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| ====2.4.5 Antigen tests====
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| 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.<ref name="MPAntigen">{{cite web |url=https://medlineplus.gov/ency/article/002224.htm |title=Antigen |work=MedlinePlus |publisher=U.S. National Library of Medicine |accessdate=07 August 2020}}</ref> 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.<ref name="Anderson5Invest20">{{cite web |url=https://www.wcvb.com/article/5-investigates-concerns-about-current-use-of-rapid-antigen-tests-for-covid-19/33538332 |title=5 Investigates: Concerns about current use of rapid antigen tests for COVID-19 |author=Anderson, K. |work=WCVB 5 ABC |date=06 August 2020 |accessdate=07 August 2020}}</ref><ref name="ServiceRadical2020">{{cite journal |title=Radical shift in COVID-19 testing needed to reopen schools and businesses, researchers say |journal=Science |author=Service, R.F. |year=2020 |doi=10.1126/science.abe1546}}</ref><ref name="KremerUWSys20">{{cite web |url=https://urbanmilwaukee.com/2020/08/07/uw-system-orders-350000-covid-19-tests/ |title=UW System Orders 350,000 COVID-19 Tests |author=Kremer, R. |work=Urban Milwaukee |date=07 August 2020 |accessdate=07 August 2020}}</ref> 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).<ref name="ServiceRadical2020" /><ref name="GuglielmiTheExp20">{{cite journal |title=The explosion of new coronavirus tests that could help to end the pandemic |journal=Nature |author=Guglielmi, G. |volume=583 |pages=506–09 |year=2020 |doi=10.1038/d41586-020-02140-8}}</ref>
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| A theory increasingly gaining traction, however, is that "[a] higher frequency of testing makes up for poor sensitivity.”<ref name="ServiceRadical2020" /><ref name="KremerUWSys20" /><ref name="ClarkCOVID20">{{cite web |url=https://www.medpagetoday.com/infectiousdisease/covid19/87930 |title=COVID Antigen Tests: Coming to Case Counts Near You? |author=Clark, C. |work=MedPage Today |date=06 August 2020 |accessdate=07 August 2020}}</ref> Several researchers have shared pre-print and published research suggesting this outcome<ref name="ServiceRadical2020" />:
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| <blockquote>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.</blockquote>
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| 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.<ref name="ServiceRadical2020" /><ref name="CourageCovid20">{{cite web |url=https://www.vox.com/2020/7/31/21336212/covid-19-test-results-delays |title=Should we be testing fewer people to stop the spread of Covid-19? |author=Courage, K.H. |work=Vox |date=31 July 2020 |accessdate=05 August 2020}}</ref> 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."<ref name="CourageCovid20" /> 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.<ref name="ClarkCOVID20" /><ref name="MervoshItsLike20">{{cite web |url=https://www.nytimes.com/2020/08/04/us/virus-testing-delays.html |title=‘It’s Like Having No Testing’: Coronavirus Test Results Are Still Delayed |author=Mervosh, S.; Fernandez, M. |work=The New York Times |date=04 August 2020 |accessdate=05 August 2020}}</ref> 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<ref name="ClarkCOVID20" />) and 2. the question of whether or not the two companies can produce enough test kits for repeat testing in those states.<ref name="ServiceRadical2020" />
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| ==3. Adding COVID-19 and other virus testing to your laboratory==
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| Does using one method make the most sense, or will your lab turn to multiple methods for virus testing?
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| What type of lab are you running? A physician office laboratory (POL) is going to have fewer options available than a CLIA moderate- or high-complexity lab.
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| ===3.1 What methodologies will you use?===
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| ====3.1.1 PCR====
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| In the previous chapter, the most common testing methodologies for [[COVID-19]] and other [[Coronavirus|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.
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| 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."<ref name="MWPoly20">{{cite web |url=https://www.marketwatch.com/press-release/polymerase-chain-reaction-market-sector-analysis-report-regional-outlook-competitive-share-forecast---2023-2020-06-23 |title=Polymerase Chain Reaction Market Sector Analysis Report, Regional Outlook & Competitive Share & Forecast - 2023 |author=Kenneth Research |work=MarketWatch |date=23 June 2020 |accessdate=06 August 2020}}</ref> 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."<ref name="DovePCR18">{{cite journal |title=PCR: Thirty-five years and counting |journal=Science |author=Dove, A. |volume=360 |issue=6389 |pages=670–672 |year=2018 |doi=10.1126/science.360.6389.673-c}}</ref> Various efforts have been made over the years to bring costs down by modifying how heating and temperature control are performed<ref name="WongARapid15">{{cite journal |title=A Rapid and Low-Cost PCR Thermal Cycler for Low Resource Settings |journal=PLoS One |author=Wong, G.; Wong, I. Chan, K. et al. |volume=10 |issue=7 |at=e0131701 |year=2015 |doi=10.1371/journal.pone.0131701}}</ref><ref name="KuznetsovDIYbio15">{{cite journal |title=DIYbio Things: Open Source Biology Tools as Platforms for Hybrid Knowledge Production and Scientific Participation |journal=Proceedings of the 33rd Annual ACM Conference on Human Factors in Computing Systems |author=Kuznetsov, S.; Doonan, C.; Wilson, N. et al. |pages=4065–68 |year=2015 |doi=10.1145/2702123.2702235}}</ref><ref name="NortonPhila16">{{cite web |url=https://www.bizjournals.com/philadelphia/news/2016/05/11/government-contract-biomeme-hiring-med-tech.html |title=Phila. med tech startup working on multimillion dollar government contract |author=Norton, D. |work=Philadelphia Business Journal |date=11 May 2016 |accessdate=06 August 2020}}</ref><ref name="AnLow20">{{cite journal |title=Low-Cost Battery-Powered and User-Friendly Real-Time Quantitative PCR System for the Detection of Multigene |journal=Micromachines |author=An, J.; Jiang, Y.; Shi, B. et al. |volume=11 |at=435 |year=2020 |doi=10.3390/mi11040435}}</ref>, but many of those system aren't typically optimal during a pandemic when turnaround time is critical.
