COVID-19 Testing: New Highly Accurate And Sensitive COVID-19 Direct RT-qPCR Test That Does Not Need Scarce Reagents

COVID-19 Testing: Researchers from University of Vermont, working in collaboration with a group from the University of Washington, have developed a platform of testing for the COVID-19 disease that does not make use of key chemicals, or reagents but still delivers an accurate result, paving the way for inexpensive, widely available testing in both developing countries and industrialized nations like the United States, where reagent supplies are again in short supply.


Dr Jason Botten & Dr Emily Bruce, who pioneered a streamlined COVID-19 test that doesn't
use scarce chemicals, in their research lab in the University of Vermont's Larner College of Medicine.
Credit: Brian Jenkins

Typically a major roadblock to large scale testing for SARS-CoV-2 infection in the developing world is a shortage of key chemicals, or reagents, needed for the test, specifically the ones used to extract the virus's genetic material, or RNA. Hence this new direct approach would be a great relief to many health authorities and institutions.
 
The current ongoing COVID-19 pandemic has created an unprecedented need for rapid diagnostic testing. The World Health Organization (WHO) recommends a standard assay that includes an RNA extraction step from a nasopharyngeal (NP) swab followed by reverse transcription–quantitative polymerase chain reaction (RT-qPCR) to detect the purified SARS-CoV-2 RNA.
 
The current global shortage of RNA extraction kits has caused a severe bottleneck to COVID-19 testing. The goal of this research was to determine whether SARS-CoV-2 RNA could be detected from NP samples via a direct RT-qPCR assay that omits the RNA extraction step altogether. The direct RT-qPCR approach correctly identified 92% of a reference set of blinded NP samples (n = 155) demonstrated to be positive for SARS-CoV-2 RNA by traditional clinical diagnostic RT-qPCR that included an RNA extraction. Importantly, the direct method had sufficient sensitivity to reliably detect those patients with viral loads that correlate with the presence of infectious virus.
 
Hence, this strategy has the potential to ease supply choke points to substantially expand COVID-19 testing and screening capacity and should be applicable throughout the world.
 
The research findings are published in the peer-reviewed journal: PLOS Biology https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3000896
 
The new diagnostic platform omits the step in the widely used reverse transcription polymerase chain reaction (RT-PCR) test where the scarce reagents are needed.
 
The sensitivity and accuracy of the new diagnostic test was evaluated by a team of researchers at the University of Washington led by Dr Keith Jerome, director of the university's Molecular Virology Lab, using 215 COVID-19 samples that RT-PCR tests had shown were positive, with a range of viral loads, and 30 that were negative.
 
 The new diagnostic platform correctly identified 92% of the po sitive samples and 100% of the negatives.
 
However the positive samples the new test failed to catch had very low levels of the virus.
 
Strangely public health experts increasingly believe that ultra-sensitive tests that identify individuals with even the smallest viral loads are not needed to slow spread of the disease.
 
Dr Jason Botten, an expert on pathogenic RNA viruses at the University of Vermont's Larner College of Medicine and senior author told Thailand Medical News, "It was a very positive result." Dr Botten's colleague Dr Emily A. Bruce is the resarch's first author.
 
Dr Botten added, "You can go for the perfect test, or you can use the one that's going to pick up the great majority of individuals and stop transmission. If the game now is focused on trying to find individuals who are infectious, there's no reason why this test shouldn't be front and center, especially in developing countries where there are often limited testing programs because of reagent and other supply shortages."
 
Typically the standard PCR test has three steps, while this simpler version of the standard test has only two, Dr Botten said.
 
He explained, "In step 1 of the RT-PCR test, you take the swab with the nasal sample, clip the end and place it in a vial of liquid, or medium. Any virus on the swab will transfer from the swab into the medium. In step 2, you take a small sample of the virus-containing medium and use chemical reagents, the ones that are often in short supply, to extract the viral RNA. In step 3, you use other chemicals to greatly amplify any viral genetic material that might be there. If virus was present, you'll get a positive signal."
 
Dr Botten added,  "It takes a sample of the medium that held the nasal swab and goes directly to the third, amplification step, removing the need for scarce RNA extraction reagents as well as significantly reducing the time, labor and costs required to extract viral RNA from the medium in step 2.”
 
Dr Botten said the test is ideally suited to screening programs, in both developed and developing countries, since it is inexpensive, takes much less processing time and reliably identifies those who are likely to spread the disease.
 
The new diagnostic test has a low cost and its efficiency could extend testing capacity to groups not currently being tested, Dr Botten said, including the asymptomatic, nursing home residents, essential workers and school children.
 
The current standard RT-PCR test could be reserved for groups, like health care workers, where close to 100% accuracy is essential.
 
The new two-step test developed by the University of Vermont team first caught the attention of the scientific community in March, when preliminary results that accurately identified six positive and three negative Vermont samples were published as a preprint. The preprint was downloaded 18,000 times in its first week; it ranked 17th among 15 million papers the site had published and the abstract was viewed 40,000 times.
 
Dr Botten heard from labs around the world who had seen the preprint and wanted to learn more about the new test.
 
Examples of queries included, “I'm from Nigeria or the West Indies. We can't test, and people's lives are at stake. Can you help us?'"
 
Dr Botten also heard from Dr Syril Pettit, the director of HESI, the Health and Environmental Sciences Institute, a non-profit that marshals scientific expertise and methods to address a range of global health challenges, who had also seen the preprint.
 
Dr Pettit asked Botten to join a think tank of likeminded scientists she was organizing whose goal was to increase global testing capacity for COVID-19. The test developed by the University of Vermont and University of Washington teams would serve as a centerpiece. To catalyze a global response, the group published a call to action in EMBO Molecular Medicine. https://www.embopress.org/doi/full/10.15252/emmm.202012634
 
The response was good as the call reached out to 10 laboratories in seven countries, including Brazil, Chile, Malawi, Nigeria and Trinidad/Tobago, as well as the U.S. and France, to see if they would be interested in giving the two-step test a trial run. "Universally, the response was yes," Dr Pettit said.
 
The global outreach led to a new HESI program called PROPAGATE. Each of the labs in the PROPAGATE Network will use the two-step test on a series of positive and negative samples sent to them by the University of Washington to see if they can replicate the results the university achieved.
 
The research has already shown promising results. One of the labs in Chile has also used the test on its own samples from the community and got accurate results.
 
Dr Pettit and her colleagues at the University of Vermont and the University of Washington as well as scientists from the 10 partner sites plan to publish the results.
 
Dr Petit added, "The goal is the make the two-step test accessible to any lab in the world facing these hurdles and see a broad uptake.”
 
The study team summed up their study, “The testing approach described here could also easily be adopted in more resource limited settings, including large portions of the developing world that at present completely lack access to RNA extraction. A testing approach that uses patient samples directly without RNA extraction would open up perhaps the only viable avenue for widespread testing in these regions. In recent months the scientific community has made enormous strides towards addressing the formidable challenge of providing ample and accessible COVID-19 testing across the globe, and yet the problem is assuredly not solved. An “all-in” approach to testing in which the global scientific community continues to develop and validate a range of testing approaches, with the goal of maximizing access and affordability across the world, represents a promising avenue to address this enormous challenge.”
 
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