In January 2020, just a few weeks after the first Covid-19 cases appeared in China, the full genome of the new coronavirus was published online. Using this genomic sequence, the scientists developed a wide range of diagnostic tests for the virus.
But the virus has mutated since then. And with the development of the coronavirus, the test landscape has also evolved. The emergence of new variants has generated great interest in the development of tests for certain virus mutations and concerns about the accuracy of some existing tests.
“With this Covid diagnostic, we had a time crisis, we had to get something out of it,” said Lorraine Lillis, scientific program manager at PATH, a global non-profit health organization that has been tracking coronavirus tests. “It usually takes a long time to diagnose and we would normally challenge it with multiple variations.” She added, “And we do it, but we do it in real time.”
The Food and Drug Administration has warned that new mutations in the coronavirus could make some tests less effective. And last week, PATH launched two online dashboards to monitor how certain variants can affect the performance of existing diagnostic tests.
So far, scientists have agreed that there is no evidence that the known worrying variants will cause tests to fail completely. “The tests are working very, very well today,” said Mara Aspinall, an expert in biomedical diagnostics at Arizona State University.
But Manufacturers and regulators need to remain vigilant to keep up with an ever-changing virus, scientists say. When variants elude detection, it can be of concern not only to individual patients who may not receive the treatment they need, but also to public health.
If a test misses someone infected with a variant, that person may not realize that they need to be isolated. “And then that person must not be quarantined, circulated in the community and possibly passed this variant on to others,” said Gary Schoolnik, doctor and infectious disease expert at Stanford University and chief medical officer of Visby Medical, a diagnostics company that does a Covid-19 test. “And so, if variants are missing, a diagnostic test can actually promote the spread of this variant.”
The risk of false negative results
Molecular tests, such as the widely used polymerase chain reaction or PCR test, are used to detect specific sequences of the coronavirus genome. If mutations occur in these “target” sequences, the tests may no longer be able to detect the virus, leading to false negative results.
“You could get into a situation where you were unlucky when you decided to take your test and something came up that made your test less effective,” said Nathan Grubaugh, a virologist at Yale University.
The gene for the virus’s signature spike protein, known as the S gene, has been particularly susceptible to mutation, and tests targeting this gene may miss certain variants. For example, Thermo Fisher’s TaqPath test fails to detect the mutated S gene of the B.1.1.7 variant, which was first identified in the UK and is now rapidly spreading in the US.
However, the test is not only based on the S gene. It has three goals, yet it can still provide accurate results by detecting two more sections of the coronavirus genome.
Only 1.3 percent of molecular tests are based solely on an S-gene target. This is based on calculations made by Rachel West, a postdoctoral fellow at the Johns Hopkins Center for Health Security. The rest either target more stable regions of the genome that are less likely to mutate, or have multiple target sequences, making them less prone to failure. “It is very unlikely that you will get mutations in all,” said Dr. Lillis.
April 14, 2021, 2:24 p.m. ET
The FDA has listed four different molecular tests “the performance of which could be affected by the variants,” but states that the tests should continue to work. Three of the tests have multiple objectives; A fourth can be a little less sensitive if the virus has a particular mutation and is present in very small amounts. (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 these four tests are significantly affected,” said Dr. Tim Stenzel, who heads the FDA’s In Vitro Diagnostic and Radiological Health Office. “We published this information out of caution and transparency.”
Antigen tests are less sensitive than molecular tests, but they are usually cheaper and faster and are widely used in coronavirus screening programs. These tests detect specific proteins on the outside of the virus. Some genetic mutations could alter the structure of these proteins and allow them to evade detection.
Most antigen tests target the nucleocapsid protein. The gene that codes for this protein, known as the N gene, is more stable and mutated than the S gene, and the FDA has not listed any antigen tests as of concern. “We didn’t find any that hoisted a red flag, nor have we received any reports,” said Dr. Stenzel.
However, experts note that not every test maker discloses the specific sequences their tests target and the virus continues to mutate. “There is no evidence that any particular molecular assay, or even an antigen test, completely misses the boat for detection.” said Neha Agarwal, the assistant director of diagnostics at PATH. “But things will change.”
The FDA continues to monitor the situation and weekly reviews the coronavirus sequence databases to see if the virus is developing in a way that may help avoid diagnostic tests. “We are very vigilant,” said Dr. Stenzel. “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, such as a PCR test, is used to determine if the virus is present. If the test is positive, a sample will be sent for genomic sequencing.
“These two tasks are currently performed in two separate workflows,” said Juan Carlos Izpisua Belmonte, developmental biologist at the Salk Institute in La Jolla, California. “This means more time, work and resources.”
Many researchers are currently working on integrated solutions – tests that can be used to determine if someone is infected with the virus and whether they may have a particular variant.
For example, Dr. Izpisua Belmonte and colleague Mo Li, a stem cell biologist at King Abdullah University of Science and Technology in Saudi Arabia, described a new test method that can be identified in a recent article Mutations in up to five different regions of the coronavirus genome.
And Dr. Grubaugh and his colleagues have developed a PCR test that can be used to detect certain combinations of mutations that characterize three questionable variants: B.1.1.7; B.1,351, which was first discovered in South Africa; and P.1, first found in Brazil. (The work has not yet been published in a scientific journal.)
Dr. Grubaugh said researchers in Brazil, South Africa and elsewhere are already using the tests to sift through a mountain of coronavirus samples and identify those that should be prioritized for full genomic sequencing. “The main interest of our group is to improve genomic surveillance through sequencing, especially in areas with limited resources,” said Dr. Grubaugh. “If you want to know if there are variants, you need a way of triage.”
A number of companies are also starting to publish coronavirus tests that they say can differentiate between certain variants, although these are for research purposes only. It is “infinitely more difficult” to create a test that can definitely diagnose someone with a particular variant, said Dr. Grubaugh.
Similar mutations come in different variants, making it difficult to differentiate between them. The mutations of interest change with the virus, and sequencing remains the best way to get a complete picture of the virus.
But tests that can look for specific mutations could be an important public health tool, Ms. Agarwal said: “These newer diagnoses that examine the variants will be really crucial to understanding the epidemiology of the virus and I think our next generation plan the efforts against it. “