Comparative Evaluation of Rapid Isothermal Amplification and Antigen Assays for Screening Testing of SARS-CoV-2
<p>Schematic of RT-RPA assay versus antigen-based test. (<b>A</b>) In RT-RTPA, viral RNA is coped to cDNA by reverse transcriptase, then degraded by RNase H. Using a forward and a FAM-labeled reverse pair of primers specific to a target sequence, the cDNA product is amplified by RPA, then denatured and hybridized to a biotinylated probe. FAM-labeled and biotin-labeled products are detected on a lateral flow strip using molecules specific for FAM and biotin and nanoparticles. (<b>B</b>) In an antigen test, protein targets are detected by a lateral flow strip using protein-specific antibodies and nanoparticles. (<b>C</b>) A mobile phone application was used to image capture, machine-read, and quantify test results. The average pixel intensity is quantified at the test line, control line, and background areas. The background-subtracted test line signal is then normalized to the background-subtracted control line and expressed at % of control. − (red), test signal below the limit of detection; + (orange), low test signal; ++ (blue), medium test signal; +++ (green), high test signal.</p> "> Figure 2
<p>Analytical sensitivity of the RT-RPA assay and the antigen test using nasal swab dilution specimens. (<b>A</b>) Lateral flow strips for the RT-RPA reactions with dilution specimens containing RNA copies ranging from 0 to 1000. (<b>B</b>) Plot from the RT-RPA assay results quantified by the mobile phone application. The x-axis corresponds to dilutions’ specimens with known input copies of SARS-CoV-2 RNA. The y-axis corresponds to background subtracted test signal normalized to the control line for each lateral flow strip. Test results (purple dots) less than 10% of control are considered negative results, which is indicated by the black dashed line. (<b>C</b>) Lateral flow strips for the antigen tests with dilution specimens containing RNA copies ranging from 0 to 200,000. (<b>D</b>) Plot from the antigen tests results quantified by the mobile phone application. The x-axis corresponds to dilutions’ specimens with known input copies of SARS-CoV-2 RNA. The y-axis corresponds to background subtracted test signal normalized to the control line for each lateral flow strip. Test results (blue dots) less than 10% of control are considered negative results, which is indicated by the black dashed line.</p> "> Figure 3
<p>Clinical performance of the RT-RPA assay and the antigen test using nasal swab specimens collected from individuals with or without COVID-19. (<b>A</b>) Comparative evaluation of the RT-RPA assay (purple) and the antigen test (blue) using nasal swab specimens from asymptomatic cases. Comparative performance between the tests was plotted according to qPCR positive (Ct values between <20 to <40) and negative results. (<b>B</b>) Comparative evaluation of the RT-RPA assay (purple) and the antigen test (blue) using nasal swab specimens from symptomatic cases. Comparative performance between the tests was plotted according to qPCR positive (Ct values between <20 to <40) and negative results.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Clinical Samples
2.2. qPCR
2.3. RT-RPA Assay
2.4. Antigen Test
2.5. Image Analysis
2.6. Statistics
3. Results
4. Discussion
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- El Sahly, H.M.; Baden, L.R.; Essink, B.; Doblecki-Lewis, S.; Martin, J.M.; Anderson, E.J.; Campbell, T.B.; Clark, J.; Jackson, L.A.; Fichtenbaum, C.J.; et al. Efficacy of the mRNA-1273 SARS-CoV-2 Vaccine at Completion of Blinded Phase. N. Engl. J. Med. 2021, 385, 1774–1785. [Google Scholar] [CrossRef] [PubMed]
- Barda, N.; Dagan, N.; Ben-Shlomo, Y.; Kepten, E.; Waxman, J.; Ohana, R.; Hernan, M.A.; Lipsitch, M.; Kohane, I.; Netzer, D.; et al. Safety of the BNT162b2 mRNA COVID-19 Vaccine in a Nationwide Setting. N. Engl. J. Med. 2021, 385, 1078–1090. [Google Scholar] [CrossRef]
- Sadoff, J.; Gray, G.; Vandebosch, A.; Cardenas, V.; Shukarev, G.; Grinsztejn, B.; Goepfert, P.A.; Truyers, C.; Fennema, H.; Spiessens, B.; et al. Safety and Efficacy of Single-Dose Ad26.COV2.S Vaccine against COVID-19. N. Engl. J. Med. 2021, 384, 2187–2201. [Google Scholar] [CrossRef] [PubMed]
- CDC. Mitigation Measures for COVID-19 in Households and Markets in Non-US Low-Resource Settings. 2021. Available online: https://www.cdc.gov/coronavirus/2019-ncov/global-covid-19/global-urban-areas.html#:~:text=%E2%80%A2%20Applying%20preventive%20measures%20that,of%20COVID%2D19 (accessed on 18 December 2021).
