Template:COVID-19 Testing, Reporting, and Information Management in the Laboratory/Adding COVID-19 and other virus testing to your laboratory/What methodologies will you use?

3. Adding COVID-19 and other virus testing to your laboratory

Maybe you've been running an environmental health laboratory and want to expand into clinical health testing. Perhaps you're in charge of an academic research lab but want to expand to the clinical diagnostic side. Or maybe you're running a physician office laboratory (POL) and are wondering if it's even possible to expand your waived testing efforts to COVID-19. Where the previous chapter discussed the "what" of COVID-19 and viral testing, this chapter aims to help you with the "how" of adding it to your laboratory offerings.

Naturally, many questions come with the "how":

• Does using one method make the most sense, or will your lab turn to multiple methods for virus testing? This may be determined by current equipment, space considerations, and budget.
• What type of lab are you running? A POL is going to have fewer options available than a CLIA moderate- or high-complexity lab.
• How interoperable are you existing laboratory and clinical informatics solutions? Research laboratories face more challenges in integrating their systems with EHRs and other clinical systems.
• What vendors and consultants are out there to help get equipped? Some vendors have very specific solutions, whereas others may have a broader range of offerings.

These questions and more are addressed in this chapter.

3.1 What methodologies will you use?

3.1.1 PCR

SARS-CoV-2 PCR screening test by nasal swab in Strasbourg on August 21, 2020.

In the previous chapter, the most common testing methodologies for COVID-19 and other coronaviruses were discussed in detail. The prevailing method (often called the "gold standard") among them all is real-time reverse-transcription polymerase chain reaction (rRT-PCR) assays for testing. Broadly speaking, PCR is useful in pharmaceutical, biotechnology, and genetic engineering endeavors, as well as clinical diagnostics. As such, labs in those industries that already have PCR infrastructure in place have a theoretical step-up over a lab that doesn't.

PCR technology has advanced to the point where it is more efficient and user-friendly than prior, yet "the high cost of the instruments, servicing contracts, and reagents pose major challenges for the market, especially to the price-sensitive academics." Writing about the thirty-fifth anniversary of PCR in 2018, science writer Alan Dove not only highlighted these cost issues but also the size and energy requirements for running the equipment. "As a result, one of the defining techniques of modern molecular biology has remained stubbornly inaccessible to educators and unusable in many remote locations." Various efforts have been made over the years to bring costs down by modifying how heating and temperature control are performed, but many of those system aren't typically optimal during a pandemic when turnaround time is critical.

Amidst the pandemic, additional challenges also exist to those wanting to conduct PCR testing for COVID-19 and other viruses. As was discussed at the end of the previous chapter, supplies of reagents and consumables are not particularly robust mid-pandemic, with shortages being reported since March 2020. These shortages may eventually work themselves out, but they highlight the need for other varying methods that don't necessarily depend on the same reagents and consumables that are in short supply.

For those labs wishing to adopt PCR testing of viruses—particularly COVID-19—into their workflow while providing reasonable turnaround times, all is not lost. However, careful planning is required. For example, you'll want to keep in mind that some PCR machines require vendor-specific reagents. If you're going to acquire a particular instrument, you'll want to do due diligence by verifying not only the supported reagents but also those reagents' overall availability (real and projected). You'll also want to consider factors such as anticipated workload (tests per day), what your workflow will look like, and how to balance overall investment with the need for reasonable turnaround times.

An increasing body of research is being produced suggesting ways to improve turnaround times with PCR testing for COVID-19, with many research efforts focusing on cutting out RNA extraction steps entirely. Alcoba-Florez et al. propose direct heating of the sample-containing nasopharyngeal swab at 70 °C for 10 minutes in place of RNA extraction. Adams et al. have proposed an "adaptive PCR" method using a non-standard reagent mix that skips RNA extraction and can act "as a contingency for resource‐limited settings around the globe." Wee et al. skip RNA extraction and nucleic acid purification by using a single-tube homogeneous reaction method run on a lightweight, portable thermocycler. Other innovations include tweaking reagents and enzymes to work with one step, skipping the reverse transcription step, and using saliva-based molecular testing that skips RNA extraction.