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| 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.<ref name="HerperShortage20">{{cite web |url=https://www.statnews.com/2020/03/10/shortage-crucial-chemicals-us-coronavirus-testing/ |title=Shortage of crucial chemicals creates new obstacle to U.S. coronavirus testing |work=STAT |author=Herper, M.; Branswell, H. |date=10 March 2020 |accessdate=10 April 2020}}</ref><ref name="HaleQiagen20">{{cite web |url=https://www.fiercebiotech.com/medtech/qiagen-aims-to-more-than-quadruple-its-covid-19-reagent-production-6-weeks |title=Qiagen aims to more than quadruple its COVID-19 reagent production in 6 weeks |work=Fierce Biotech |author=Hale, C. |date=18 March 2020 |accessdate=10 April 2020}}</ref><ref name="MehtaMystery20">{{cite web |url=https://www.chemistryworld.com/mystery-surrounds-uk-claim-of-covid-19-test-reagent-shortage/4011457.article |title=Mystery surrounds UK claim of Covid-19 test reagent ‘shortage’ |work=Chemistry World |author=Mehta, A. |date=03 April 2020 |accessdate=10 April 2020}}</ref><ref name="RocheIrish20">{{cite web |url=https://www.irishtimes.com/news/science/irish-scientists-develop-reagent-in-effort-to-ease-covid-19-testing-delays-1.4223897 |title=Irish scientists develop reagent in effort to ease Covid-19 testing delays |work=The Irish Times |author=Roche, B. |date=08 April 2020 |accessdate=10 April 2020}}</ref><ref name="PadmaEfforts20">{{cite web |url=https://www.chemistryworld.com/news/efforts-to-combat-covid-19-in-india-hit-by-imported-reagent-shortages/4011718.article#/ |title=Efforts to combat Covid-19 in India hit by imported reagent shortages |author=Padma, T.V. |work=Chemistry World |date=13 May 2020 |accessdate=19 May 2020}}</ref><ref name="DavidSurvey20">{{cite web |url=https://abcnews.go.com/Health/survey-shows-resources-covid-19-diagnostic-testing-limited/story?id=71341885 |title=Survey shows resources for COVID-19 diagnostic testing still limited months later |author=David, E.; Farber, S.E. |work=ABC News |date=20 June 2020 |accessdate=08 July 2020}}</ref><ref name="JohnsonNCLabs20">{{cite web |url=https://patch.com/north-carolina/charlotte/nc-labs-facing-shortages-covid-19-testing-chemicals |title=NC Labs Facing Shortages In COVID-19 Testing Chemicals |author=Johnson, K. |work=Patch |date=02 July 2020 |accessdate=08 July 2020}}</ref><ref name="MervoshItsLike20">{{cite web |url=https://www.nytimes.com/2020/08/04/us/virus-testing-delays.html |title=‘It’s Like Having No Testing’: Coronavirus Test Results Are Still Delayed |author=Mervosh, S.; Fernandez, M. |work=The New York Times |date=04 August 2020 |accessdate=05 August 2020}}</ref><ref name="CourageCovid20">{{cite web |url=https://www.vox.com/2020/7/31/21336212/covid-19-test-results-delays |title=Should we be testing fewer people to stop the spread of Covid-19? |author=Courage, K.H. |work=Vox |date=31 July 2020 |accessdate=05 August 2020}}</ref> 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.
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| 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.
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| 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.'' [[Journal:Fast SARS-CoV-2 detection by RT-qPCR in preheated nasopharyngeal swab samples|propose]] direct heating of the sample-containing nasopharyngeal swab at 70 °C for 10 minutes in place of RNA extraction.<ref name="Alcoba-FlorezFast20">{{cite journal |title=Fast SARS-CoV-2 detection by RT-qPCR in preheated nasopharyngeal swab samples |journal=International Journal of Infectious Diseases |author=Alcoba-Florez, J.; González-Montelongo, R.; Íñigo-Campos, A.; García-Martínezde Artola, D.; Gil-Campesino, H.;<br />The Microbiology Technical Support Team; Ciuffreda, L.; Valenzuela-Fernández, A.; Flores, C. |volume=97 |pages=66–68 |year=2020 |doi=10.1016/j.ijid.2020.05.099}}</ref> 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."<ref name="ShapiroStream20">{{cite web |url=https://news.vanderbilt.edu/2020/07/29/streamlined-diagnostic-approach-to-covid-19-can-avoid-potential-testing-logjam/ |title=Streamlined diagnostic approach to COVID-19 can avoid potential testing logjam |author=Shapiro, M. |work=Research News @ Vanderbilt |date=29 July 2020 |accessdate=06 August 2020}}</ref><ref name="AdamsCOVID20">{{cite journal |title=COVID‐19 diagnostics for resource‐limited settings: Evaluation of “unextracted” qRT‐PCR |journal=Journal of Medical Virology |author=Adams, N.M.; Leelawong, M.; Benton, A. et al. |year=2020 |doi=10.1002/jmv.26328}}</ref> Wee ''et al.'' skip RNA extraction and nucleic acid purification by using a single-tube homogeneous reaction method run on a lightweight, portable thermocycler.<ref name="MeharImproving20">{{cite web |url=https://www.techexplorist.com/improving-speed-gold-standard-covid-19-diagnostic-test/34069/ |title=Improving the speed of gold-standard COVID-19 diagnostic test |author=Mehar, P. |work=Tech Explorist |date=27 July 2020 |accessdate=06 August 2020}}</ref><ref name="WeeRapid20">{{cite journal |title=Rapid Direct Nucleic Acid Amplification Test without RNA Extraction for SARS-CoV-2 Using a Portable PCR Thermocycler |journal=Genes |author=Wee, S.K.; Sivalingam, S.P.; Yap, E.P.H. |volume=11 |issue=6 |at=664 |year=2020 |doi=10.3390/genes11060664}}</ref> Other innovations include tweaking reagents and enzymes to work with one step, skipping the reverse transcription step,<ref name="CSIRFaster20">{{cite web |url=https://www.engineeringnews.co.za/article/faster-local-covid-19-test-kits-could-be-ready-by-year-end-2020-07-30/ |title=Faster, local COVID-19 test kits could be ready by year-end |author=Council for Scientific and Industrial Research |work=Engineering News |publisher=Creamer Media |date=30 July 2020 |accessdate=07 August 2020}}</ref> and using saliva-based molecular testing that skips RNA extraction.<ref name="RanoaSaliva20">{{cite journal |title=Saliva-Based Molecular Testing for SARS-CoV-2 that Bypasses RNA Extraction |journal=bioRxiv |author=Ranoa, D.R.E.; Holland, R.L.; Alnaji, F.G. et al. |year=2020 |doi=10.1101/2020.06.18.159434}}</ref>
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| ====3.1.2 Pooled testing====
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| 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."<ref name="RohdeCOVID20">{{cite web |url=https://asm.org/Articles/2020/July/COVID-19-Pool-Testing-Is-It-Time-to-Jump-In |title=COVID-19 Pool Testing: Is It Time to Jump In? |author=Rohde, R. |publisher=American Society for Microbiology |date=20 July 2020 |accessdate=06 August 2020}}</ref> 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.<ref name="RohdeCOVID20" /><ref name="MashaPooled20">{{cite web |url=https://asiatimes.com/2020/08/pooled-virus-tests-help-stretched-health-services/ |title=Pooled virus tests help stretched health services |author=Masha, M.; Chau, S. |work=Asia Times |date=04 August 2020 |accessdate=06 August 2020}}</ref><ref name="CitronerHowPool20">{{cite web |url=https://www.healthline.com/health-news/how-pooled-testing-can-help-us-fight-spread-of-covid-19 |title=How Pooled Testing Can Help Us Fight Spread of COVID-19 |author=Citroner, G. |work=Healthline |date=03 August 2020 |accessdate=06 August 2020}}</ref>
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| 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.<ref name="RohdeCOVID20" /><ref name="MashaPooled20" /><ref name="CitronerHowPool20" />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.<ref name="RohdeCOVID20" /> (Validation of pooled methods may differ in other countries.<ref name="MashaPooled20" />) The U.S. Centers for Disease Control and Prevention (CDC) has published [https://www.cdc.gov/coronavirus/2019-ncov/lab/pooling-procedures.html interim guidance] on pooled testing strategies for [[SARS-CoV-2]].