- CDC. Testing Strategies for SARS-CoV-2. 2021. Available online: https://www.cdc.gov/coronavirus/2019-ncov/lab/resources/sars-cov2-testing-strategies.html (accessed on 18 December 2021).
- Erster, O.; Beth-Din, A.; Asraf, H.; Levy, V.; Kabat, A.; Mannasse, B.; Azar, R.; Shifman, O.; Lazar, S.; Mandelboim, M.; et al. Specific Detection of SARS-CoV-2 B.1.1.529 (Omicron) Variant by Four Rt-Qpcr Differential Assays. medRxiv 2021. [Google Scholar] [CrossRef]
- Boger, B.; Fachi, M.M.; Vilhena, R.O.; Cobre, A.F.; Tonin, F.S.; Pontarolo, R. Systematic review with meta-analysis of the accuracy of diagnostic tests for COVID-19. Am. J. Infect. Control 2021, 49, 21–29. [Google Scholar] [CrossRef] [PubMed]
- Binnicker, M.J. Challenges and Controversies to Testing for COVID-19. J. Clin. Microbiol. 2020, 58, e01695-20. [Google Scholar] [CrossRef] [PubMed]
- Vandenberg, O.; Martiny, D.; Rochas, O.; van Belkum, A.; Kozlakidis, Z. Considerations for diagnostic COVID-19 tests. Nat. Rev. Microbiol. 2021, 19, 171–183. [Google Scholar] [CrossRef] [PubMed]
- Bwire, G.M.; Majigo, M.V.; Njiro, B.J.; Mawazo, A. Detection profile of SARS-CoV-2 using RT-PCR in different types of clinical specimens: A systematic review and meta-analysis. J. Med. Virol. 2021, 93, 719–725. [Google Scholar] [CrossRef]
- LeBlanc, J.J.; Heinstein, C.; MacDonald, J.; Pettipas, J.; Hatchette, T.F.; Patriquin, G. A combined oropharyngeal/nares swab is a suitable alternative to nasopharyngeal swabs for the detection of SARS-CoV-2. J. Clin. Virol. 2020, 128, 104442. [Google Scholar] [CrossRef]
- Kahn, R.; Kennedy-Shaffer, L.; Grad, Y.H.; Robins, J.M.; Lipsitch, M. Potential Biases Arising From Epidemic Dynamics in Observational Seroprotection Studies. Am. J. Epidemiol. 2021, 190, 328–335. [Google Scholar] [CrossRef]
- Hay, J.A.; Kennedy-Shaffer, L.; Kanjilal, S.; Lennon, N.J.; Gabriel, S.B.; Lipsitch, M.; Mina, M.J. Estimating epidemiologic dynamics from cross-sectional viral load distributions. Science 2021, 373, 6552. [Google Scholar] [CrossRef] [PubMed]
- Tom, M.R.; Mina, M.J. To Interpret the SARS-CoV-2 Test, Consider the Cycle Threshold Value. Clin. Infect. Dis. 2020, 71, 2252–2254. [Google Scholar] [CrossRef]
- Peccia, J.; Zulli, A.; Brackney, D.E.; Grubaugh, N.D.; Kaplan, E.H.; Casanovas-Massana, A.; Ko, A.I.; Malik, A.A.; Wang, D.; Wang, M.; et al. Measurement of SARS-CoV-2 RNA in wastewater tracks community infection dynamics. Nat. Biotechnol. 2020, 38, 1164–1167. [Google Scholar] [CrossRef] [PubMed]
- Randazzo, W.; Truchado, P.; Cuevas-Ferrando, E.; Simon, P.; Allende, A.; Sanchez, G. SARS-CoV-2 RNA in wastewater anticipated COVID-19 occurrence in a low prevalence area. Water Res. 2020, 181, 115942. [Google Scholar] [CrossRef] [PubMed]
