COVID-19 Variants—Diagnostics and Surveillance

Several variants of concern are circulating in the US—labs must remain vigilant of variants that affect testing

Suzanne Leech, PhD

As the coronavirus pandemic continues to threaten lives worldwide, researchers and clinicians are closely monitoring SARS-CoV-2 variants over concerns that mutations could lead to reduced protection from convalescent immunity or COVID-19 vaccines and false positive test results.

Viruses with RNA-based genomes, including SARS-CoV-2, are known to have high mutation rates. Although SARS-CoV-2 is not as mutationally active as some RNA viruses (it mutates approximately half as quickly as influenza), the emergence of variants of this deadly pathogen has been a serious concern. 

So far, almost 29,000 mutations have been identified in the SARS-CoV-2 genome. Although the vast majority are unlikely to impact human health, some can. For example,  S-gene mutations alter the virus’s spike (S) protein, which can influence cell receptor and antibody binding. S-gene mutations can change the virus’s infectivity, transmission potential, and capacity to evade post-infection immunity and vaccine defenses, as well as affect visibility in diagnostic tests that target the S protein or gene. 

Several variants of concern (VOCs)—including B.1.1.7, a variant first isolated in the UK; B.1.351, first reported in South Africa; P.1, a B.1.1.28-lineage variant that was first isolated from Brazilian travelers in Japan; and the B.1.427 and B.1.429 variants detected in Southern California, among others—have been confirmed to be circulating in the US. In May 2021, the CDC estimated that 66 percent of patient specimens contained the B.1.1.7 variant.

Last month, researchers at Duke University in Durham, North Carolina, examined how well the SARS-CoV-2 mutant B.1.429 and B.1.351 variants were neutralized by antibodies from people who were immunized with the Moderna or Novavax vaccines or had recovered from COVID-19. They reported that B.1.429 was two to three times less likely, and B.1.351 was nine to 14 times less likely, to be inhibited by antibodies from the Moderna-vaccinated, Novavax-vaccinated, and recovered groups than a variant isolated earlier in the pandemic. 

Some preliminary trials have found vaccines to be highly effective against the B.1.1.7 variant, e.g., in Israel, where the prevalence of B.1.1.7 was estimated to be 94.5 percent at the time, the Pfizer–BioNTech mRNA COVID-19 vaccine BNT162b2 was found to have 95.3 percent efficacy. However, the AstraZeneca vaccine, ChAdOx1 nCoV-19 (AZD1222), showed 70.4 percent clinical efficacy against B.1.1.7 compared with 81.5 percent for non-B.1.1.7 variants. In a May 5 press release, Moderna Inc. announced preliminary findings from the Phase 2 trial of the protective efficacy of mRNA-1273 and mRNA-1273.351 (B.1.351-specific) booster vaccines against B.1.351 and P.1 in previously vaccinated individuals. Antibody titers against the B.1.351 and P.1 variants increased to levels equal to, or higher than, those raised to the ancestral variant D614G after primary vaccination. 

In vaccine research funded by the National Institutes of Health, two out of 417 participants from the Rockefeller University campus, NY, developed symptoms of COVID-19 and tested positive for SARS-CoV-2 after they were fully vaccinated with the Pfizer–BioNTech or Moderna vaccine. The vaccines appeared to induce adequate immune responses; however, sequencing revealed the presence of unrecognized SARS-CoV-2 variants. Researchers reported these as breakthrough cases, as the vaccines are not 100 percent effective.

The impact of variants on COVID-19 testing

The two main types of SARS-CoV-2 diagnostics are lateral flow and molecular (PCR) tests. The accuracy of both can be impacted by mutations in the SARS-CoV-2 genome, depending on the mutation site and test design. Mutations have been found throughout the RNA genome of SARS-CoV-2, with a number of mutation hotspots have been identified, including sites encoding nucleocapsid phosphoprotein, S protein, and RNA-dependent RNA polymerase (an important component of replication/transcription). Molecular tests, which are highly specific for a target RNA sequence, are vulnerable to changes in the genome. Antibody tests that recognize specific protein antigens (or antibodies in the case of serological tests) can also be affected by changes to the protein structure and amino acid sequence. 

Lateral flow immunological tests are considered to be less accurate, in general, than molecular tests, although sensitivity and specificity vary widely among brands. The main benefits of lateral flow assays are that they work well for people with high viral burdens, and their speed and ease of use at home, clinics, and testing stations means that those most likely to become gravely ill and transmit the virus to others can be identified and quickly isolated. Early evidence of the ability of some lateral flow assays to detect the most prevalent new variants has been encouraging. 

"Apart from diagnostic purposes, molecular tests can be designed to specifically detect known variants and can, therefore, be used in surveillance efforts and to alert clinicians to possible outbreaks of new forms of the virus."