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| ====3.1.3 Rapid antigen testing====
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| 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.<ref name="ServiceRadical2020">{{cite journal |title=Radical shift in COVID-19 testing needed to reopen schools and businesses, researchers say |journal=Science |author=Service, R.F. |year=2020 |doi=10.1126/science.abe1546}}</ref><ref name="GuglielmiTheExp20">{{cite journal |title=The explosion of new coronavirus tests that could help to end the pandemic |journal=Nature |author=Guglielmi, G. |volume=583 |pages=506–09 |year=2020 |doi=10.1038/d41586-020-02140-8}}</ref> 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.<ref name="FDAEmerg20">{{cite web |url=https://www.fda.gov/medical-devices/coronavirus-disease-2019-covid-19-emergency-use-authorizations-medical-devices/vitro-diagnostics-euas |title=In Vitro Diagnostics EUAs |publisher=U.S. Food and Drug Administration |date=11 August 2020 |accessdate=12 August 2020}}</ref> With six U.S. states already contracted to purchase hundreds of thousands of the two companies' test kits<ref name="ClarkCOVID20">{{cite web |url=https://www.medpagetoday.com/infectiousdisease/covid19/87930 |title=COVID Antigen Tests: Coming to Case Counts Near You? |author=Clark, C. |work=MedPage Today |date=06 August 2020 |accessdate=07 August 2020}}</ref><ref name="MervoshItsLike20">{{cite web |url=https://www.nytimes.com/2020/08/04/us/virus-testing-delays.html |title=‘It’s Like Having No Testing’: Coronavirus Test Results Are Still Delayed |author=Mervosh, S.; Fernandez, M. |work=The New York Times |date=04 August 2020 |accessdate=05 August 2020}}</ref>, it's not clear how well they'll manage to meet demand.
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| ====3.1.4 LAMP and CRISPR====
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| 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 test|nucleic acid amplification]] technique that allows for RNA amplification, was also quietly being discussed<ref name="LambRapid20">{{cite journal |title=Rapid Detection of Novel Coronavirus (COVID-19) by Reverse Transcription-Loop-Mediated Isothermal Amplification |journal=medRxiv |author=Lamb, L.E.; Barolone, S.N.; Ward, E. et al. |year=2020 |doi=10.1101/2020.02.19.20025155}}</ref><ref name="Schmid-BurgkLAMP20">{{cite journal |title=LAMP-Seq: Population-Scale COVID-19 Diagnostics Using Combinatorial Barcoding |journal=bioRxiv |author=Schmid-Burgk, J.L.; Li, D.; Feldman, D. et al. |year=2020 |url=https://www.biorxiv.org/content/10.1101/2020.04.06.025635v2.article-info |doi=10.1101/2020.04.06.025635}}</ref>, and it has since gained more attention.<ref name="YuRapid20">{{cite journal |title=Rapid Detection of COVID-19 Coronavirus Using a Reverse Transcriptional Loop-Mediated Isothermal Amplification (RT-LAMP) Diagnostic Platform |journal=Clinical Chemistry |author=Yu, L.; Wu, S.; Hao, X. et al. |volume=66 |issue=7 |pages=975–77 |year=2020 |doi=10.1093/clinchem/hvaa102 |pmid=32315390 |pmc=PMC7188121}}</ref><ref name="ParkDevelop20">{{cite journal |title=Development of Reverse Transcription Loop-Mediated Isothermal Amplification Assays Targeting Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) |journal=Journal of Molecular Diagnostics |author=Park, G.-S.; Ku, K.; Baek, S.-H. et al. |volume=22 |issue=6 |pages=729–35 |year=2020 |doi=10.1016/j.jmoldx.2020.03.006 |pmid=32276051 |pmc=PMC7144851}}</ref><ref name="KellnerARapid20">{{cite journal |title=A rapid, highly sensitive and open-access SARS-CoV-2 detection assay for laboratory and home testing |journal=bioRxiv |author=Kellner, M.J.; Ross, J.J.; Schnabl, J. et al. |year=2020 |doi=10.1101/2020.06.23.166397}}</ref><ref name="ThiAColor20">{{cite journal |title=A colorimetric RT-LAMP assay and LAMP-sequencing for detecting SARS-CoV-2 RNA in clinical samples |journal=Science Translational Medicine |author=Thi, V.L.D.; Herbst, K.; Boerner, K. et al. |at=eabc7075 |year=2020 |doi=10.1126/scitranslmed.abc7075 |pmid=32719001}}</ref><ref name="EsbinOver20">{{cite journal |title=Overcoming the bottleneck to widespread testing: a rapid review of nucleic acid testing approaches for COVID-19 detection |journal=RNA |author=Esbin, M.N.; Whitney, O.N.; Chong, S. et al. |volume=26 |issue=7 |pages=771–83 |year=2020 |doi=10.1261/rna.076232.120 |pmid=32358057 |pmc=PMC7297120}}</ref><ref name="HaleOxford20">{{cite web |url=https://www.fiercebiotech.com/medtech/oxford-researchers-develop-portable-covid-19-test-costing-less-than-25 |title=Oxford researchers develop portable COVID-19 test costing less than $25 |author=Hale, C. |work=Fierce Biotech |date=09 July 2020 |accessdate=07 August 2020}}</ref> 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."<ref name="HaleOxford20" /> 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."<ref name="ThiAColor20" /> And California-based Color Genomics have set up their own proprietary RT-LAMP system, capable of handling up to 10,000 tests per day.<ref name="SheridanThisCal20">{{cite web |url=https://www.statnews.com/2020/08/06/better-simpler-faster-covid-19-test/ |title=This California company has a better version of a simpler, faster Covid-19 test |author=Sheridan, K. |work=STAT |date=06 August 2020 |accessdate=08 August 2020}}</ref>
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| 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<ref name="SheridanThisCal20" /><ref name="GuglielmiTheExp20">{{cite journal |title=The explosion of new coronavirus tests that could help to end the pandemic |journal=Nature |author=Guglielmi, G. |volume=583 |pages=506–09 |year=2020 |doi=10.1038/d41586-020-02140-8}}</ref><ref name="HeidtSaliva20">{{cite web |url=https://www.the-scientist.com/news-opinion/saliva-tests-how-they-work-and-what-they-bring-to-covid-19-67720 |title=Saliva Tests: How They Work and What They Bring to COVID-19 |author=Heidt, A. |work=The Scientist |date=09 July 2020 |accessdate=08 August 2020}}</ref>, 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.<ref name="EsbinOver20" /> Esbin ''et al.'' add<ref name="EsbinOver20" />:
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| <blockquote>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.</blockquote>
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| CRISPR methods are also being used in conjunction with RT-LAMP.<ref name="GuglielmiTheExp20" /><ref name="EsbinOver20" /><ref name="BroughtonCRISPR20">{{cite journal |title=CRISPR–Cas12-based detection of SARS-CoV-2 |journal=Nature Biotechnology |author=Broughton, J.P.; Deng, X.; Yu, G. et al. |volume=38 |pages=870–74 |year=2020 |doi=10.1038/s41587-020-0513-4 |pmid=32300245}}</ref> 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<ref name="GuglielmiTheExp20" /><ref name="GDHCRISPR20">{{cite web |url=https://www.medicaldevice-network.com/comment/crispr-biotechnology-disrupt-covid-19-testing-market/ |title=CRISPR biotechnology set to disrupt Covid-19 testing market |author=GlobalData Healthcare |work=Verdict Medical Devices |date=14 July 2020}}</ref>, 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.<ref name="EsbinOver20" /><ref name="GDHCRISPR20" />
