COVID-19 Tests | Wish List

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Re: COVID-19 Tests | Wish List

Post by trader32176 »

that comes as no surprise to me either .
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Re: COVID-19 Tests | Wish List

Post by trader32176 »

UVA-designed nasopharyngeal swabs to support high-priority COVID-19 testing across Virginia

8/25/20 ... ginia.aspx

The Commonwealth of Virginia will receive 60,000 nasopharyngeal swabs weekly to support high-priority COVID-19 testing across the state, thanks to a collaboration led by University of Virginia faculty to design this key testing supply.

A total of 75,000 swabs are slated for production each week, with 15,000 swabs remaining at UVA Health to support testing at the health system.

"There have been critical shortages across Virginia and across the country of nasopharyngeal swabs for COVID-19 testing," said Amy Mathers, MD, associate director of clinical microbiology at UVA, whose lab can perform up to 750 tests per day. "These swabs will allow our supply chain team to focus on other areas where testing supplies remain limited."

Mathers worked with a group that included William Guilford, a biomedical engineer in the UVA School of Engineering, and local engineer Andy Homyk to design the swabs as part of a university- and community-wide effort to produce needed medical supplies.

The unusually strong spirit of collaboration that we enjoy at UVA was immeasurably helpful when it came to meeting this challenge. We all understood the importance of working together to get this done, and to do it right."

-William Guilford, Biomedical Engineer, UVA School of Engineering

Beginning with a 3D-printed swab prototype, the team switched gears to create an injection-molded plastic swab that Mathers successfully tested for safety and effectiveness in a clinical trial at UVA Medical Center.

With approval from the U.S. Food and Drug Administration, UVA is working with private companies to manufacture, sterilize and package the swabs for use. Other hospitals and health systems could replicate the swab's design and begin using them after conducting a similar clinical trial at their own facility, Mathers said. Along with their use for COVID-19 testing, nasopharyngeal swabs are also critical for influenza testing.

"We are proud to support the Commonwealth of Virginia's COVID-19 testing efforts, which are a key element in battling this pandemic," said K. Craig Kent, MD, UVA's executive vice president for health affairs. "I am very appreciative of the hard work of this UVA team to develop this vital testing supply, which will also support UVA's ongoing testing efforts in communities around the Charlottesville area."
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Re: COVID-19 Tests | Wish List

Post by trader32176 »

New COVID-19 antigen test can be directly read from the testing card

8/27/20 ... -card.aspx

Today, the U.S. Food and Drug Administration issued an emergency use authorization for the first antigen test where results can be read directly from the testing card, a similar design to some pregnancy tests. This simple design is fast and efficient for healthcare providers and patients and does not need the use of an analyzer.

This new COVID-19 antigen test is an important addition to available tests because the results can be read in minutes, right off the testing card. This means people will know if they have the virus in almost real-time. Due to its simpler design and the large number of tests the company anticipates making in the coming months, this new antigen test is an important advancement in our fight against the pandemic."

-Jeff Shuren, MD, JD, Director, Center for Devices and Radiological Health, Food and Drug Administration

How it works:

A healthcare provider swabs the patient's nose and twirls that sample on a test card with a testing reagent added. After waiting 15 minutes, the healthcare provider reads the results directly from the testing card. One line indicates a negative result; two lines indicate a positive result.

Where it can be used:

This test could be used at point-of-care settings, like a doctor's office, emergency room or some schools. This test has been authorized for use in patients suspected of COVID-19 by their healthcare provider within seven days of symptom onset.

Given the simple nature of this test, it is likely that these tests could be made broadly available. According to the test manufacturer, Abbott, it plans to make up to 50 million tests available monthly in the U.S. at the beginning of October 2020.

Test details:

In general, antigen tests are very specific but are not as sensitive as molecular tests. Due to the potential for decreased sensitivity compared to molecular assays, negative results from an antigen test may need to be confirmed with a molecular test prior to making treatment decisions.

Negative results from an antigen test should be considered in the context of clinical observations, patient history and epidemiological information.