So far, all of the important variants have S gene mutations that confer cell-invading advantages to the virus. The manufacturers of lateral flow tests that detect antigens of or antibodies to the SARS-CoV-2 nucleocapsid, a structural protein that forms a complex with the viral RNA, claim their tests are resilient to common S gene mutations. Abbot Diagnostics, which provides the FDA-approved BinaxNOWTM COVID-19 Ag Card, regularly validates the assay through its variant surveillance program and is confident of the test’s ability to detect emerging variants. The CEO of Cellex agrees that their product, the Cellex qSARS-CoV-2 Antigen Rapid Test, also approved for use in the US, is likely to remain sensitive to the B.1.1.7 (UK), B.1.351 (South Africa), and P.1 (Brazil) variants, as it targets the nucleocapsid (N) protein. However, a study showed that mutations can occur throughout the genome and the nucleocapsid gene is actually a mutation hotspot. Therefore, vigilance is needed to ensure that tests designed to all protein targets, not just S protein antigens, are sensitive to emerging variants.

On June 3, the FDA advised caution when interpreting the outcomes of four commercial molecular tests. For example, the FDA said the Linea COVID-19 Assay Kit (Applied DNA Sciences, Inc.) and the TaqPath COVID-19 Combo Kit (Thermo Fisher Scientific, Inc.) could be impacted by a mutation of the B.1.1.7 variant. However, all four kits are designed to detect multiple genomic sites, and the FDA stresses that molecular tests that target multiple genomic markers are more resilient to the increase in circulating variants. 

Apart from diagnostic purposes, molecular tests can be designed to specifically detect known variants and can, therefore, be used in surveillance efforts and to alert clinicians to possible outbreaks of new forms of the virus. For example, the Cobas SARS-CoV-2 Variant Set 1 Test by Roche can determine the presence of B.1.1.7, B.1.351, and P.1.

Tracking variants of interest and concern

The SARS-CoV-2 Interagency Group, involving collaborations among the CDC, National Institutes of Health, FDA, Biomedical Advanced Research and Development Authority, and Department of Defense, closely monitors the emergence of variants and their significance to human health. Variants are classified by the US government into variants of interest (VOIs), VOCs, and variants of high consequence. 

VOI mutations are related to changes to receptor binding, a lower susceptibility to antibodies and treatments, or reduced visibility in tests, or are predicted to have increased transmission rates or virulence. VOIs in the US include P.2, B.1.525, and B.1.526, the latter of which was first detected in New York. When there is evidence that mutations increase the speed of transmission or disease severity, significantly reduce antibody neutralization or treatment efficacy, or increase the likelihood of false negative test results, the virus is considered a VOC. According to the CDC, five VOCs are circulating in the US, including B.1.1.7. Fortunately, there have been no reports of variants of high consequence—those that cause a severe disease course and for which current testing and treatment strategies are known to be ineffective—in the US or worldwide. 

"According to the CDC, five VOCs are circulating in the US, including B.1.1.7. Fortunately, there have been no reports of variants of high consequence."

There are reports of variants emerging that carry combinations of mutations seen in older variants. This may occur by chance or through recombination events, when two viral variants merge to form a new variant with mutations from both. A recent VOC, variant B.1.618, which has been implicated in the severe epidemic currently affecting India, may have emerged through several recombination events, although this is still speculation. B.1.618 carries both the E484K and D614G mutations of the B.1.1.7 and B.1.351 variants, as well as two amino acid deletions of the spike protein gene. However, there have been no reports to date of B.1.618 in the US.

The CDC leads the National SARS-CoV-2 Strain Surveillance system and enlists the help of federal laboratories, public health laboratories, private corporations, academic institutions, and non-profit health and research laboratories via the SARS-CoV-2 Sequencing for Public Health Emergency Response Epidemiology and Surveillance (SPHERES) scheme to continually sequence SARS-CoV-2 samples and provide information on mutations to public-health response teams. 

SPHERES is still operating at a relatively small scale, as the majority of the sequencing is performed by smaller academic laboratories, rather than larger genomic centers; however, there are hopes that increased federal resources will be allocated to these efforts. Laboratories working to sequence as many samples as possible are finding it difficult to obtain sufficient patient specimens, as diagnostic laboratories tend to dispose of samples after testing or have concerns over patient confidentiality or the regulations governing the dissemination and use of patient specimens. Therefore, the CDC is working to increase coordination between diagnostic teams and sequencing laboratories to address these issues.

The emergence of new COVID-19 variants is placing extra pressure on health systems, epidemiological efforts, and diagnostic teams. Therefore, stringent prevention and control measures must be maintained. It is imperative that laboratories remain vigilant of changes that may affect SARS-CoV-2 testing and use a diversity of tests combining multiple targets to avoid false negative results when confirming diagnoses.