| |
|
| |
| ====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.<ref name="WHOCOVIDTarget20">{{cite web |url=https://www.who.int/publications/m/item/covid-19-target-product-profiles-for-priority-diagnostics-to-support-response-to-the-covid-19-pandemic-v.0.1 |title=COVID-19 Target product profiles for priority diagnostics to support response to the COVID-19 pandemic v.0.1 |author=World Health Organization |publisher=World Health Organization |date=05 August 2020 |accessdate=12 August 2020}}</ref> Addressing POC testing, the WHO recommends that such assays<ref name="WHOCOVIDTarget20" /><ref name="PeplowRapid20">{{cite web |url=https://cen.acs.org/analytical-chemistry/diagnostics/Rapid-COVID-19-testing-breaks/98/web/2020/08 |title=Rapid COVID-19 testing breaks free from the lab |author=Peplow, M. |work=Chemical & Engineering News |date=10 August 2020 |accessdate=12 August 2020}}</ref>:
| |
|
| |
| * 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.<ref name="PeplowRapid20" /><ref name="KriegerCorona20">{{cite web |url=https://www.mercurynews.com/2020/08/10/coronavirus-how-to-test-everyone-all-the-time/ |title=Coronavirus: How to test everyone, all the time |author=Krieger, L.M. |work=The Mercury News |date=10 August 2020 |accessdate=12 August 2020}}</ref><ref name="KriegerCorona20">{{cite web |url=https://www.mercurynews.com/2020/08/10/coronavirus-how-to-test-everyone-all-the-time/ |title=Coronavirus: How to test everyone, all the time |author=Krieger, L.M. |work=The Mercury News |date=10 August 2020 |accessdate=12 August 2020}}</ref><ref name="BrownPoint20">{{cite web |url=https://www.mcknights.com/news/point-of-care-testing-could-be-biggest-advance-in-covid-19-fight/ |title=Point-of-care testing could be ‘biggest advance’ in COVID-19 fight |author=Brown, D. |work=McKnight's |date=10 August 2020 |accessdate=12 August 2020}}</ref><ref name="WissonCOVID20">{{cite web |url=https://www.healtheuropa.eu/covid-19-and-effective-cohorting-rapid-point-of-care-triage-testing/101696/ |title=COVID-19 and effective cohorting: Rapid point of care triage testing |author=Wisson, J. |work=Health Europa |date=28 July 2020 |accessdate=12 August 2020}}</ref> 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."<ref name="TrombergRapid20">{{cite journal |title=Rapid Scaling Up of Covid-19 Diagnostic Testing in the United States — The NIH RADx Initiative |journal=New England Journal of Medicine |author=Tromberg, B.J.; Schwetz, T.A.; Pérez-Stable, E.J. et al. |year=2020 |doi=10.1056/NEJMsr2022263}}</ref> 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.<ref name="NIHDelivering20">{{cite web |url=https://www.nih.gov/news-events/news-releases/nih-delivering-new-covid-19-testing-technologies-meet-us-demand |title=NIH delivering new COVID-19 testing technologies to meet U.S. demand |author=National Institutes of Health |publisher=National Institutes of Health |work=News Releases |date=31 July 2020 |accessdate=12 August 2020}}</ref> 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<ref name="FDAEmerg20" />, 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<ref name="EsbinOver20" /><ref name="WissonCOVID20" /><ref name="Leichman10Ways20">{{cite web |url=https://www.israel21c.org/how-israeli-scientists-are-improving-corona-testing/ |title=10 ways Israeli scientists are improving corona testing |author=Leichman, A.K. |work=Isael21c |date=27 July 2020 |accessdate=11 August 2020}}</ref>:
| |
|
| |
| * 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 bead-based kits;
| |
| * 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 kind of space, equipment, and supplies will you need?===
| |
| ====3.2.1 Laboratory space arrangements====
| |
| '''PCR considerations'''
| |
|
| |
| Whether adding PCR into your existing laboratory, modifying existing PCR workflows, or starting from scratch, preventing contamination is a top priority. As PCR can effectively amplify even the tiniest of quantities of DNA and RNA, the risk of amplifying a contaminant and ruining the validity of an assay is very real.<ref name="MifflinSetting03">{{cite book |url=http://www.biosupplynet.com/pdf/01_pcr_primer_p.5_14.pdf |format=PDF |chapter=Chapter 1: Setting Up a PCR Laboratory |title=PCR Primer |author=Mifflin, T.E. |editor=Dieffenbach, C.; Dveksler, G. |edition=2nd |publisher=Cold Spring Harbor Laboratory Press |pages=5–14 |year=2003 |isbn=9780879696542 |accessdate=13 August 2020}}</ref><ref name="RochePCR06">{{cite book |url=https://www.gene-quantification.de/ras-pcr-application-manual-3rd-ed.pdf |format=PDF |chapter=Chapter 2: General Guidelines |title=PCR Applications Manual |editor=Degen, H.-J.; Deufel, A.; Eisel, D. et al. |edition=3rd |publisher=Roche Diagnostics GmbH |pages=19–38 |year=2006 |accessdate=13 August 2020}}</ref><ref name="AhmedSetting14">{{cite book |url=http://grcpk.com/wp-content/uploads/2014/10/12.-Setting-up-PCR-Lab.pdf |format=PDF |chapter=Chapter 12: Setting-up a PCR Lab |title=Manual of PCR |author=Ahmed, S. |publisher=Genetics Resource Centre |year=2014 |accessdate=13 August 2020}}</ref><ref name="RedigTheDevil14">{{cite web |url=https://bitesizebio.com/19880/the-devil-is-in-the-details-how-to-setup-a-pcr-laboratory/ |title=The Devil is in the Details: How to Setup a PCR Laboratory |author=Refig, J. |work=BiteSizeBio |date=01 August 2014 |accessdate=13 August 2020}}</ref><ref name="BioChekTheBasics18">{{cite web |url=https://www.biochek.com/wp-content/uploads/2018/05/BioChek-E-book-The-basics-of-PCR.pdf |format=PDF |title=The basics of PCR: Detecting viruses and bacteria red-handed |publisher=BioChek BV |date=May 2018 |accessdate=13 August 2020}}</ref><ref name="DasMitig18">{{cite journal |title=Mitigating PCR /Amplicon Contamination in a High Risk High Burden Mycobacterial Reference Laboratory in a Resource Limited Setting |journal=Mycobacterial Diseases |author=Das, P.K.; Ganguly, S.B.; Mandal, B. |volume=8 |issue=2 |at=261 |year=2018 |doi=10.4172/2161-1068.1000261}}</ref><ref name="WHODos18">{{cite web |url=https://www.who.int/malaria/areas/diagnosis/molecular-testing-dos-donts/en/ |title=Dos and Don'ts for molecular testing |author=World Health Organization |publisher=World Health Organization |date=31 January 2018 |accessdate=14 August 2020}}</ref> Contamination typically comes from non-amplified environmental substances such as aerosols, and from carryover contamination of amplicons from earlier PCR cycles. As such, not only do best-practice processes and procedures (P&P) need to be followed (e.g., unidirectional workflow, thorough cleaning procedures, proper preparation and disposal), but also where to place PCR-related equipment must be carefully considered.<ref name="MifflinSetting03" /><ref name="RochePCR06" /><ref name="RedigTheDevil14" /><ref name="DasMitig18" />