The emergency use authorization was issued to Abbott Diagnostics Scarborough, Inc for its BinaxNOW COVID-19 Ag Card.
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Re: COVID-19 Tests | Wish List

Post by Howzitgoing »

COVID Breathalyzer

Breathalyzers work for Happy Hours. Why not for self-administered COVID-19 tests since we know that airborne transmission is a critical route to infection. Anyway, woke up with that thought hoping that TSOI was already perfecting a sealed, disposable device in a secret lab. Fortunately researchers are way ahead of me.
Coronavirus breathalyzer

Diagnostic medical device enabling the user to determine with 90% or greater accuracy the presence of coronavirus in an exhaled breath. As of the first half of 2020, the idea of a practical coronavirus breathalyzer was concomitantly developed by unrelated research groups in the United States, Finland, Israel, England, Australia and Germany.

Here's one description: ... 120714.htm
Toward a coronavirus breathalyzer test
> The authors acknowledge funding from the Technion-Israel Institute of Technology
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Re: COVID-19 Tests | Wish List

Post by trader32176 »


Very interesting , and timely information !
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Re: COVID-19 Tests | Wish List

Post by trader32176 »

Are these fast, cheap coronavirus tests the game-changer everyone is waiting for?

8/28/20 ... index.html

Move over, slowpoke coronavirus tests that take a week or more to return a result -- there's a new generation of rapid tests coming to town and they're poised to transform the landscape.

They are the antigen tests
. But are they really all they're cracked up to be?

The US Food and Drug Administration granted emergency use authorization to another antigen test this week, bringing the total to four.

Compared to the most commonly used type of coronavirus test in the country -- molecular diagnostic tests, also called PCR tests -- antigen tests don't need complicated chemicals, viral transport media or RNA extraction kits. They don't necessarily require appointments at specialized labs, highly trained technicians, or certain machines.

And they can provide an answer in minutes, rather than hours or days.
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Re: COVID-19 Tests | Wish List

Post by trader32176 »

Covid-19 tests may be detecting traces of DEAD virus, giving ‘false positives’ and EXAGGERATING pandemic – research

5 Sep, 2020 13:03 / Updated 1 day ago ... -pandemic/

New research has discovered that coronavirus tests may be finding dead traces from weeks-old infections, resulting in false positives that inflate the scale of the pandemic.

The study was carried out by experts from the University of Oxford’s Centre for Evidence-Based Medicine and the University of the West of England. It found there was a risk of “false positives” because of how Covid-19 testing is being conducted.

The scientists discovered that, despite people with Covid-19 being infectious for only around a week, one test used to detect the disease can still give a positive reading weeks after the patient has recovered.

The team examined 25 studies on the widely used polymerase chain reaction test, which is used to determine if someone has the virus in their system. The test takes a sample from a suspected Covid-19 case and uses a process that increases the amount of DNA, or genetic material, in the sample, to enable it to be examined.

The research found that the tests can amplify coronavirus genetic material that is not a viable virus and no longer capable of causing an infection.

Professor Carl Heneghan, one of the authors of the study, said there was a risk that a surge in testing across the UK was increasing the risk of this sample contamination occurring and it may explain why the number of Covid-19 cases is rising but the number of deaths is static.

“Evidence is mounting that a good proportion of ‘new’ mild cases and people re-testing positives after quarantine or discharge from hospital are not infectious, but are simply clearing harmless virus particles which their immune system has efficiently dealt with,” he wrote in The Spectator magazine.

Professor Heneghan said an “international effort” was required to avoid “the dangers of isolating non-infectious people or whole communities”.
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Re: COVID-19 Tests | Wish List

Post by trader32176 »

Pooled saliva testing increases COVID-19 testing capacity

9/7/20 ... acity.aspx

As the COVID-19 pandemic in the US continues to spread aggressively, testing remains somewhat dysfunctional and uncoordinated. This has made it challenging to test for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and to obtain results in time for action to be taken to contain the spread of the virus. Now, a new study by researchers at Yale School of Medicine and Yale School of Public Health and published on the preprint server medRxiv* in September 2020 shows that pooled saliva samples can save resources and increase testing capacity, making a significant difference in the management of the pandemic.

Pool Testing

The most significant constraint in stopping the virus has been the limited availability of tests and testing kits, reagents, and laboratories. However, testing is crucial to not only diagnose but also to screen for the disease and to monitor and predict outbreaks. To increase the testing capacity in all places where gatherings of people are necessary for the return to near-normal life, such as schools and workplaces, pooled samples have become the way to go.

