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2.4.5 Antigen tests

An antigen is a substance—often a protein but may also be an environmental like a virus—that provokes the immune system to produce an antibody against it. As such, another approach to testing for the presence of a virus in a specimen is to test for the antigen rather than the antibody. An antigen test is useful as a repeated surveillance test, but it has drawbacks as a one-time diagnostic test. For COVID-19 and other viral infections, an antigen test has the advantage that specimen collection can typically be done with a simple nasal swab rather than a more invasive nasopharyngeal swab. Another advantage, on one hand, is that antigen testing is more rapid and convenient because the extraction and amplification steps of PCR are not used. On the other, antigen testing is less sensitive for the same reason: you test only what's there (rather than amplifying the amount for greater sensitivity).

A theory increasingly gaining traction, however, is that "[a] higher frequency of testing makes up for poor sensitivity.” Several researchers have shared pre-print and published research suggesting this outcome:

Larremore and his colleagues have modeled the benefits of more frequent tests, even ones that are less accurate than today’s. Fast tests repeated every three days, with isolation of people who test positive, prevents 88% of viral transmission compared with no tests; a more sensitive test used every two weeks reduced viral transmission by about 40%, they report in a 27 June preprint on medRxiv. Paltiel and his colleagues reached much the same conclusion when they modeled a variety of testing regimes aimed at safely reopening a 5000-student university. In a 31 July paper in JAMA Network Open, they found that, with 10 students infected at the start of the semester, a test that identified only 70% of positive cases, given to every student every two days, could limit the number of infections to 28 by the end of the semester. Screening every seven days allowed greater viral spread, with the model predicting 108 infections.

As such, the 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. The end result, in theory, would be few people who are target-positive would be missed, positives could be isolated and verified with a more sensitive test, and more target-positive people would be identified and isolated before reaching peak infectivity. To be clear, it's not a perfect solution, but as Harvard epidemioligist Michael Mina and Boston University economist Laurence Kotlikoff suggest, "[w]e need the best means of detecting and containing the virus, not a perfect test no one can use." A coalition of six U.S. state governors has bought into that concept and agreed to work together with the Rockefeller Foundation, as well as the Quidel Corporation and Becton, Dickinson and Company, which have received FDA EUAs to market antigen tests for SARS-CoV-2. However, it's not clear how those six states will best put the tests to use despite 1. their moderate sensitivity (and thus a greater chance of false negatives) and 2. the question of whether or not the two companies can produce enough test kits for repeat testing in those states.

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

Does using one method make the most sense, or will your lab turn to multiple methods for virus testing?

3.1 What methodologies will you use?

3.1.1 PCR

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 was 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 ifrastructure 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 requirments 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 investent with the need for reasonable turnaround times.

As of August 2020, 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.

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 dillution, 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., an 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 great. However, as of August 2020, only two vendors have EUAs for antigen diagnostic tests: Quidel Corporation and Becton, Dickinson and Company. With six U.S. states already contracted to purchase hundreds of thousands of the two companies' test kits, it's not clear how well they'll manage to 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 test. 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 August 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 couples 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

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." As of August 2020, RADx has 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. 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 fingerstick 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 beads;
• the addtional use of an artificial intelligence (AI) application to better scrutenize 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 thinking that have some kind of chance at playing a greater role in the clinical laboratory or point of care settings in the future.

3.2 What equipment and supplies will you need?

Instruments

Eppendorf Mastercycler Pro S, a thermal cycler for PCR and other applications

Thermal cyclers (a.k.a. PCR machines) - standard PCR systems, RT PCR - advantages of digital PCR systems such as precision, sensitivity, accuracy, reproducibility, direct quantification and multiplexing, and speed of the analysis systems, and digital PCR systems "The market is witnessing an emerging trend of digital and droplet digital PCR technology, which is sensitive and accurate than the traditional method."

https://www.thermofisher.com/search/browse/category/us/en/602552/PCR+Machines+%28Thermal+Cyclers%29+%26+Accessories https://blog.biomeme.com/how-do-you-test-for-covid-19

Reagents

template DNA, PCR primers and probes, dNTPs, PCR buffers, enzymes, and master mixes

Consumables

PCR tubes, plates, and other accessories https://www.sigmaaldrich.com/labware/labware-products.html?TablePage=9577275

Vendors

Major players operating in the global PCR market are Bio-Rad Laboratories, Inc., QIAGEN N.V., F. Hoffmann-La Roche AG, Thermo Fisher Scientific, Inc. Becton, Dickinson and Company, Abbott, Siemens Healthcare GmbH (Siemens AG), bioMérieux SA, Danaher Corporation, and Agilent Technologies. Merck KGaA, Promega