| |
|
| |
| Where possible, separate rooms for sample preparation, PCR setup, and post-PCR activities, each with their own airflow control, are encouraged.<ref name="MifflinSetting03" /><ref name="RochePCR06" /><ref name="BioChekTheBasics18" /><ref name="DasMitig18" /><ref name="WHODos18" /> However, the laboratory attempting to add PCR to an already small clinical diagnostic lab may not have the luxury of having multiple rooms. In that case, a single-room setup may suffice, if the workflow areas remain demarcated or physically partitioned. Additionally, a single-room setup must also have stricter P&P and design controls to offset the space constraints. For example, the sample preparation area of the room should have a laminar flow hood with UV light that is regularly cleaned, and post-PCR analysis may need to occur later in the day after cleanup from prior steps.<ref name="MifflinSetting03" /><ref name="AhmedSetting14" /><ref name="WHODos18" /> Of course, always maintaining unidirectional workflow—regardless of number of rooms—is also criticial to minimizing contamination. For example, technicians shouldn't be transporting amplified materials into the DNA extraction area.
| |
|
| |
| Although dated, Roche Diagnostics' 2006 ''PCR Applications Manual''<ref name="RochePCR06" /> provides a detailed breakdown of setting up the laboratory for PCR. Das ''et al.''<ref name="DasMitig18" /> and Dr. Jennifer Redig<ref name="RedigTheDevil14" /> provide additional valuable insight. The World Health Organization (WHO) also provides guidance for molecular testing.<ref name="WHODos18" />
| |
|
| |
| '''Isothermal amplification considerations'''
| |
|
| |
| Similarly, because DNA and RNR amplification is involved, contamination concerns exist with isothermal amplification techniques. Multiple pipetting steps and repeated freezing and thawing of reagents can still lead to cross-contamination<ref name="DiegoProgress19">{{cite journal |title=Progress in loop-mediated isothermal amplification assay for detection of Schistosoma mansoni DNA: Towards a ready-to-use test |journal=Scientific Reports |author=Diego, J. G.-B.; Fernández-Soto, P.; Crego-Vicente, B. et al. |volume=9 |at=14744 |year=2019 |doi=10.1038/s41598-019-51342-2 |pmid=31611563 |pmc=PMC6791938}}</ref>, as does opening the reaction chamber after reaction is completed.<ref name="MartzyChall19">{{cite journal |title=Challenges and perspectives in the application of isothermal DNA amplification methods for food and water analysis |journal=Analytical and Bioanalytical Chemistry |author=Martzy, R.; Kolm, C.; Krska, R. et al. |volume=411 |pages=1695–1702 |year=2019 |doi=10.1007/s00216-018-1553-1 |pmid=30617408 |pmc=PMC6453865}}</ref> However, the advent of microfluidics and lateral flow technologies in isothermal amplification processes has seen the development of "fully enclosed microstructured devices into which performing the isothermal amplification reduces the risk of sample contamination and allows integration and portable device realization."<ref name="ZanoliIsotherm13">{{cite journal |title=Isothermal Amplification Methods for the Detection of Nucleic Acids in Microfluidic Devices |journal=Biosensors |author=Zanoli, L.M.; Spoto, G. |volume=3 |issue=1 |pages=18–43 |year=2013 |doi=10.3390/bios3010018 |pmid=25587397 |pmc=PMC4263587}}</ref><ref name="RoskosSimple13">{{cite journal |title=Simple System for Isothermal DNA Amplification Coupled to Lateral Flow Detection |journal=PLoS One |author=Roskos, K.; Hickerson, A.I.; Lu, H.-W. et al. |volume=8 |issue=7 |at=e69355 |year=2013 |doi=10.1371/journal.pone.0069355 |pmid=23922706 |pmc=PMC3724848}}</ref> Even more cutting-edge techniques to reduce contamination such as the CUT-LAMP technique of Bao ''et al.''<ref name="BaoCUT20">{{cite journal |title=CUT-LAMP: Contamination-Free Loop-Mediated Isothermal Amplification Based on the CRISPR/Cas9 Cleavage |journal=ACS Sensors |author=Bao, Y.; Jiang, Y.; Xiong, E. et al. |volume=5 |issue=4 |pages=1082–91 |year=2020 |doi=10.1021/acssensors.0c00034 |pmid=32242409}}</ref> or the dUTP/UDG system for COVID-19 RT-LAMP reactions of Kellner ''et al.''<ref name="KellnerARapid20" /> hold further promise in making isothermal amplification processes in the laboratory easier to manage. That said, labs running isothermal amplification processes such as LAMP requiring analysis with agarose gel electrophoresis or a method requiring the opening of reaction vessels will preferably have a secondary area set up for analysis steps so as to minimize the chances of contamination.<ref name="NEBLoop14">{{cite web |url=https://www.neb.com/protocols/2014/06/17/loop-mediated-isothermal-amplification-lamp |title=Loop-mediated Isothermal Amplification (LAMP) |publisher=New England BioLabs |date=17 June 2014 |accessdate=14 August 2020}}</ref><ref name="Fernández-SotoDevelop14">{{cite journal |title=Development of a Highly Sensitive Loop-Mediated Isothermal Amplification (LAMP) Method for the Detection of ''Loa loa'' |journal=PLoS One |author=Fernández-Soto, P.; Mvoulouga, P.O.; Akue, J.P. et al. |volume=9 |issue=4 |at=e94664 |year=2014 |doi=10.1371/journal.pone.0094664 |pmid=24722638 |pmc=PMC3983228}}</ref>
| |
|
| |
| ====3.2.2 Instruments and assays====
| |
| [[File:Eppendorf Mastercycler Pro S.jpg|right|400px|thumb|Eppendorf Mastercycler Pro S, a thermal cycler for PCR and other applications]]
| |
| '''High and moderate CLIA testing'''
| |
|
| |
| Thermal cyclers are the standard instruments for PCR testing. Today, real-time or quantitative (qPCR) systems largely fill this niche. However, [[Digital polymerase chain reaction|digital and droplet digital PCR]] systems are emerging, and they have the benefit of producing even more rapid, precise, sensitive, accurate, and reproducable results, and they are capable of direct quantification and multiplexing. Other instruments and accessories for PCR workflows include proper power supplies, analytical balances, electrophoresis chambers, water and/or dry baths, and mini/micro centrifuges. However, if you're considering the addition of PCR workflow to your laboratory, the thermal cycler is typically where the largest up-front cost will be. As such, it's important to ask yourself critical questions to help guide your acquisition decisions.
