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Brown University Researchers Develop RNA-Based Breath Test for COVID-19

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NEW YORK – Differing from more traditional diagnostic tests that detect metabolic changes in breath, a research team at Brown University has developed a method that relies on viral RNA detection to diagnose SARS-CoV-2.

Breath tests developed by companies like Canary Health Technologies, Owlstone Medical, Avisa Dx, and Breathonix feature methods such as mass spectrometry, gas chromatography, and biosensors that measure volatile organic compounds and biomarkers that indicate the presence of a virus or disease.

Belgian R&D firm Imec is also working on a SARS-CoV-2 test using silicon chips to collect aerosols and droplets that are then placed into a qPCR machine.

Many of these companies have developed tests for SARS-CoV-2, including Canary Health Technologies and Imec, although none have received Emergency Use Authorization from the US Food and Drug Administration.

But the research team at Brown, led by biology professor William Fairbrother, has developed a unique method for detecting SARS-CoV-2 in breath that relies on an enzymatic reaction mixture to detect viral RNA and convert it to DNA, where it can be measured using a PCR instrument.

In a paper published in late September in the Journal of Molecular Diagnostics, Fairbrother's team laid out the development of the Bubbler — a device that not only reverse transcribes RNA from airborne virus particles into DNA to be tested via PCR but can also barcode that DNA, allowing samples to be linked directly to the patient they've come from and be used for sequencing.

That sequencing use is another facet of the Bubbler, beyond its diagnostic use to determine whether a sample is positive or negative, Fairbrother said in an interview.

Within the Bubbler, a sample is collected by a patient breathing into the device for 10 seconds, which creates the bubbling sound the device is named for. Then, the enzymatic reaction and mineral oil mixture developed by the team is activated, transcribing the RNA into DNA that is then loaded into a conventional PCR instrument, where a positive or negative result is returned.

The mineral oil is used to show a visual reaction that the mixture has been discharged, as it gets cloudy when it interacts with the breath sample, Fairbrother said.

Fairbrother was determined to develop a less invasive test that doesn't require a nasal swab after seeing the struggles children had tolerating the swab, he said. It "can be quite a distressing experience" for kids, he said.

And when looking at the SARS-CoV-2 pandemic, he said he saw a "classic RNA biology problem" that he could potentially contribute a solution to.

He noted that viral RNA can be found in cells for months after the virus is active, meaning a nasal swab that relies on detecting RNA in cells doesn't necessarily mean the person has an active infection. The breath test his team developed, however, measures only the active viral particles, so it can provide a more accurate portrait of whether a person is contagious.

"If you have been infected with COVID, the RNA can persist in your cells for a long time," he said. A swab test "is not really an indication of an active infection … because you may have already recovered from COVID," he added.

Beyond the basic determination of a positive or negative result, the team decided to add barcode primers to the reaction mixture that would append a random nucleotide signature to the patient's DNA, allowing someone to analyze test kits "en masse" and figure out which sample belongs to which patient, Fairbrother said.

The benefit of this technology is the ability to use it with pooled testing, he said. Traditional pooled testing requires a positive pool to have each sample retested to determine which sample is positive.

With the unique barcode, a user could tell which specific sample or samples were positive, requiring no retesting and offering the chance to do more high-throughput, quicker testing. It can also determine which strain of the virus a person has, he said.

In developing the barcode technique, his lab raised the question "How can we pool samples together but still retain their identity so we can go back and see which ones are positive?" Fairbrother said.

"During the beginning of the pandemic, there was a huge problem with throughput," Fairbrother said. "One of the things that we wanted to do was, anticipating another event like this, is [ask] are there ways to be able to analyze thousands of kits in one reaction?"

However, in order to see the specific barcode, sequencing is required, he said. The specific kits could work in areas where high-throughput testing is a necessity, although Fairbrother noted that the testing turnaround time would still be impacted by both the speed of a PCR instrument, which could take multiple hours to provide results, and the sequencing, which adds another nine hours to get results.

Comparable to nasal swabs?

In the clinical trial from the September paper Fairbrother and his team published, 70 patients were tested using the Bubbler, with results compared to samples obtained from nasopharyngeal swabs and saliva or mouth scrapes.

Subjects for the trial provided a breath sample and two tongue scrapings that served as controls, along with the nasopharyngeal sample originally collected by the hospital. Sensitivity of the Bubbler compared to the nasopharyngeal swab was 89 percent, and specificity was 82 percent.

"The novelty is treating human breath as a new bio-sample," he said. "We do swabs, we do blood samples, but now we're introducing human breath."

Fairbrother said he believes the Bubbler and its related PCR test are comparable to or cheaper than tests using nasopharyngeal swabs, since there's no RNA extraction or stabilization needed.

Another potential application for the device is using it as an attachment to a hospital ventilator to monitor someone's COVID-19 levels from the intensive care unit, Fairbrother added. It can also help predict damage done to the lungs that can be seen in X-rays and be used for environmental testing, applications further laid out in the JMD paper.

Raed Dweik, the chair of the Cleveland Clinic's Respiratory Institute who has also developed a breath test using mass spectrometry and pattern recognition and is now working on a similar device to the Bubbler, said he considers the Bubbler a "novel way of collecting samples of breath."

Breath contains multiple components, including the gas, or volatile organic compounds, fluid, and droplets, and the Brown test — as well as the device Dweik is developing — is trying to find ways to capture the droplets, he said. One issue with condensate is that researchers aren't sure where it's coming from in the body, but that isn't a concern for COVID-19 testing, he said.

While he thinks the test has to be verified by other researchers, he said the concept is viable and it's another way to get a noninvasive sample. And with more ways to sample patients, there are "more ways to make COVID tests available sooner," he said.

The key difference between the Bubbler and other breathalyzer tests are that other tests are using metabolites to measure the body's reaction to the virus, Fairbrother said.

Currently, the Brown team has a patent application in the works and is looking to license the test to a partner that could help commercialize it, with "some leads" on partners right now, Fairbrother said.

He added that the researchers are also looking to get FDA EUA and are "partway through the application." Data from the paper published in September followed FDA EUA protocol and would be used in the EUA application, he added.

His team also has plans to develop tests for other viruses, including a respiratory panel. To develop a respiratory panel, Fairbrother said the primers in the reaction mixture would need to be changed, and a different PCR test would need to be used. There are "15 or 20 different respiratory viruses" that are "obvious targets" for the team to consider developing tests for, he said.

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