proprietary test formulations. For the SARS-CoV-2 pandemic, the issue surfaced this summer upon discovery of a dropout-causing mutation in a pan-SARS E-gene target in rare cases in Belgium.

That dropout affected assays from Roche, and the firm declined to disclose the sequence information or other formulations for the test, as is standard practice in the industry. Like with the S-gene dropouts to date, individual patient test results were not impacted by the failure of E-gene target in the multi-target test.

However, in that case as well as well as the current case, researchers have suggested that the drive to protect intellectual property can slow down labs' ability to understand discrepant results.

Thermo Fisher publicly disclosed the primer sequences for its S-gene target in late December, "so labs can sequence and identify the deletion," Ghandi said. The information is available on the firm's website. "It should also be noted that the location of the S-gene 69-70 deletion and the entire sequence around it is in the public GISAID database," he added.

Researchers have also begun developing tests to specifically target the S-gene variants. An assay to detect the B.1.1.7 variant was published by an international team in bioRxiv last week, for example.

Grubaugh and his team at Yale recently developed an RT-qPCR test targeting B.1.1.7 and they have posted a draft protocol online. They developed the test to monitor for the presence of B.1.1.7 variants and to screen samples for sequencing confirmation, Grubaugh said.

They chose not to use the TaqPath test for this purpose because they wanted to conserve resources for clinical diagnostic use. Furthermore, the S dropouts are not specific for the B.1.1.7 variant, since several other SARS-CoV-2 lineages have the same deletion that causes the dropout, he said.

The Yale protocol is publicly and freely available, and although it hasn't been fully validated yet, the team expects to be able to share validation data early next week.

"Our intention was for this to be used by public health labs to screen a subset of SARS-CoV-2-positive samples to monitor the prevalence of B.1.1.7 in the community," Grubaugh said. In addition, the "protocol and sequences are open if any company would like to incorporate them in their LDTs or EUA submissions." 

According to Grubaugh, the fact that Thermo disclosed its sequence and primer formulations but did not disclose the probe formulation led to an inability to quickly recreate the assay.

"While it isn't difficult to design new probes, we know that the Thermo Fisher probe is already validated for the detection of a specific deletion," Grubaugh said. "It is surprising that the company continues to not be completely helpful during a public health emergency," he said, adding, "I would hope that other companies would do better."

Greninger at UW noted that Thermo Fisher may be a bit of a disadvantage as a manufacturer of reagent kits, since its tests are "eminently jailbreakable if sequences are available."

That said, "The virus does move and, especially when those sequences are baked into a sample-to-answer instrument like a Roche, Cepheid, Hologic, Abbott ... I think you can more easily argue that those [primers and probes] could be available, since it's the fluidics that are more of the special sauce," Greninger said.

He himself initially encountered this issue while investigating aberrant lab results that were caused by herpes simplex virus fusions. "We couldn't figure out where the primers and probes were for pretty much all HSV commercial assays. [Manufacturers] would just tell you the gene it's in," Greninger said. "It's a CAP requirement to recheck your primers every year to look for drift, which is not possible with these FDA-[authorized] tests," he added.

Although currently it's hard to make an argument that a lack of primer and probe information is a major public health issue, "Overall, I agree with those looking for more transparency," Greninger said.

Altona's Ölschläger said that his firm does not have a contingency plan if an important mutation began to cause dropouts for its test.

"To be honest we have not considered such a case, but I am very positive that we would share the data if we learned that this would be of help for public health," he said. The firm also shares its sequence data under non-disclosure agreements with reference laboratories and specific customers on a regular basis.

And, perhaps luckily, the SARS-CoV-2 genome seems to be a bit more stable than other pathogens, he said.

"From an objective viewpoint, there is really no need to be particularly concerned about SARS-CoV-2 diagnostics, as long as two targets are used," Ölschläger said.