| |
|
| |
| As part of their June 2018 survey on PCR equipment, ''Lab Manager'' poses five questions potential buyers should ask before making PCR purchases<ref name="KerkhofPCR18">{{cite web |url=https://www.labmanager.com/surveys/what-to-look-for-in-a-pcr-system-2154 |title=PCR Equipment Survey Results |author=Kerkhof, J. |work=Lab Manager |date=13 June 2018 |accessdate=14 August 2020}}</ref>:
| |
|
| |
| # Do your current and long-term needs require basic PCR systems, qPCR systems, or digital PCR systems?
| |
| # What sample formats do you anticipate using?
| |
| # What throughput requirements do you have now and anticipate in the near future?
| |
| # What are you willing to sacrifice in regards to temperature ramp up and cool down times and accuracies?
| |
| # Do you anticipate needing to run more than one independent PCR at the same time (multiblock PCR)?
| |
|
| |
| Given the considerable investment that goes into these and other life science instruments, you may want to seek vendors who have a strong track record of supporting and supplying parts for instruments they manufacture and distribute years after the instruments are introduced.<ref name="LMResults20">{{cite web |url=https://www.labmanager.com/surveys/results-from-the-lab-manager-life-science-technology-survey-22257 |title=Results from the Lab Manager Life Science Technology Survey |author=Lab Manager |work=Lab Manager |date=07 April 2020 |accessdate=14 August 2020}}</ref>
| |
|
| |
| As for PCR-based assays, the U.S. FDA has issued EUAs for more than 100 of them. The most up-to-date listing is of course found at the [https://www.fda.gov/medical-devices/coronavirus-disease-2019-covid-19-emergency-use-authorizations-medical-devices/vitro-diagnostics-euas FDA website]. However, sorting through the extra details can be tedious. The Center for Systems Biology at Harvard has been maintaining a [https://csb.mgh.harvard.edu/covid contextual PDF chart] of the various COVID-19 diagnostic tests, which includes information such as run time, manufacturer-supplied data, and published clinical data (when available). This may prove useful in deciding on one or more particular tests.
| |
|
| |
| Isothermal amplification techniques have the advantage of not requiring an expensive thermal cycler.<ref name="ThiAColor20" /> Instrument-appropriate reaction vessels, baths, heating units, turbidimeters, thermocyclers, etc. may be required, depending on what type of amplification you're doing. Companies like Meridian Bioscience offer LAMP-based molecular platforms, though they may not offer a specific COVID-19 assay to run on the platform.<ref name="MBAlethia">{{cite web |url=https://www.meridianbioscience.com/platform/molecular/alethia/ |title=Alethia |publisher=Meridian Bioscience |accessdate=14 August 2020}}</ref> As can be seen in Table 1, two isothermal amplification assays that run on their own proprietary instrument have received EUAs and are CLIA-waved, with a third potentially on the way. Using these systems and their COVID-19 assays at the point of care provides an somewhat more attractive option for laboratories wanting to add COVID-19 or even multiplex viral assays to their offerings.
| |
|
| |
| '''CLIA-waived testing'''
| |
|
| |
| If you're running a POL, or attempting to provide COVID-19 testing at the point of care, you'll be looking at the following assay and instrument options:
| |
|
| |
| {|
| |
| | STYLE="vertical-align:top;"|
| |
| {| class="wikitable" border="1" cellpadding="5" cellspacing="0" width="80%"
| |
| |-
| |
| | style="background-color:white; padding-left:10px; padding-right:10px;" colspan="7"|'''Table 1.''' CLIA-waived COVID-19-related ''in vitro'' diagnostic tests receiving U.S. FDA Emergency Use Authorizations (EUAs)
| |
| |-
| |
| ! style="background-color:#e2e2e2; padding-left:10px; padding-right:10px;"|Date EUA issued
| |
| ! style="background-color:#e2e2e2; padding-left:10px; padding-right:10px;"|Manufacturer
| |
| ! style="background-color:#e2e2e2; padding-left:10px; padding-right:10px;"|Name of test or assay
| |
| ! style="background-color:#e2e2e2; padding-left:10px; padding-right:10px;"|Required instrument
| |
| ! style="background-color:#e2e2e2; padding-left:10px; padding-right:10px;"|Technology (method)
| |
| ! style="background-color:#e2e2e2; padding-left:10px; padding-right:10px;"|RADx-funded?