Saliva as a specimen

The saliva molecular tests in particular are intriguing. Talk of the potential utility of using saliva as a specimen for COVID-19 was occurring as early as April, and the first saliva-based COVID-19 test, produced by Spectrum Solutions in cooperation with RUCDR Infinite Biologics Laboratory and Vault Health, was given an FDA EUA in April. On August 15, Yale School of Public Health was given an EUA for it SalivaDirect molecular test. Although still PCR-based (and a CLIA high-complexity test), SalivaDirect is being touted as a means to improve specimen collection safety, consume fewer reagents, prove compatible with high-throughput workflow, and cut overall turnaround time. Not only is saliva easier to collect and safer for healthcare staff, the test is essentially "open sourced," not requiring proprietary equipment from Yale, making the test more flexible by being validated to reliably function with a wider array of reagents and instruments. When compared to using a nasopharyngeal swab specimen using the ThermoFisher Scientific TaqPath COVID-19 combo kit, results were comparable 94.1% of the time. While sensitivity and specificity may be slightly less comparable to other PCR options, the overall advantages during reagent shortages and a definitive need for broader testing likely outweigh the slightly lesser sensitivity and specificity. As of November 2020, public health agencies in Arizona and Minnesota have already begun running trials of free saliva-based molecular testing.

3.1.2 Pooled testing

Another method some labs are taking to speed up turnaround time is using pooled testing. The general concept involves placing two or more test specimens together and testing the pool as one specimen. The most obvious advantage to this is that the process saves on reagents and other supplies, particularly when supply chains are disrupted. This methodology is best used "in situations where disease prevalence is low, since each negative pool test eliminates the need to individually test those specimens and maximizes the number of individuals who can be tested over a given amount of time." However, it's best left to situations where expectations are that less than 10 percent of the population being tested is affected by what's being tested for.

The downside of pooled testing comes with the issues of dilution, contamination, and populations with 10 or more percent infected. A target-positive specimen that comingles with other target-free specimens is itself diluted and in some cases may cause issues with the limit of detection for the assay. Additionally, if the pool tests positive, target-free specimens may become contaminated by a target-positive specimen. This may cause issues with any individual specimen assays that get ran. And the workflows involving pooling must be precise, as a technician working with multiple specimens at the same time increases the chance of lab errors. Finally, at least in the U.S., a Food and Drug Administration (FDA) emergency use authorization (EUA) for a validated pooled testing method is required. (Validation of pooled methods may differ in other countries.) The U.S. Centers for Disease Control and Prevention (CDC) has published interim guidance on pooled testing strategies for SARS-CoV-2.

3.1.3 Rapid antigen testing

As mentioned in the previous chapter, the benefits of antigen testing For COVID-19 and other viral infections are 1. specimen collection can typically be done with a simple nasal swab rather than a more invasive nasopharyngeal swab, 2. testing is more rapid and convenient, and 3. it takes some pressure off the PCR supply chain. However, antigen testing only tests what's there, rather than amplifying the amount, resulting in generally lower sensitivities. As such, the real utility of antigen testing, despite its lower sensitivity, appears to be surveillance situations where a large group of individuals who are at risk can be screened at regularly scheduled intervals of two to four days. If your lab is able to support this sort of testing, then this type of testing may be an option. As of November 2020, six vendors have EUAs for antigen diagnostic tests. With six U.S. states already contracted to purchase hundreds of thousands of two of those companies' test kits, it remains to be seen how well they'll meet demand.

3.1.4 LAMP and CRISPR

Early on in the pandemic, while PCR was getting most of the attention, reverse transcription loop-mediated isothermal amplification (RT-LAMP), an isothermal nucleic acid amplification technique that allows for RNA amplification, was also quietly being discussed, and it has since gained more attention. The University of Oxford, for example, is in the process of getting a rapid, affordable, clinically-validated RT-LAMP test approved for the European market. Oxford also notes that "[a]n advantage of using LAMP technology is that it uses different reagents to most laboratory-based PCR tests." Thi et al. have tested a two-color RT-LAMP assay with an N gene primer set and diagnostic validation using LAMP-sequencing, concluding that the pairing of the two "could offer scalable testing that would be difficult to achieve with conventional qRT-PCR based tests." And California-based Color Genomics have set up their own proprietary RT-LAMP system, capable of handling up to 10,000 tests per day.

