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After almost a year of a global pandemic and a fall resurgence of the virus in many places, new strategies are needed to try to bring back some semblance of normal life. An approach on multiple pathways is needed to do this effectively and rapid, accurate testing, combined with measures such as contact tracing, is one way to achieve this.

Reverse transcriptase polymerase chain reaction or RT-PCR, which amplifies small amounts of viral RNA in swab samples taken from the nose and throat, started as and remains the main “gold standard” test for detecting infection across the world. But labs running these testing services have been under enormous pressure and delays are common.

Will Blake
Will Blake, Sherlock Biosciences

“In normal times, results are typically reported to patients in less than 48 hours. However, due to an exponential increase in demand, it’s taking significantly longer for patients to receive results,” says Will Blake, Ph.D., chief technical officer at Sherlock BioSciences, a company based in Massachusetts developing fast diagnostics for SARS-CoV-2.

California-based Color Genomics moved into the COVID-19 diagnostics space early in 2020. “At the beginning of the pandemic, here in the U.S., we had a lot of labs returning results in five days or more. It’s very clear from an epidemiological model we ran that if your turnaround time gets too long, it actually becomes almost useless to even do the testing, because you’re not able to isolate and trace as effectively,” explains Alicia Zhou, Ph.D., chief scientific officer at Color.

Problems such as these have inspired many researchers, both based in academia and in biotech or medtech companies, to develop new types of tests or adapt old methods for a new purpose, namely, developing SARS-CoV-2 tests that are fast, cheap, and simple to carry out.

Navigating the testing maze

The COVID-19 pandemic has triggered what amounts to a research and development frenzy with many companies and research groups churning out hundreds of new tests, vaccines and treatments for patients infected with the virus.

The rapid testing space is no exception. Tests in this area largely fall into molecular (including RT-LAMP) or antigen tests, which use antibodies against SARS-CoV-2 to pick up traces of viral antigens in patient samples. There are other types of fast tests in development using other technologies, but these are largely at an early stage and are not currently being used as widely as antigen or molecular tests.

Table source: FDA website, accessed 12.22.20

A frontrunner in the fast molecular testing space is reverse transcription loop-mediated isothermal amplification, or RT-LAMP, a simplified method for amplifying small amounts of RNA.

This is the technique Color chose when it decided to develop a SARS-CoV-2 test early in 2020, and indeed the company secured the first emergency use approval (EUA) from the FDA for their test.

“One of the reasons we chose RT-LAMP was because there was a lot of supply constraint on RT-PCR reagents,” says Zhou. “It was also incredibly attractive to us, because of the prior work that had already been done. For a lot of other viruses like Zika and dengue, they use LAMP as a point of care test.”

The technique was developed in 2000 as a simple way of amplifying DNA, but can also be used to amplify RNA. It uses similar technology to PCR, using several short primer sequences to amplify small samples of DNA or RNA into larger, detectable ones, but is less complex and does not require skilled personnel to complete.

A key difference is that RT-LAMP can be carried out in a simple water bath and does not need specialized machinery. RNA extraction from samples prior to testing is not required and the mixture can be primed to change color depending on the test result.

Also providing quick and inexpensive results are antigen tests. While these tests vary, many operate in a similar way to pregnancy tests simply involving some form of test strip that changes color if an antigen protein—generated by exposure to the virus—is present in a sample.

Ellume’s test
Ellume’s test sends results directly to users’ cell phones in as little
as 15 minutes.

So far, antigen tests have all been designed to be used as ‘point of care’ single use tests to allow quick detection in clinics or at home. For example, Abbott’s BinaxNOW test and Ellume’s recently approved over the counter home test.

What does “rapid” really mean?

An important question to answer is how do you define “rapid” in terms of testing? Point of care tests are designed to be one shot tests that can be used quickly. For example, at a patient’s bedside, at home, or if someone comes into a clinic with a suspected infection.

While many of these tests will produce results very quickly, often within 15 minutes, they are not designed for testing large groups of people. RT-PCR, RT-LAMP and antigen tests all have the potential to be scaled up, but so far only the two molecular tests have been scaled up in this way.

“One of the things about RT-LAMP and the way we do it in a centralized lab is that it’s really thinking about doing rapid tests at huge scale,” says Zhou.

“That is something that’s very different than thinking about doing single tests very quickly. There is important utility to both categories. But I think that sometimes the narrative around what rapid means gets convoluted,” she adds.

For providers doing rapid tests such as RT-LAMP on a large scale, another important point to consider is logistics, as this can have a dramatic impact on overall test timings.

“We realized that in centralized lab testing it is, of course, important to make sure that your lab protocols are efficient. But actually, most of the efficiencies are gained by all the other wrapping logistics like courier shipments,” explains Zhou. “We’re literally timing our lab runs to when FedEx drop offs happen, so that we don’t actually lose any time between when those samples arrive, and when we run them. Those are the types of things that actually turned out to make a big difference.”

Living up to accuracy expectations

When trying to assess the accuracy of a diagnostic test, two terms are important, namely, sensitivity and specificity. A test’s sensitivity refers to how likely a test is to correctly identify someone as having the infection, whereas its specificity indicates how likely the test is to correctly identify someone as not having the infection.

For an infectious disease like SARS-CoV-2 it’s important to keep the sensitivity high and the number of false negatives to a minimum to ensure people don’t spread infection further.

Michael Knop, Ph.D., a researcher at the University of Heidelberg in Germany, and colleagues developed an RT-LAMP test to help with testing demand in the spring of 2020. He urges caution when assessing the accuracy of fast SARS-CoV-2 tests.