Also called batched testing, the pooling of test samples allows multiple people to be tested together to detect the presence of the virus in one or more of the samples. If the result is negative, all the individuals are reported to be negative, while if positive, each sample must then be individually re-tested.

So far, pooled samples have used reverse transcriptase-polymerase chain reaction (RT-PCR) to detect viral RNA, after first extracting it from individual samples. However, pooled testing can be initiated after mixing the samples and extracting the pooled RNA just as well. Earlier studies have shown that these approaches save resources, but laboratory data is still necessary to back up future modifications.

Saliva for Viral RNA Detection

Recently, saliva has been shown by the current team of researchers to be an efficient option for viral RNA detection. Not only is it easy to self-collect, but it does not require either stabilizing buffers or cold chain maintenance during transport.

Different Pool Sizes

The current study focuses on pooling 5, 10, and 20 saliva samples for a high-throughput testing workflow. The samples came from COVID-19 inpatients and healthcare professionals. The samples containing the virus were combined for all cycle threshold values below 38 Ct with negative saliva. Subsequently, they performed RNA extraction and detection.

All samples included in the pool had already been tested individually. The comparison between the pool testing results and the latter shows that with increasing pool size, test sensitivity declined by ~2, 3, and 3.6 Ct with a pool of 5, 10, and 15, respectively. This effect does not vary with the Ct value for the undiluted sample, and therefore will not cause a decline in sensitivity at different Ct values. In other words, it remains equally sensitive at various viral loads.

These results agree with previous pooled swab testing research findings. However, the current study also used increased extraction volume, from 300 μL to 400 μL, but without changing the elution volume, to enhance sensitivity to a level comparable to that for undiluted samples.

Again, the effect of pooling the samples after extraction of RNA, and of using pooled RNA templates from undiluted saliva samples, in sets of 5 and 10, showed the decrease in sensitivity to be similar to that obtained with pre-extraction pooling. However, with the latter, the individual sample variance was smaller.

Analysis of the Ct values for all positive samples from their previous study showed that with a pool of 5, 10, and 20 samples, about 93%, 90% and 85% of samples would be correctly detected, respectively, in comparison to the individual detection of the samples. In other words, the sensitivity reduces by 12-15% with pooled testing relative to individual testing.

The Cost-Effectiveness of Pooled Saliva Testing

A broad-based testing technique must also be inexpensive in order to achieve its objective of identifying infectious people to isolate them from the population. The cost-effectiveness of this strategy was, therefore, compared to individual sample testing. The researchers modeled the number of tests required for a population of 10,000 at varying levels of prevalence, using pools of 5, 10, or 20.

This showed that the number of positive pools would increase with rising prevalence, which means more individual tests will be required to confirm the presence of the infection in individuals.

Reduced Number of Tests

When the prevalence is 3% or more, therefore, smaller pools of 5 will result in the optimal number of tests overall. But with lower prevalence, larger pool sizes of 10 or 20 will reduce the number of tests required, saving on both cost and test supplies. For instance, with a prevalence of 0.8%, a pool size of 20 would be ideal to ensure that ongoing population surveillance and early identification of new outbreaks are feasible.

In general, once the tested population has a prevalence of 30% or less, pooled samples would reduce the number of tests needed. Beyond this prevalence, pooled sizes were not tested. However, with reduced prevalence, the cost savings associated with pooled testing increases.

To illustrate this, the researchers found that with a prevalence of 0.5%, just over 1,300 tests would be enough to cover a population of 10,000 people. This would mean saving over $260,000 by pooled testing vs. individual testing, given that tests typically cost $30 each, while still identifying 43-50 infections.

Increased Testing Frequency

An even greater advantage is that increased testing frequency is allowed, which means more infectious individuals are identified and isolated, preventing viral spread. Thus, even in areas of low prevalence, pooled testing still has significant economic benefits, promoting continued surveillance.

The researchers comment, “Our model demonstrates that as local outbreaks fluctuate, varying pool sizes in response will have resource-savings benefits. Taken together, pooled testing of the non-invasive and cost-effective saliva sample type facilitates extended duration and breadth of screening test strategies.”

Screening for surveillance, not Diagnosis for Treatment

The pooled testing strategy approved by the US Food and Drug Administration (FDA) will be of greatest value in a population with a high prevalence of infection. However, since the sensitivity drops by ~12-15% with pools of 10 or 20 samples, FDA authorization may occur only if this is recognized to be a screening strategy rather than a diagnostic tool, and if the capability to offer more frequent and repeated testing is also considered.