| |
| ! style="background-color:#e2e2e2; padding-left:10px; padding-right:10px;"|Additional comments
| |
| |-
| |
| | style="background-color:white; padding-left:10px; padding-right:10px;"|20 March 2020
| |
| | style="background-color:white; padding-left:10px; padding-right:10px;"|Cepheid
| |
| | style="background-color:white; padding-left:10px; padding-right:10px;"|[https://www.cepheid.com/coronavirus Xpert Xpress SARS-CoV-2 test]
| |
| | style="background-color:white; padding-left:10px; padding-right:10px;"|[https://www.cepheid.com/en_US/systems/GeneXpert-Family-of-Systems/GeneXpert-Xpress GeneXpert Xpress System (Tablet and Hub Configurations)]
| |
| | style="background-color:white; padding-left:10px; padding-right:10px;"|Molecular (RT-PCR)
| |
| | style="background-color:white; padding-left:10px; padding-right:10px;"|No
| |
| | style="background-color:white; padding-left:10px; padding-right:10px;"|Has largely received positive review of sensitivity and specificity<ref name="MoranDetect20">{{cite journal |title=Detection of SARS-CoV-2 by Use of the Cepheid Xpert Xpress SARS-CoV-2 and Roche cobas SARS-CoV-2 Assays |journal=Journal of Clinical Microbiology |author=Moran, A.; Beavis, K.G.; Matushek, S.M. et al. |volume=58 |issue=8 |at=e00772-20 |year=2020 |doi=10.1128/JCM.01072-20 |pmid=32303565 |pmc=PMC7383516}}</ref><ref name="LoeffelholzMulti20">{{cite journal |title=Multicenter Evaluation of the Cepheid Xpert Xpress SARS-CoV-2 Test |journal=Journal of Clinical Microbiology |author=Loeffelholz, M.J.; Alland, D.; Butler-Wu, S.M. et al. |volume=58 |issue=8 |at=e00926-20 |year=2020 |doi=10.1128/JCM.00926-20 |pmid=32366669 |pmc=PMC7383535}}</ref><ref name="GoldenbergerBrief20">{{cite journal |title=Brief validation of the novel GeneXpert Xpress SARS-CoV-2 PCR assay |journal=Journal of Virological Methods |author=Goldenberger, D.; Leusinger, K.; Sogaard, K.K. et al. |volume=284 |at=113925 |year=2020 |doi=10.1016/j.jviromet.2020.113925 |pmid=32659240 |pmc=PMC7351036}}</ref>; company is reportedly working on a multiplex assay for SARS-CoV-2, Flu A, Flu B, and RSV<ref name="PRNCepheidAnn20">{{cite web |url=https://www.prnewswire.com/news-releases/cepheid-announces-development-of-four-in-one-combination-test-for-sars-cov-2-flu-a-flu-b-and-rsv-301072489.html |title=Cepheid Announces Development of Four-in-One Combination Test for SARS-CoV-2, Flu A, Flu B and RSV |author=Cepheid |work=PR Newswire |date=09 June 2020 |accessdate=13 August 2020}}</ref>
| |
| |-
| |
| | style="background-color:white; padding-left:10px; padding-right:10px;"|23 March 2020
| |
| | style="background-color:white; padding-left:10px; padding-right:10px;"|Mesa Biotech Inc.
| |
| | style="background-color:white; padding-left:10px; padding-right:10px;"|[https://www.mesabiotech.com/coronavirus Accula SARS-CoV-2 test]
| |
| | style="background-color:white; padding-left:10px; padding-right:10px;"|[https://www.mesabiotech.com/products Accula Dock] or [https://www.sekisuidiagnostics.com/product-category/point-of-care-testing/?wpv_view_count=5737&wpv_post_search=silaris Silaris Dock]
| |
| | style="background-color:white; padding-left:10px; padding-right:10px;"|Molecular (RT-PCR)
| |
| | style="background-color:white; padding-left:10px; padding-right:10px;"|Yes
| |
| | style="background-color:white; padding-left:10px; padding-right:10px;"|Has received only minor scrutiny<ref name="HoganComp20">{{cite journal |title=Comparison of the Accula SARS-CoV-2 Test with a Laboratory-Developed Assay for Detection of SARS-CoV-2 RNA in Clinical Nasopharyngeal Specimens |journal=Journal of Clinical Microbiology |author=Hogan, C.A.; Garamani, N.; Lee, A.S. et al. |volume=58 |issue=8 |at=e01072-20 |year=2020 |doi=10.1128/JCM.01072-20 |pmid=32461285 |pmc=PMC7383558}}</ref>, with no formal FDA complaints<ref name="FDAMAUDE">{{cite web |url=https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfMAUDE/TextSearch.cfm |title=MAUDE - Manufacturer and User Facility Device Experience |publisher=U.S. Food and Drug Administration |accessdate=13 August 2020}}</ref>
| |
| |-
| |
| | style="background-color:white; padding-left:10px; padding-right:10px;"|27 March 2020
| |
| | style="background-color:white; padding-left:10px; padding-right:10px;"|Abbott Diagnostics Scarborough, Inc.
| |
| | style="background-color:white; padding-left:10px; padding-right:10px;"|[https://www.globalpointofcare.abbott/en/product-details/id-now-covid-19.html ID NOW COVID-19]
| |
| | style="background-color:white; padding-left:10px; padding-right:10px;"|[https://www.globalpointofcare.abbott/en/product-details/id-now.html ID NOW]
| |
| | style="background-color:white; padding-left:10px; padding-right:10px;"|Molecular (isothermal amplification)
| |
| | style="background-color:white; padding-left:10px; padding-right:10px;"|No
| |
| | style="background-color:white; padding-left:10px; padding-right:10px;"|Targets "a unique region of the RNA-dependent RNA polymerase (RdRP) gene"<ref name="RaviDiag20">{{cite journal |title=Diagnostics for SARS-CoV-2 detection: A comprehensive review of the FDA-EUA COVID-19 testing landscape |journal=Biosensors and Bioelectronics |author=Ravi, N.; Cortade, D.L.; Ng, E. et al. |volume=165 |at=112454 |year=2020 |doi=10.1016/j.bios.2020.112454 |pmid=32729549 |pmc=PMC7368663}}</ref>; as of August 2020, the sensitivity of the test is still under scrutiny<ref name="DevineCorona20">{{cite web |url=https://www.cnn.com/2020/07/03/politics/coronavirus-white-house-test-abbott/index.html |title=Coronavirus test used by White House has questionable accuracy |author=Devine, C. |work=CNN Politics |date=03 July 2020 |accessdate=08 July 2020}}</ref><ref name="PerroneFDA20">{{cite web |url=https://apnews.com/c8ab010e8e02dfe7beb34a5e5df11279 |title=FDA probes accuracy issue with Abbott’s rapid virus test |author=Perrone, M. |work=Associated Press |date=14 May 2020 |accessdate=19 May 2020}}</ref><ref name="BasuPerform20">{{cite journal |title=Performance of Abbott ID Now COVID-19 Rapid Nucleic Acid Amplification Test Using Nasopharyngeal Swabs Transported in Viral Transport Media and Dry Nasal Swabs in a New York City Academic Institution |journal=Journal of Clinical Microbiology |author=Basu, A.; Zinger, T.; Inglima, K. et al. |volume=58 |issue=8 |at=e01136-20 |year=2020 |doi=10.1128/JCM.01136-20 |pmid=32471894 |pmc=PMC7383552}}</ref><ref name="MitchellEval20">{{cite journal |title=Evaluation of the COVID19 ID NOW EUA assay |journal=Journal of Clinical Virology |author=Mitchell, S.L.; St. George, K. |volume=128 |at=104429 |year=2020 |doi=10.1016/j.jcv.2020.104429 |pmid=32425657 |pmc=PMC7227587}}</ref>
| |
| |-
| |
| | style="background-color:white; padding-left:10px; padding-right:10px;"|08 May 2020
| |
| | style="background-color:white; padding-left:10px; padding-right:10px;"|Quidel Corporation
| |
| | style="background-color:white; padding-left:10px; padding-right:10px;"|[https://www.quidel.com/immunoassays/rapid-sars-tests/sofia-sars-antigen-fia Sofia SARS Antigen FIA]
| |
| | style="background-color:white; padding-left:10px; padding-right:10px;"|[https://www.quidel.com/immunoassays/sofia-tests-kits/sofia-2-analyzer Sofia 2]
| |
| | style="background-color:white; padding-left:10px; padding-right:10px;"|Antigen
| |
| | style="background-color:white; padding-left:10px; padding-right:10px;"|Yes
| |
| | style="background-color:white; padding-left:10px; padding-right:10px;"|With a comparatively lower specificity, best used as surveillance, repeat screening tool<ref name="ServiceRadical2020" /><ref name="GuglielmiTheExp20" />
| |
| |-
| |
| | style="background-color:white; padding-left:10px; padding-right:10px;"|10 June 2020
| |
| | style="background-color:white; padding-left:10px; padding-right:10px;"|Cue Health Inc.
