In January 2020, just weeks after the first Covid-19 cases emerged in China, the full genome of the new coronavirus was published online. Using this genomic sequence, scientists scrambled to design a large assortment of diagnostic tests for the virus.
But the virus has mutated since then. And as the coronavirus has evolved, so has the landscape of testing. The emergence of new variants has sparked a flurry of interest in developing tests for specific viral mutations and prompted concerns about the accuracy of some existing tests.
“With these Covid diagnostics, we were on a time crunch, we had to get something out there,” said Lorraine Lillis, the scientific program officer at PATH, a global health nonprofit that has been tracking coronavirus tests. “Normally, diagnostics take a long, long time, and we’d normally challenge them with multiple variants.” She added: “And we’re doing that, but we’re doing it in real time.”
The Food and Drug Administration has warned that new mutations in the coronavirus could render some tests less effective. And last week, PATH launched two online dashboards to monitor how certain variants might affect the performance of existing diagnostic tests.
So far, scientists have agreed, there is no evidence that the known variants of concern are causing tests to fail completely. “The tests today work very, very well,” said Mara Aspinall, an expert in biomedical diagnostics at Arizona State University.
But manufacturers and regulators will need to remain vigilant to ensure they keep pace with a constantly changing virus, scientists say. If variants begin to evade detection, that could be consequential not only for individual patients, who may not receive the treatment they need, but also for public health.
If a test misses someone who is infected by a variant, then that person may not realize they need to isolate. “And that person is allowed then to be unquarantined, to circulate in the community and possibly spread that variant to others,” said Gary Schoolnik, a physician and infectious disease expert at Stanford University and the chief medical officer of Visby Medical, a diagnostics company that makes a Covid-19 test. “And that’s how a diagnostic test, if it’s missing variants, can actually promote the spread of that variant.”
The risk of false negatives
Molecular tests, like the widely used polymerase chain reaction, or P.C.R., test, are designed to detect specific sequences of the coronavirus genome. If mutations appear in these “target” sequences, the tests may no longer be able to detect the virus, yielding false negatives.
“You could run into a situation where you just got unlucky with where you chose to target your test, and something popped up there that then made your test less effective,” said Nathan Grubaugh, a virologist at Yale University.
The gene for the virus’s characteristic spike protein, known as the S gene, has been particularly prone to mutation, and tests that target this gene may miss certain variants. For instance, Thermo Fisher’s TaqPath test fails to detect the mutated S gene of the B.1.1.7 variant, which was first identified in Britain and is now spreading rapidly through the United States.
But the test does not rely on the S gene alone; it has three targets and can still return accurate results by detecting two other stretches of the coronavirus genome.
Just 1.3 percent of molecular tests rely solely on an S gene target, according to calculations performed by Rachel West, a postdoctoral associate at the Johns Hopkins Center for Health Security. The rest either target more stable regions of the genome, which are less likely to mutate, or have multiple target sequences, which makes them less susceptible to failure. “It’s very unlikely that you’re going to get mutations in all of them,” Dr. Lillis said.
The F.D.A. has listed four different molecular tests “whose performance could be impacted” by the variants, but notes that the tests should still work. Three of the tests have multiple targets; a fourth may be slightly less sensitive when the virus has one particular mutation and is present at very low levels. (The four tests are the TaqPath Covid-19 Combo Kit, the Linea Covid-19 Assay Kit, the Xpert Xpress and Xpert Omni SARS-CoV-2, and the Accula SARS-CoV-2 Test.)
“We don’t think that those four assays are significantly impacted,” said Dr. Tim Stenzel, who directs the F.D.A.’s office of in vitro diagnostics and radiological health. “It was more out of an abundance of caution and transparency that we made that information public.”
Antigen tests are less sensitive than molecular tests, but they are typically cheaper and faster, and they are being deployed widely in coronavirus screening programs. These tests detect specific proteins on the outside of the virus. Some genetic mutations could change the structure of these proteins, allowing them to escape detection.
Most antigen tests target the nucleocapsid protein. The gene that codes for this protein, known as the N gene, is more stable and less likely to mutate than the S gene, and the F.D.A. has not listed any antigen tests as being of concern. “We haven’t found one that raises a red flag nor have we had any reports of such,” Dr. Stenzel said.
Still, experts note, not every test manufacturer discloses the specific sequences that their tests target, and the virus will continue to mutate. “There hasn’t been any evidence to show that a particular molecular assay or even an antigen test completely misses the boat in terms of detection,” said Neha Agarwal, the associate director of diagnostics at PATH. “But things are going to change.”
The F.D.A. is continuing to monitor the situation, checking coronavirus sequence databases weekly to see if the virus is evolving in ways that may help it evade diagnostic tests. “We’re being very vigilant,” Dr. Stenzel said. “And we will stay vigilant.”
Screening for specific variants
As the variants spread, researchers are also working to develop and improve tests to detect them. At the moment, identifying a variant is typically a two-step process. First, a standard coronavirus test, like a P.C.R. test, is used to determine whether the virus is present. If the test comes back positive, a sample is then sent for genomic sequencing.
“These two tasks are currently done in two separate workflows,” said Juan Carlos Izpisua Belmonte, a developmental biologist at the Salk Institute in La Jolla, Calif. “This means more time, labor and resources.”
Many researchers are now working to create integrated solutions — tests that can determine both whether someone is infected with the virus and whether they might have a particular variant.
For instance, in a recent paper, Dr. Izpisua Belmonte and his colleague, Mo Li, a stem cell biologist at King Abdullah University of Science and Technology in Saudi Arabia, described a new testing method that can identify mutations in up to five different regions of the coronavirus genome.
And Dr. Grubaugh and his colleagues have developed a P.C.R. test that can detect specific combinations of mutations that characterize three variants of concern: B.1.1.7; B.1.351, which was first detected in South Africa; and P.1, first found in Brazil. (The work has not yet been published in a scientific journal.)
Dr. Grubaugh said that researchers in Brazil, South Africa and elsewhere are already using the tests to sift through a mountain of coronavirus samples, identifying those that should be prioritized for full genomic sequencing. “Our group’s primary interest is enhancing genomic surveillance through sequencing, especially in resource-limited areas,” Dr. Grubaugh said. “If you want to know if there’s variants that are circulating, you need a way to triage.”
A number of companies are also beginning to release coronavirus tests that they say can differentiate between certain variants, although these are intended for research purposes only. Creating a test that can definitively diagnose someone with a particular variant is “infinitely harder,” Dr. Grubaugh said.
Similar mutations are springing up in different variants, which makes distinguishing among them more difficult. The mutations of interest will change as the virus does, and sequencing remains the best way to get a complete picture of the virus.
But tests that can screen for certain mutations could be an important public health tool, Ms. Agarwal said: “These newer diagnostics that are looking across the variants, I think will be really key in understanding the epidemiology of the virus and planning our next generation of efforts against it.”