Again, they say, “The probability of a false negative should not be considered per test, but rather for a given testing regime over a specified period of time.” They also consider the real number of tests required to be probably still lower, as there are likely to be more than one positive result in a single pool. Test protocols can also be tweaked to yield the most significant number of true positives by reducing the effect of sample dilution. For instance, the sample volume could be increased to the maximum, or the RNA elution volume reduced, while an order of magnitude increases the Ct positivity threshold. The RNA extraction step may also be omitted until after pooling.

The study concludes, “Together with the ease of saliva collection, this strategy should be considered as an effective testing strategy to expand the breadth of testing and continued surveillance during the ongoing COVID-19 pandemic.”
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Re: COVID-19 Tests | Wish List

Post by trader32176 »

Scientists develop a rapid, reliable and low-cost COVID-19 antibody test

9/8/20 ... -test.aspx

Robust and widespread antibody testing has emerged as a key strategy in the fight against SARS-CoV-2, the virus responsible for the COVID-19 pandemic. However current testing methods are too inaccurate or too expensive to be feasible on a global scale. But now, scientists at the Okinawa Institute of Science and Technology Graduate University (OIST) have developed a rapid, reliable and low-cost antibody test.

The device, described in a proof-of-concept study published this week in Biosensors and Bioelectronics, uses portable lab-on-a-chip technology to accurately measure the concentration of antibodies present in diluted blood plasma.

Antibodies are proteins produced by the immune system to neutralize the virus. Research has found that COVID-19 antibodies are present in the later stages of infection and can linger in the blood after the infection has cleared, allowing previously infected individuals to be identified. Antibody tests are thus an important means of determining the full spread of the coronavirus - information that is crucial to guide public health policies.

And yet many nations have so far failed to employ large-scale antibody testing.

" Many existing platforms for antibody tests are accurate and reliable, but they are costly and need to be carried out in a lab by trained operators. This means that it can take hours, or even days, to obtain results. Other tests are easier to use, portable and rapid, but are not sufficiently accurate, which hampers testing efforts."

- Dr. Riccardo Funari, first author and postdoctoral researcher in the Micro/Bio/Nanofluidics Unit at OIST

The researchers avoided this trade-off between accuracy and accessibility by developing an alternative antibody testing platform that combines a powerful light-sensing technology with a microfluidic chip. The chip provides results within 30 minutes and is highly sensitive, detecting even the lowest clinically-relevant antibody concentration. Each chip is cheap to manufacture and negates the need for a lab or trained operators, increasing the feasibility of nation-wide testing.

And there's another distinctive advantage of this newly developed platform. "The test doesn't just detect whether the antibodies are present or absent - it also provides information about the quantity of antibodies produced by the immune system. In other words, it's quantitative," said Professor Amy Shen, who leads the Micro/Bio/Nanofluidics Unit. "This greatly expands its potential applications, from treating COVID-19 to use in developing vaccines."

Illuminating the antibodies

The antibody testing platform consists of a microfluidic chip which is integrated with a fiber optic light probe. The chip itself is made from a gold-covered glass slide with an embedded microfluidic channel. Using an electric voltage, the team fabricated tens of thousands of tiny spiky gold structures, each one smaller than the wavelength of light, on a glass slide.

The researchers then modified these gold nanospikes by attaching a fragment of the SARS-CoV-2 spike protein. This protein is crucial for helping the coronavirus infect cells and causes a strong reaction from an infected person's immune system.

In this proof-of concept study, the scientists demonstrated the principle behind how the test detects antibodies by using artificial human plasma sample spiked with COVID-19 antibodies that are specific to the spike protein.

Using a syringe pump, the sample is drawn through the chip. As the plasma flows past the protein-coated gold nanospikes, the antibodies bind to the spike protein fragments. This binding event is then detected by the fiber optic light probe.

"The detection principle is simple but powerful," said Dr. Funari. He explained that is it based on the unique behavior of electrons on the surface of the gold nanospikes, which oscillate together when hit by light. These resonating electrons are highly sensitive to changes in the surrounding environment, such as the binding of antibodies, which causes a shift in the wavelength of light absorbed by the nanospikes.