In most cases, LAMP-based testing is much simpler than PCR, lacking the requirement of specialized instruments. Despite LAMP generally being thought of as less sensitive than PCR, the recent explosion of research into RT-LAMP methods for testing for the presence of SARS-CoV-2 seems to gradually indicate that "under optimized conditions," RT-LAMP methods may actually be able to rival the sensitivity and specificity of many RT-PCR COVID-19 tests. Esbin et al. add:

These methods allow for faster amplification, less specialized equipment, and easy readout. LAMP methods also benefit from the ability to multiplex targets in a single reaction and can be combined with other isothermal methods, like [recombinase polymerase amplification] in the RAMP technique, to increase test accuracy even more. These techniques may be particularly useful for rapid, point-of-care diagnoses or for remote clinical testing without the need for laboratory equipment.

CRISPR methods are also being used in conjunction with RT-LAMP. RT-LAMP creates complementary double-stranded DNA (cDNA) from specimen RNA and then copies (amplifies) it. Then CRISPR methods are used to detect a predefined coronavirus sequence (from a cleaved molecular marker) in the resulting amplified specimen. Though as of November 2020 approved assays using CRISPR-based detection of SARS-CoV-2 are limited to a handful of companies, the technology has some promise as an alternative testing method. It has the additional advantage of being readily coupled with lateral flow assay technology to be deployed in the point-of-care (POC) setting.

3.1.5 Point-of-care and other alternative testing

Example of a microfluidic chip used in point-of-care medical devices

On August 5, 2020, the WHO published a draft blueprint for what they call Target Product Profiles (TPP), which "describe the desirable and minimally acceptable profiles" for four difference COVID-19 test categories. Addressing POC testing, the WHO recommends that such assays:

• have a sensitivity (true positive rate) or at least 70 percent;
• have a specificity (true negative rate) of at least 97 percent;
• provide results in less than 40 minutes;
• require diagnostic machines that cost less than $3,000 U.S.;
• individually cost less than $20 for the patient;
• be simple enough that only a few hours of training are required to run the test; and
• operate reliably outside a clean laboratory environment.

While few of the available test systems can meet all these requirements, it's clear the push to expand COVID-19 testing to the point of care is accelerating. The U.S. National Institutes of Health's Rapid Acceleration of Diagnostics (RADx) funding program has sought to speed up innovation in COVID-19 testing and promote "truly nontraditional approaches for testing that have a slightly longer horizon." In August 2020, RADx had chosen to fund seven biomedical diagnostic companies making new lab-based and POC tests that could significantly ramp up overall testing in the U.S. into September 2020. Four offerings are lab-based (from Ginkgo Bioworks, Helix OpCo, Fluidigm, and Mammoth Biosciences) and three are POC tests (from Mesa Biotech, Quidel, and Talis Biomedical), all using varying technologies and methods such as next-generation sequencing, CRISPR, microfluidic chips, nucleic acid testing, antigen testing, and saliva testing. Both Mesa Biotech's rapid, cartridge-based RT-PCR Accula System and Quidel's rapid Sofia SARS Antigen FIA test are already EUAed and CLIA-waived, with Talis' Talis One LAMP-based lateral flow immunoassay kit still awaiting EUA and CLIA status approval. As of October 28, 2020, RADx has added an additional 15 biomedical diagnostics projects for funding, for a total of 22. Whether or not these POC and lab-based tests make it to the average physician office laboratory remains to be seen, however.

Outside the RADx program, enterprising researchers in other parts of the world are also attempting non-traditional approaches to improving COVID-19 testing options. Examples include:

• a method of DNA nanoswitch detection of virus particles;
• a dual biomarker-based finger-stick test for acute respiratory infections;
• a rapid breath test to detect volatile organic chemicals from the lungs;
• an affordable, hand-held spectral imaging device to detect virus in blood or saliva in seconds;
• an ultrahigh frequency spectroscopic scanning device to see virus particles resonating;
• a method that combines optical devices and magnetic particles to detect virus RNA;
• an RNA extraction protocol that uses magnetic bead-based kits;
• a nanotube-based electrochemical biosensor for detecting biomarkers in a sample in less than a minute;
• the additional use of an artificial intelligence (AI) application to better scrutinize test results; and
• the miniaturization of PCR technology to make it more portable and user-friendly.

Of course, most of these are largely experimental technologies, and realistically getting them into the lab may be far out. But they represent out-of-the-box ideas that have some kind of chance at playing a greater role in the clinical laboratory or in point-of-care settings in the future.