- Salcedo, N.; Harmon, A.; Herrera, B.B. Pooling of Samples for SARS-CoV-2 Detection Using a Rapid Antigen Test. Front. Trop. Dis. 2021, 2, 7865. [Google Scholar] [CrossRef] [PubMed]
- Mina, M.J.; Parker, R.; Larremore, D.B. Rethinking COVID-19 Test Sensitivity—A Strategy for Containment. N. Engl. J. Med. 2020, 383, e120. [Google Scholar] [CrossRef] [PubMed]
- He, X.; Lau, E.H.Y.; Wu, P.; Deng, X.; Wang, J.; Hao, X.; Lau, Y.C.; Wong, J.Y.; Guan, Y.; Tan, X.; et al. Temporal dynamics in viral shedding and transmissibility of COVID-19. Nat. Med. 2020, 26, 672–675. [Google Scholar] [CrossRef] [Green Version]
- Zanza, C.; Racca, F.; Longhitano, Y.; Piccioni, A.; Franceschi, F.; Artico, M.; Abenavoli, L.; Maiese, A.; Passaro, G.; Volonnino, G.; et al. Risk Management and Treatment of Coagulation Disorders Related to COVID-19 Infection. Int, J. Environ. Res. Public Health 2021, 18, 1268. [Google Scholar] [CrossRef]
- Maiese, A.; Frati, P.; Del Duca, F.; Santoro, P.; Manetti, A.C.; La Russa, R.; Di Paolo, M.; Turillazzi, E.; Fineschi, V. Myocardial Pathology in COVID-19-Associated Cardiac Injury: A Systematic Review. Diagnostics 2021, 11, 1647. [Google Scholar] [CrossRef]
- Larremore, D.B.; Wilder, B.; Lester, E.; Shehata, S.; Burke, J.M.; Hay, J.A.; Tambe, M.; Mina, M.J.; Parker, R. Test sensitivity is secondary to frequency and turnaround time for COVID-19 screening. Sci. Adv. 2021, 7, eabd5393. [Google Scholar] [CrossRef]
- Nash, B.; Badea, A.; Reddy, A.; Bosch, M.; Salcedo, N.; Gomez, A. Validating and Modeling the Impact of High-Frequency Rapid Antigen Screening on COVID-19 Spread and Outcomes. J. Clin. Trials 2021, 11, 483. [Google Scholar]
- Harmon, A.; Chang, C.; Salcedo, N.; Sena, B.; Herrera, B.B.; Bosch, I.; Holberger, L.E. Validation of an At-Home Direct Antigen Rapid Test for COVID-19. JAMA Netw. Open 2021, 4, e2126931. [Google Scholar] [CrossRef] [PubMed]
- Chen, C.C.; Lu, S.C.; Bai, C.H.; Wang, P.Y.; Lee, K.Y.; Wang, Y.H. Diagnostic Accuracy of SARS-CoV-2 Antigen Tests for Community Transmission Screening: A Systematic Review and Meta-Analysis. Int. J. Environ. Res. Public Health 2021, 18, 11451. [Google Scholar] [CrossRef]
- Pagano, A.M.; Maiese, A.; Izzo, C.; Maiese, A.; Ametrano, M.; De Matteis, A.; Attianese, M.R.; Busato, G.; Caruso, R.; Cestari, M.; et al. COVID-19 Risk Management and Screening in the Penitentiary Facilities of the Salerno Province in Southern Italy. Int. J. Environ. Res. Public Health 2020, 17, 8033. [Google Scholar] [CrossRef] [PubMed]
- FDA. In Vitro Diagnostics EUAs—Antigen Diagnostic Tests for SARS-CoV-2; FDA: Silver Spring, ML, USA, 2021. [Google Scholar]
- Corman, V.M.; Haage, V.C.; Bleicker, T.; Schmidt, M.L.; Muhlemann, B.; Zuchowski, M.; Jo, W.K.; Tscheak, P.; Moncke-Buchner, E.; Muller, M.A.; et al. Comparison of seven commercial SARS-CoV-2 rapid point-of-care antigen tests: A single-centre laboratory evaluation study. Lancet Microbe 2021, 2, e311–e319. [Google Scholar] [CrossRef]
