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The Impact of Emerging SARS-CoV-2 Variants on Diagnostic Testing

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Like other viruses, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can mutate and evolve over time, leading to genetic variants such as B.1.1.7, first identified in the UK in December 2020. In most cases, the tiny genetic changes “end up being inconsequential,” Arvind Kothandaraman from PerkinElmer told Technology Networks. However, “there are other cases in which these mutations could lead to the virus becoming more infectious and threatening to the host,” he added.

SARS-CoV-2 variants are continually monitored by the World Health Organization (WHO) SARS-CoV-2 Virus Evolution Working Group and several national authorities, to enable the rapid identification of relevant mutations and study their potential impacts on viral transmissibility and disease severity. The effectiveness of current countermeasures, such as diagnostics, therapeutics and vaccines is also evaluated, as well as investigations into how negative effects could be reduced.

Studies of the B.1.1.7 variant found that the lineage contains multiple mutations in the gene that codes for the spike (S) protein, which could impact the effectiveness of some diagnostic tests. “The 69–70 deletion in S gene of the UK [B.1.1.7] variant has been reported to cause a negative result owing to a complete dropout of the genetic region targeted by certain RT-PCR assays,” Kothandaraman said.

What is an RT-PCR test?

RT-PCR stands for reverse transcription polymerase chain reaction. It is a molecular method widely used to detect genetic material from viruses and other pathogens.

“An RT-PCR test is the gold standard for COVID-19 diagnostics, as this molecular assay offers a highly reliable method to detect the presence of SARS-CoV-2 in a human sample,” commented Kothandaraman.

Samples for testing are obtained via an oropharyngeal or nasopharyngeal swab, or saliva. Any SARS-CoV-2 genetic material present in the sample is isolated and the RNA is converted to complimentary DNA by reverse transcription. Primers targeting nucleic acid sequences specific to the SARS-CoV-2 genome are used to define the region of the DNA that will be amplified.

On January 8, the US Food and Drug Administration (FDA) issued an alert to clinical laboratory staff and healthcare providers highlighting “that false negative results may occur with any molecular test for the detection of SARS-CoV-2 if a mutation occurs in the part of the virus’ genome assessed by that test.”

For the B.1.1.7 variant, the main area of concern are tests that target the S gene. “Labs using an assay that targets the spike protein should review performance closely and watch for false-negative results,” commented Kothandaraman. Tests that target other genomic regions of the virus such as the ORF1ab gene and envelope (E) gene are not affected by S gene mutations and so their performance against the B.1.1.7 variant should not be reduced.

Since the majority of SARS-CoV-2 PCR tests use multiple genetic targets rather than a single target, the impact of the B.1.1.7 variant on diagnostics is not anticipated by the WHO to be significant. It has also been suggested by the FDA that the detection pattern on tests which do target the S gene, in addition to other targets, could help with early identification of new variants in patients. Samples demonstrating an S gene dropout could then be further characterized by genetic sequencing.

What about the impact of the variant on other diagnostic methods, such as those used in rapid mass screening? Most antigen tests target the C-terminus of the N protein, encoded by the N gene, so should be minimally impacted. Public Health England reports that evaluations confirm that lateral flow devices are effective in detecting the B.1.1.7 variant, although all devices tested work by detecting the N rather than the S protein. The performance of antibody assays has not yet been evaluated, but assays detecting antibodies to the S protein could potentially be affected.

In addition to B.1.1.7, other SARS-CoV-2 variants are circulating globally, including B.1.351, first identified in South Africa, and P.1, first identified in four travelers from Brazil. It is likely that further variants of interest will emerge, which could see the introduction of mutations in other areas of the genome that may impact the performance of current diagnostic approaches and other countermeasures. Many diagnostic providers are actively monitoring SARS-CoV-2 variants to ensure their tests remain effective, both by examining the viral genetic sequence and testing real-world samples.

“The UK and South African variants are just two instances of viral evolution. The scientific community will continue to track mutations, study viral evolution and adjust tests, treatments and vaccines accordingly,” Kothandaraman concluded.

Arvind Kothandaraman, General Manager, Specialty Diagnostics at PerkinElmer, was speaking to Anna MacDonald, Science Writer for Technology Networks.