"The more antibodies that bind, the larger the shift in the wavelength of the absorbed light," added Dr. Funari. "The fiber optic probe is connected to a light detector which measures this shift. Using that information, we can determine the concentration of antibodies within the plasma sample."

A bright future

The large-scale roll-out of a quantitative test could greatly impact how COVID-19 is treated.

For example, quantitative tests could help doctors track how effectively a patient's immune system is fighting the virus. It could also be used to help identify suitable donors for a promising experimental treatment, called plasma transfusion therapy, where a recovered patient's antibody-rich blood is donated to currently infected patients to help them fight the virus.

Being able to measure the level of immune response can also aid vaccine development, allowing researchers to determine how effectively a trial vaccine triggers the immune system.

However, the researchers emphasized that the device is still undergoing active development. The unit aims to reduce the chip size to cut manufacturing costs and is also working on improving the reliability of the test.

"We have shown that the device works to detect different concentrations of the spike protein antibody in artificial human plasma samples. We now want to expand the test so that the chip can detect multiple different antibodies at the same time," said Dr. Funari. "Once the device is optimized, we plan to collaborate with local hospitals and medical institutions to perform tests on real patient samples."
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Re: COVID-19 Tests | Wish List

Post by trader32176 »

Saliva Test for COVID-19: Researchers Enable Alternative to Nasal Swabs

9/8/20 ... asal-swabs

Saliva samples instead of deep, "brain-tickling" nasal swabs: A new wave of noninvasive, less tech-intensive testing is the latest advance in the battle against COVID-19. Dedicated researchers like Anne Wyllie and Andrew Berglund are making it possible.

The need couldn't be more urgent. University students are taking virtual classes yet moving back on campus. Professional athletes have been sequestered to bubbles and are playing their sports in front of zero live fans.

Preventing the spread of COVID-19 among healthy people living and working in close quarters is tricky. Saliva testing may represent the newest hope for staying ahead of the novel coronavirus.

Anne Wyllie, an associate research scientist at Yale School of Public Health in New Haven, Connecticut, has been studying saliva for the past 10 years. But she never expected the fruits of her research to draw attention from a professional sports league reacting to a global pandemic.

In late June, Yale announced a partnership with the National Basketball Association and the National Basketball Players Association to launch a study for testing players, coaches and staff with both standard nasal swab testing and Yale's newly devised saliva approach, and then comparing the results for accuracy. They wanted to see if the saliva test could be as effective as the nasal swab route.

“This whole pandemic, just so many bizarre things have happened,” Wyllie says. “It’s been like putting one fire out after another. But the NBA was completely unexpected.”

Early on, COVID-19 testing using nasal swabs became the standard for most testing labs. But compared to saliva, nasal swab testing is relatively high-tech for labs to run – and uncomfortable for those being tested.

With SalivaDirect, the testing protocol developed by Wyllie and her Yale colleagues, labs can eliminate a cumbersome processing step. Instead of having to extract viral RNA material from a nasal swab sample, lab workers mix the saliva sample with a simple chemical reagent. Heating the mixture releases the virus particles.

On Aug. 4, the Yale team released preprint findings on its saliva-testing approach. The Yale researchers found more than 90% agreement between nasal swab and saliva testing results. Preprint articles are shared publicly before they have been peer-reviewed and so require further scientific evaluation.

However, the new method demonstrated potential to meet the nationwide need for inexpensive, accessible testing. On Aug. 15, the Food and Drug Administration issued an emergency use authorization for SalivaDirect, to allow wider use of the method.

Wyllie was in her lab when she got the message about the FDA announcement. “I was just sitting here alone, already working on that Saturday morning,” she says. “The enormity of it sort of dawned on me and I had a little quiet moment.” But she didn’t have the luxury of time for dwelling. “I dived back into work. There was just so much to do,” says Wyllie, whose previous saliva-related research includes other infectious organisms like pneumonia-causing bacteria.

As it was, a sense of urgency had sent the research team into overdrive. “We were already in the middle of the pandemic,” Wyllie says. “We’ve been working 8 a.m. to midnight, if not longer, almost seven days a week, regardless of what we were finding. We were working to respond to this.”