“Between what companies promise and what scientists measure afterwards, there are huge gaps. Many of these tests don’t live up to their promises,” Knop says.

He advises checking the details of the reference group used to set specificity and sensitivity, as this can have a big influence on how accurate a test will be.

“If you go to a place where you have a high number of freshly infected people with a high viral load, you’re going to get a fantastic specificity and sensitivity,” says Knop, “but if you go to an area where there’s a lower incidence rate, your specificity and sensitivity will be poor.”

Another issue that often confuses discussions about accuracy is that there are essentially two types of test sensitivity.

“One is the analytic sensitivity of your assay, meaning if there is SARS-CoV-2 RNA in the sample can this test detect it? And then the other is clinical sensitivity, which means if I stick this swab up your nose, can I get SARS-CoV-2 on that swab?” explains Zhou.

Clinical accuracy can be impacted by test timing, as there is a period of a few days between exposure and someone developing symptoms when it is harder to collect viral samples. Poor collection technique has been found to be a problem with tests that require nasal or throat swabs, such as Innova’s lateral flow antigen test. In a recent community testing pilot in Liverpool in the U.K., this test only picked up 50% of the positive cases identified by RT-PCR.

In contrast, analytical sensitivity is more of a direct reflection on the technology and labs carrying out the tests. Automation is one way to improve this, a route Color has taken. RT-LAMP lends itself to this, due to its simplicity and the fact that tests can be designed to change color and thus be read by plate readers.

Sherlock Biosciences is one of two companies, the other being Mammoth Biosciences, that have souped up their RT-LAMP tests using the gene editing tool CRISPR-Cas9.

“The Sherlock CRISPR SARS-CoV-2 test kit includes a Cas-based detection step that generates a signal only when the target nucleic acid sequence is present,” says Blake. “This is designed to improve specificity and sensitivity.”

Antigen tests generally have lower sensitivity than RT-PCR or RT-LAMP, something that has put many healthcare professionals and organizations off from using them. However, this has not held back test developers.

Ellume’s antigen test
Ellume’s antigen test uses a small device to detect multi-quantum dot fluorescent particles, which emit light in the presence of virus proteins. The device sends test results to the user’s smartphone in less than 15 minutes.

Ellume is an Australian diagnostics company that has developed a home antigen test, which was recently approved by the FDA. It will be available without a prescription and will cost around $30, according to CEO and Founder Sean Parsons, Ph.D.

The test uses a small device to detect multi-quantum dot fluorescent particles, which emit light in the presence of virus proteins. The device then sends the test result to the user’s smartphone via Bluetooth in less than 15 minutes.

“While lateral flow technology is typically limited in sensitivity, our patented detection system can overcome this by detecting minute fluorescence signals,” says Parsons.

Sean Parsons, Ph.D.
Sean Parsons, Ph.D., founder and CEO, Ellume

“Our tests are designed to mitigate past concerns about home testing. These include misinterpreting results without the expertise of a healthcare professional, a lack of reporting for the benefit of public health, and the possibility of false positives or negatives.”

Making tests more accessible and affordable

COVID-19 is such a widespread problem that in order to have a real impact, tests need to be as accessible as possible. This means making them affordable, simple to carry out, and making sure people use them.

“One of the things we’ve noticed here in the U.S. is that there isn’t actually a shortage of test capacity,” emphasizes Zhou. “The problem is that people who really need the tests don’t necessarily get access, and that’s because the capacity might be in different parts of the country from where the patients are, or they just don’t know how to get access to the test.”

For centralized testing facilities, this can be improved by digitization of patient scheduling and results to make the process less confusing and more efficient.

Ellume’s test is available to consumers without a prescription.

For point of care tests, in terms of price and simplicity, antigen tests come out top. So, if developers such as Ellume’s claims about improved accuracy hold true, then their home-based test could be a winner.

RT-LAMP tests are generally a bit more expensive than antigen tests, but cheaper and simpler to do than RT-PCR tests.

Brian Cunningham, Ph.D., is a professor at the University of Illinois at Urbana-Champaign and an engineer focused on developing new health technology. He and his team are working to make RT-LAMP technology more accessible and have developed a prototype point-of-care test that can give a reading from a cell phone.

“We use a $50 clip on device that allows the phone’s rear-facing camera to measure the test, so the results are completely integrated with an app and cloud-based service system,” Cunningham says. “The camera can sense the fluorescent light generated by the assay when it turns positive.  The fluorescence is green, and it becomes visible when the test sample is illuminated by blue LEDs.”

Testing our way back to normality

The COVID-19 testing space is large and can be confusing, but can rapid tests help us get back to normal life? It is probably too late now for testing to significantly change the pandemic as a whole, but on a localized city or large organization level it has the potential to have a positive impact and help prevent future deaths and hospitalizations.

The speed of development of many of these tests means that inevitably some will be better than others, but the science continues to improve and we are already seeing improvements on earlier iterations of the antigen and RT-LAMP tests.

Good sensitivity is key for fast tests to have an impact and antigen tests still have a way to go on this front. RT-LAMP has the advantage of being almost as sensitive as RT-PCR, but is cheaper, easier, and quicker to carry out.

“I really do think that being able to massively scale testing with a short turnaround time is really important for SARS-CoV-2 testing,” says Zhou. “RT-LAMP helps us strike that balance of being able to do 10,000 tests a day, and still keep turnaround times at 24 hours or below. I think it is that kind of Goldilocks solution. Certainly not as fast as a single point of care test. But you can’t do a single point of care test at massive scale.”

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