Two months into the pandemic, the number of COVID-19 molecular diagnostic tests across the U.S. remains woefully inadequate. More than 4 million coronavirus tests had been conducted in total as of Monday and about 150,000 per day in the last couple of weeks, according to some accounts.
Experts say this is not nearly enough to track new infections adequately, which would be needed to gradually lift stay-at-home orders and other restrictions on public life and businesses. While there are several reasons for the continuing testing shortage, one challenge has been a shortage of reagents, in particular RNA extraction kits. Many SARS-CoV-2 assays also are difficult to perform in high throughput to allow for a real boost in test numbers.
Such kit shortages may not affect tests from vendors that sell fully automated systems like Roche, Cepheid, Hologic, Abbott, or GenMark, but they do apply to the dozens of labs around the country that have developed their own tests or run the Centers for Disease Control and Prevention's assay.
To try to relieve this bottleneck, several labs and at least one company have been working on assays that add samples directly to the PCR reaction and don't require RNA extraction, a time-consuming step.
A group at the University of Vermont College of Medicine and their collaborators at the University of Washington, for example, published a paper this month in which they described an RNA-extraction-free SARS-CoV-2 test. Validating it on 155 known positive nasopharyngeal samples, they found that it correctly called 92%of them.
Jason Botten, an associate professor at the Vermont Center for Immunology and Infectious Diseases and co-corresponding author of the study said his virology research lab didn't expect to get into COVID-19 PCR diagnostics but wanted to help testing labs in Vermont that were short of RNA extraction kits.
"During the discussions, a couple of us said, 'What if we just exclude the extraction step?'," he recalled. Doing this would not only eliminate the kit shortage but also speed up the test. "This extraction step is the most limiting step of the whole assay," he said. "It takes more time to run the extractions than to do the downstream PCR."
Within less than a week, his team designed an RNA extraction-free assay, tested it on two samples from positive patients to show that it works, and posted the results. After testing it on a few more samples, his group teamed up with Keith Jerome, who runs a clinical testing lab at UW, and validated the assay with 150 additional samples from patients with high, medium, and low viral loads. They found that the only samples the test missed had low levels of virus, a category that about a quarter of all samples that tested positive at UW fell into.
In the meantime, at least two other groups, based in Chile and Denmark, have independently reported similar RNA-extraction-free protocols, and one of them showed it also worked with nasal swabs.
Botten said he could envision the RNA-extraction-free assay being used as a screening tool that could pick up the great majority of people who are virus-positive while saving time and labor, and not requiring extraction kits. Individuals who test negative but are suspected of having the disease would need to be retested with an assay that does use RNA extraction, he added.
Testing labs in Vermont ultimately did not adopt his protocol, he said, because they were able to start using large automated systems, for which they could obtain sufficient reagent kits. Likewise, the UW collaborators have not seen the need to adopt the assay yet.
However, Botten has received dozens of emails from labs around the world saying they could not start testing because of a lack of RNA extraction kits. "I think the real benefit of this might be in all of the areas in the world that just don't have access to these kits or don't have access to these really high-throughput automated machines to do the testing for them," Botten said.
"You are going to want as many of your big, automated, robust systems as you can have online, but that's only going to cover a certain amount [of labs]," he said. "For everybody else, which might be the majority, you're going to need something as simple as you can make, so that all these constraints are removed and people can just rapidly test."
The assay could also be further optimized to increase its sensitivity, he said, for example by using less swab medium, which would increase the concentration of viral particles in the sample.
Nathan Ledeboer, medical director of the microbiology laboratory at the Medical College of Wisconsin, agreed that RNA extraction "is a significant challenge for clinical laboratories" doing SARS-CoV-2 testing, at least for those that run their own lab-developed tests or the CDC test, rather than fully automated systems.
His own lab started out with the CDC assay and was initially unable to get RNA extraction reagents for its two extraction systems. Other labs across the country, he said, had the same experience.
"The fact that in this particular study, they were able to demonstrate a very high degree of sensitivity without having to do extraction is a really a promising alternative to having to extract, particularly as those reagents are very, very limited," he said.
While the extraction kit shortage has improved somewhat over time, he said, there is still a need to test more people and "as we start to expand that testing capability, we will see additional stress being put on the system."
While more and more labs appear to be shifting away from lab-developed tests and towards commercial platforms, Ledeboer said, and manufacturers are ramping up production, "there is still a tremendous need and the vendors can't entirely supply the number of tests that are needed."
Labs that want to adopt the RNA-extraction-free protocol, he said, would need to validate it internally and submit it to the Food and Drug Administration as a modification to their current emergency use authorization.
Meanwhile, other labs have been exploring ways to relieve coronavirus testing bottlenecks. One lab at Rutgers University, for example, recently started using saliva samples as input material because they are much easier to collect than nasopharyngeal swabs and don't require swabs and viral transport medium, which have also become scarce. However, a lack of time to validate modifications like these in their own labs might be holding others back from implementing some of these newer workflows.
"As the first wave of the outbreak begins to slow down, many of us that would otherwise be doing those studies will get the time to breathe and be able to start thinking about doing that," Ledeboer said. "One of the challenges up until now has really been, clinical laboratories have been running full throttle, if you will, focused on being able to provide as many tests as they can, and as broadly as they can. That has largely been driven by the very rapid expansion of the number of cases that we're detecting. As we start to see this first phase begin to slow down and wane, we really have the opportunity to begin those studies, doing them in well-designed fashions so we can get useful viable data."
This story first appeared in our sister publication, Genomeweb.