- Pickering, S.; Batra, R.; Merrick, B.; Snell, L.B.; Nebbia, G.; Douthwaite, S.; Reid, F.; Patel, A.; Kia Ik, M.T.; Patel, B.; et al. Comparative performance of SARS-CoV-2 lateral flow antigen tests and association with detection of infectious virus in clinical specimens: A single-centre laboratory evaluation study. Lancet Microbe 2021, 2, e461–e471. [Google Scholar] [CrossRef]
- Ganguli, A.; Mostafa, A.; Berger, J.; Aydin, M.Y.; Sun, F.; Ramirez, S.A.S.; Valera, E.; Cunningham, B.T.; King, W.P.; Bashir, R. Rapid isothermal amplification and portable detection system for SARS-CoV-2. Proc. Natl. Acad. Sci. USA 2020, 117, 22727–22735. [Google Scholar] [CrossRef]
- Baek, Y.H.; Um, J.; Antigua, K.J.C.; Park, J.H.; Kim, Y.; Oh, S.; Kim, Y.I.; Choi, W.S.; Kim, S.G.; Jeong, J.H.; et al. Development of a reverse transcription-loop-mediated isothermal amplification as a rapid early-detection method for novel SARS-CoV-2. Emerg. Microbes Infect. 2020, 9, 998–1007. [Google Scholar] [CrossRef] [Green Version]
- Rabe, B.A.; Cepko, C. SARS-CoV-2 detection using isothermal amplification and a rapid, inexpensive protocol for sample inactivation and purification. Proc. Natl. Acad. Sci. USA 2020, 117, 24450–24458. [Google Scholar] [CrossRef]
- Qian, J.; Boswell, S.A.; Chidley, C.; Lu, Z.X.; Pettit, M.E.; Gaudio, B.L.; Fajnzylber, J.M.; Ingram, R.T.; Ward, R.H.; Li, J.Z.; et al. An enhanced isothermal amplification assay for viral detection. Nat. Commun. 2020, 11, 5920. [Google Scholar] [CrossRef]
- Mancuso, C.P.; Lu, Z.X.; Qian, J.; Boswell, S.A.; Springer, M. A Semi-Quantitative Isothermal Diagnostic Assay Utilizing Competitive Amplification. Anal. Chem. 2021, 93, 9541–9548. [Google Scholar] [CrossRef] [PubMed]
- Piepenburg, O.; Williams, C.H.; Stemple, D.L.; Armes, N.A. DNA detection using recombination proteins. PLoS Biol. 2006, 4, e204. [Google Scholar] [CrossRef] [PubMed]
- Donato, L.J.; Trivedi, V.A.; Stransky, A.M.; Misra, A.; Pritt, B.S.; Binnicker, M.J.; Karon, B.S. Evaluation of the Cue Health point-of-care COVID-19 (SARS-CoV-2 nucleic acid amplification) test at a community drive through collection center. Diagn. Microbiol. Infect. Dis. 2021, 100, 115307. [Google Scholar] [CrossRef] [PubMed]
- Ptasinska, A.; Whalley, C.; Bosworth, A.; Poxon, C.; Bryer, C.; Machin, N.; Grippon, S.; Wise, E.L.; Armson, B.; Howson, E.L.A.; et al. Diagnostic accuracy of loop-mediated isothermal amplification coupled to nanopore sequencing (LamPORE) for the detection of SARS-CoV-2 infection at scale in symptomatic and asymptomatic populations. Clin. Microbiol. Infect. 2021, 27, e1341–e1347. [Google Scholar] [CrossRef] [PubMed]