Depending on factors including chemical reagent costs, shipping of samples, testing equipment and personnel, labs could be charging anywhere from about $15 to $35 per saliva testing kit, Wyllie says.

That could represent significant savings if the method becomes widespread. “Our main motivation was this: We need tests to be affordable to everyone,” Wyllie says. “There’s no reason they need to be $100 or $150. We just wanted a test that could be would be more accessible to all communities across the country and not just those that could pay for it.”

On Aug. 24, to the consternation of many health experts, the Centers for Disease Control and Prevention scaled back its recommendations on COVID-19 testing for people who don’t have symptoms. The CDC now advises that you do not need a test if you’re asymptomatic and have not been in close contact with someone known to have a COVID-19 infection. On Aug. 26, CDC director Dr. Robert Redfield clarified the message with a statement that testing "may be considered" for asymptomatic contacts of people with confirmed or probable cases of COVID-19.

Because asymptomatic spread is thought to be a key driver of COVID-19 infection, many organizations are moving in the opposite direction – ramping up COVID-19 testing to identify cases among seemingly healthy people, to prevent the spread of the coronavirus and stave off disastrous outbreaks among those under their responsibility.

Andrew Berglund, director of the RNA Institute at the University at Albany in New York, has devoted much of his career to studying myotonic dystrophy, a common form of muscular dystrophy. But in 2020 his laboratory made a sharp pivot to pandemic work – developing pooled saliva testing to allow widespread COVID-19 surveillance among university students, faculty and staff. Pooled testing allows the university to get a better snapshot of COVID-19 prevalence among those showing no signs, but without the expense of diagnostic testing for every single person.

After reading about the SalivaDirect testing method from Yale, along with a similar method from University of Illinois at Urbana-Champaign, Berglund and his team were inspired to build a modified version of those two protocols that was optimal for their lab setting.

Surveillance refers to collecting data and producing information to identify the extent of a health threat like COVID-19, and then using those insights to develop an effective public health response. This is different from diagnostic testing (which Wyllie's team developed) that can confirm whether an individual has the specific health condition.

Berglund emphasizes that pooled testing is not diagnostic. Instead, it pinpoints asymptomatic people who might be positive for COVID-19. It does so by detecting RNA signs of the coronavirus in saliva samples from multiple small groups of participants. To that end, the RNA Institute, in partnership with the UAlbany School of Public Health, is in the process of building 25,000 test kits, with three saliva collection test tubes per kit. Many have already been distributed across campus as part of pooled testing.

All students studying, working or visiting at any UAlbany campus during the fall 2020 semester must participate in the pooled surveillance testing program, according to the university website. All faculty and staff present on any campus, for any reason, are strongly encouraged to do so.

Students might find these are the easiest tests they take all semester. Tests are done by random sampling, and students are notified that “today’s the day” by email. Using the small plastic test tubes in their kits, Berglund says, “The individual will spit or drool into that tube and then wipe it down with an alcohol wipe, put it in the bag and then deposit that sample into a collection site.”

Students can fill their test tubes at home or on campus, but they should be alone. “We don’t want people congregating to do tests together,” Berglund says. “They do it in their bathroom, on their own.” They can then wrap the tube in a small biohazard bag, slip that into their backpack and just drop it off on campus.

Once the institute collects all the samples, about 1,000 or more per day, the staff groups them into pools of four. “We just have four students, or four faculty and staff, in a pool,” Berglund says. “That way, you can test in a more cost-effective way.” The majority of the pools are presumed negative, he notes, with COVID-19 positivity rates now very low in New York state.

For students, faculty or staff members in a “presumed-positive” pool, where testing indicates likelihood that at least one participant is positive, all four individuals are strongly recommended for diagnostic testing and quarantine, Berglund says. “But these are asymptomatic individuals,” he notes. “So, normally, you wouldn’t be picking them up at all.”

As the COVID-19 pandemic continues, Berglund has been most surprised at how contagious the virus is and how quickly it spreads. “I find it just fascinating,” he says. “As a person, it’s a little scary. But as a scientist, understanding that is interesting.”

The scientists describe the satisfaction of seeing their laboratory efforts become practical resources in the effort against COVID-19. “I’m doing research on a broader level that really translates to the population,” Wyllie says. “And I like that translation of it.”

As for Berglund, “I really enjoy helping people,” he says. “I like seeing how my science can have a direct impact.”
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