| |
| | style="background-color:white; padding-left:10px; padding-right:10px;"|[https://www.cuehealth.com/covid-19 Cue COVID-19 Test Cartridge]
| |
| | style="background-color:white; padding-left:10px; padding-right:10px;"|[https://www.cuehealth.com/covid-19 Cue Health Monitoring System]
| |
| | style="background-color:white; padding-left:10px; padding-right:10px;"|Molecular (isothermal amplification)
| |
| | style="background-color:white; padding-left:10px; padding-right:10px;"|No
| |
| | style="background-color:white; padding-left:10px; padding-right:10px;"|"Test primers amplify the nucleocapsid (N) region of the gene"<ref name="CueCOVID19Inst">{{cite web |url=https://www.cuehealth.com/s/IN9100003-1_10_IFU-Cue-COVID-19-Test-Cartridge-Pack-Professional.pdf |format=PDF |title=Cue COVID-19 Test Instructions for Use |publisher=Cue Health, Inc |date=June 2020 |accessdate=13 August 2020}}</ref>
| |
| |-
| |
| | style="background-color:white; padding-left:10px; padding-right:10px;"|02 July 2020
| |
| | style="background-color:white; padding-left:10px; padding-right:10px;"|Becton, Dickinson and Company
| |
| | style="background-color:white; padding-left:10px; padding-right:10px;"|[https://www.bd.com/en-us/offerings/capabilities/microbiology-solutions/point-of-care-testing/bd-veritor-plus-system-for-rapid-covid-19-sars-cov-2-testing BD Veritor System for Rapid Detection of SARS-CoV-2]
| |
| | style="background-color:white; padding-left:10px; padding-right:10px;"|[https://www.bd.com/en-us/offerings/capabilities/microbiology-solutions/point-of-care-testing/veritor-plus-system BD Veritor Plus]
| |
| | style="background-color:white; padding-left:10px; padding-right:10px;"|Antigen
| |
| | style="background-color:white; padding-left:10px; padding-right:10px;"|No
| |
| | style="background-color:white; padding-left:10px; padding-right:10px;"|With a comparatively lower specificity, best used as surveillance, repeat screening tool<ref name="ServiceRadical2020" /><ref name="GuglielmiTheExp20" />
| |
| |-
| |
| | style="background-color:white; padding-left:10px; padding-right:10px;"|N/A (Anticipated)
| |
| | style="background-color:white; padding-left:10px; padding-right:10px;"|Talis Biomedical
| |
| | style="background-color:white; padding-left:10px; padding-right:10px;"|[https://talis.bio/technology/ Talis One Cartridge]
| |
| | style="background-color:white; padding-left:10px; padding-right:10px;"|[https://talis.bio/technology/ Talis One Instrument]
| |
| | style="background-color:white; padding-left:10px; padding-right:10px;"|Molecular (RT-LAMP)
| |
| | style="background-color:white; padding-left:10px; padding-right:10px;"|Yes
| |
| | style="background-color:white; padding-left:10px; padding-right:10px;"|Expectations are that it will receive an FDA EUA and be CLIA-waived<ref name="TalisOne">{{cite web |url=https://talis.bio/technology/ |title=The Talis Advantage |publisher=Talis Biomedical |accessdate=13 August 2020}}</ref>, but yet to be determined.
| |
| |-
| |
| |}
| |
| |}
| |
|
| |
|
| |
| ====3.2.3 Reagents====
| |
| '''High and moderate CLIA testing'''
| |
|
| |
| '''CLIA-waived testing'''
| |
|
| |
| template DNA, PCR primers and probes, dNTPs, PCR buffers, enzymes, and master mixes
| |
|
| |
| ====3.2.4 Consumables====
| |
| '''High and moderate CLIA testing'''
| |
|
| |
| '''CLIA-waived testing'''
| |
|
| |
| PCR tubes, plates, and other accessories
| |
| https://www.sigmaaldrich.com/labware/labware-products.html?TablePage=9577275
| |
|
| |
| ====3.2.5 Software and services====
| |
| Tie into the "System interoperability" section of the current Chapter 3
| |
|
| |
| ====3.2.6 Major 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
| |
|
| |
| https://www.thermofisher.com/search/browse/category/us/en/602552/PCR+Machines+%28Thermal+Cyclers%29+%26+Accessories
| |
| https://www.thermofisher.com/us/en/home/life-science/dna-rna-purification-analysis.html
| |
| https://blog.biomeme.com/how-do-you-test-for-covid-19
| |
| https://www.fishersci.com/us/en/browse/90217014/thermal-cyclers
| |
| https://www.biocompare.com/PCR-Real-Time-PCR/6482-PCR-Equipment/
| |
|
| |
| ===3.3 What other considerations should be made?===
| |
| ====3.3.1 Regulatory compliance====
| |
| ====3.3.2 Reporting====
| |
|
| |
| ==References==
| |
| {{Reflist|colwidth=30em}}
| |