- Dong, Y.; Wu, X.; Li, S.; Lu, R.; Li, Y.; Wan, Z.; Qin, J.; Yu, G.; Jin, X.; Zhang, C. Comparative evaluation of 19 reverse transcription loop-mediated isothermal amplification assays for detection of SARS-CoV-2. Sci. Rep. 2021, 11, 2936. [Google Scholar] [CrossRef] [PubMed]
- Paltiel, A.D.; Zheng, A.; Walensky, R.P. Assessment of SARS-CoV-2 Screening Strategies to Permit the Safe Reopening of College Campuses in the United States. JAMA Netw. Open 2020, 3, e2016818. [Google Scholar] [CrossRef]
- Ramdas, K.; Darzi, A.; Jain, S. ‘Test, re-test, re-test’: Using inaccurate tests to greatly increase the accuracy of COVID-19 testing. Nat. Med. 2020, 26, 810–811. [Google Scholar] [CrossRef]
- Pavelka, M.; Van-Zandvoort, K.; Abbott, S.; Sherratt, K.; Majdan, M.; CMMID COVID-19 Working Group; Inštitút Zdravotných Analýz; Jarcuska, P.; Krajci, M.; Flasche, S.; et al. The impact of population-wide rapid antigen testing on SARS-CoV-2 prevalence in Slovakia. Science 2021, 372, 635–641. [Google Scholar] [CrossRef]
Asymptomatic Phase | ||||||||
---|---|---|---|---|---|---|---|---|
qPCR | 95% CI | |||||||
+ | − | Total | PPA | 100.00% | 90.00% | 100.00% | ||
RT-RPA | + | 35 | 0 | 35 | NPA | 100.00% | 95.26% | 100.00% |
− | 0 | 76 | 76 | PPV | 100.00% | |||
Total | 35 | 76 | 111 | NPV | 100.00% | |||
qPCR | 95% CI | |||||||
+ | − | Total | PPA | 82.86% | 66.35% | 93.44% | ||
Antigen | + | 29 | 1 | 30 | NPA | 98.68% | 92.89% | 99.97% |
− | 6 | 75 | 81 | PPV | 96.67% | 80.45% | 99.51% | |
Total | 35 | 76 | 111 | NPV | 92.59% | 85.78% | 96.28% |
Symptomatic Phase | ||||||||
---|---|---|---|---|---|---|---|---|
qPCR | 95% CI | |||||||
+ | − | Total | PPA | 95.83% | 78.88% | 99.89% | ||
RT-RPA | + | 23 | 0 | 23 | NPA | 100.00% | 90.75% | 100.00% |
− | 1 | 38 | 39 | PPV | 100.00% | |||
Total | 24 | 38 | 62 | NPV | 97.44% | 84.80% | 99.62% | |
qPCR | 95% CI | |||||||
+ | − | Total | PPA | 91.67% | 73.00% | 98.97% | ||
Antigen | + | 22 | 0 | 22 | NPA | 100.00% | 90.75% | 100.00% |
− | 2 | 38 | 40 | PPV | 100.00% | |||
Total | 24 | 38 | 62 | NPV | 95.00% | 83.45% | 98.62% |
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Salcedo, N.; Sena, B.F.; Qu, X.; Herrera, B.B. Comparative Evaluation of Rapid Isothermal Amplification and Antigen Assays for Screening Testing of SARS-CoV-2. Viruses 2022, 14, 468. https://doi.org/10.3390/v14030468
Salcedo N, Sena BF, Qu X, Herrera BB. Comparative Evaluation of Rapid Isothermal Amplification and Antigen Assays for Screening Testing of SARS-CoV-2. Viruses. 2022; 14(3):468. https://doi.org/10.3390/v14030468
Chicago/Turabian StyleSalcedo, Nol, Brena F. Sena, Xiying Qu, and Bobby Brooke Herrera. 2022. "Comparative Evaluation of Rapid Isothermal Amplification and Antigen Assays for Screening Testing of SARS-CoV-2" Viruses 14, no. 3: 468. https://doi.org/10.3390/v14030468