Point-of-care COVID-19 antigen and nucleic acid amplification tests

This article will compare several Clinical Laboratory Improvement Amendments (CLIA) 88-waived COVID-19 nucleic acid amplification tests (NAATs) and antigen-based assays that can provide a diagnosis of COVID-19 before a patient leaves your office. We will also discuss how best to interpret results in relation to symptom onset and history of COVID-19 exposure. Note that we will not discuss COVID-19 antibody tests in this review.

COVID-19 point-of-care assays in perspective

One needs to understand the transmission of SARS- CoV-2 infections, as well as the concept of “viral load” to put COVID-19 point-of-care-tests (POCTs) in perspective.

The incubation period for COVID-19 ranges from 2 to 14 days following exposure, with most cases showing symptoms approximately 5 days after exposure. Individuals are most contagious just prior to or just after symptom onset.^1-5 This means that the timing of obtaining a specimen relative to exposure or symptom onset is critical in interpreting the results of COVID-19 POCTs.

Secondly, the reference standard for SARS-CoV-2 assays is a quantitative reverse transcription-polymerase chain reaction (RT-qPCR) assay. These are performed at large, centralized laboratories. The accuracy of COVID-19 POCTs is determined by comparing their results with those produced by these assays.

Finally, when a sample is obtained from the throat, sputum, saliva, anterior nares, or nasopharynx, the specimen is placed in a viral transport medium and sent to a

lab. The amount of virus in the sample is critical to the ability of the test to detect SARS-CoV-2 and depends on:

• The viral load of the patient, ie, the amount of virus available to be captured by sampling. The viral load in asymptomatic patients may be as high as in those with symptoms.^1-5
• The quantity of viruses captured by the swab. The skill and technique of the individuals obtaining specimens likely affects this. More viruses can be captured if a double-swab technique is used (ie, both nares are swabbed).
• The specimen source. Viral loads are generally higher when nasopharyngeal specimens are obtained, compared with nares, throat, and saliva specimens.

COVID-19 antigen POCTs vs nucleic acid antigen tests

No matter which POCT you perform, you need to comply with the Centers for Disease Control and Prevention guidelines regarding using appropriate personal protective equipment (PPE) when obtaining a specimen, as this is considered a procedure that can produce aerosolized virus.⁶ For personnel, this means, when collecting specimens, maintaining proper infection control and use recommended PPE. Follow manufacturers’ guidelines regarding processing specimens.

Theoretically, SARS-CoV-2 nucleocapsid proteins can be detected via antigen POCTs once an individual is infected. However, the sensitivity of antigen detection is lower than that of RT-PCR methods and are accurate for a few days after symptom onset. Therefore, a positive SARS-CoV-2 antigen POCT in practice should be considered a “true” positive, but a negative antigen does not rule out a COVID-19, and a NAAT or reference assay should be performed. Advantages of COVID-19 antigen POCTs are their low cost and rapid results. One needs to consider a negative COVID-19 antigen test in the context of the time of symptom onset in an ill patient and whether the asymptomatic patient was exposed to someone with a COVID-19 infection.

Sensitivities varied significantly between COVID-19 antigen POCTs in 48 studies, according to a Cochrane review published earlier this year.⁷ Average sensitivity was high in the first week when symptomatic patients were tested, whereas sensitivity of antigen tests were 78% compared with 51% when patients were tested after 1 week. Asymptomatic sensitivities were low, averaging 58%.⁷

The Cochrane review reported 33 evaluations of several different NAATs, with most relating to the Abbott ID NOW and Cepheid Xpert Xpress assays.⁷ The average sensitivity of ID NOW was 73% and average specificity was 99.7%, whereas the Xpert Xpress system had a sensitivity of 100% and an average specificity of 97.2%.⁷ These studies did not distinguish between symptomatic and asymptomatic patients.⁷ Furthermore, it was noted that some of the ID NOW studies involved placement of the swab in viral culture media, which is no longer recommended by the manufacturer. In a recent study, the ID NOW COVID-19 NAAT had a positive predictive agreement of 95% and a negative predictive agreement of 97%, when the test is performed according to the current recommendations.⁸ In another study, the sensitivity of the ID NOW was 98% and the specificity was 97%.⁹

Popular options for COVID-19 antigen tests include the Abbott Binax-Now COVID-19 antigen test, the BD Veritor Plus system for rapid detection of SARS-CoV-2, and the Quidel Sofia 2 Fluorescent Immunoassay Analyzer.

These tests have many features in common as they all use lateral flow technology to detect the SARS-CoV-2 nucleocapsid protein antigen and take about the same time to perform. They all require nasal specimens, cost about $10 to $20 per test, and are reimbursed at about $35 per test. The BD Veritor system and the Quidel systems use dedicated readers to improve the accuracy of their assays.

There are several CLIA 88-waived COVID-19 PCR POCTs (generally referred to as NAATs) available, which vary considerably regarding time to yield results, complexity of performing an assay, additional viruses included in the test, and the costs of the test. Many offices are already using ID NOW or the Roche Cobas Liat tests for identifying patients with strep, influenza, or respiratory syncytial virus (RSV). Office NAATs are more sensitive assays compared with COVID-19 antigen POCTs and can yield positive results in the presymptomatic period and beyond 1 week of infection. Office NAATs can produce results in as little as 5 minutes and as long as an hour, with most producing results in 20 to 30 minutes. The Cepheid, BioFire, and Roche tests require that a specimen swab be placed in viral transport medium before testing (purchased separately), whereas the specimen swab is tested directly with the Abbott and Visby Medical tests. Although the costs of the tests vary with the volume of testing, one would expect to pay at least $50 per cartridge for the tests listed, except for the BioFire assay, which tests for many viruses and bacteria. The BioFire test is the first (and only, as of this writing) to have full US Food & Drug Administration De Novo classification (and not just emergency use authorization for identifying SARS-CoV-2). Payments for the tests listed depend on insurance coverage, but generally exceed $70 per test, except for the BioFire test, which generally will be paid at $500 or higher. Unlike the other tests listed, the Visby Medical COVID-19 assay is a self-contained testing system.

A prudent plan for COVID-19 POCTs

Given that it is advantageous to diagnose COVID-19 infections before a patient leaves the office, I recommend that all pediatricians utilize the inexpensive SARS-CoV-2 antigen test as a first-line test. Consider a positive test a true positive, but also consider that a negative antigen may need serial antigen testing, or testing with an office NAAT, or with a more sensitive hospital or lab service assay. If you already have a NAAT in your office that you use for influenza, RSV, and strep testing, purchase a quantity of the SARS-CoV-2 cartridges to be used if the antigen test is negative, but still suspect a COVID-19 infection. If you do not own a NAAT device, consider purchasing a quantity of the Visby Medical COVID-19 tests to use when antigen tests are negative.

The future

There are many exciting SARS-CoV-2 tests that are forthcoming. At least 1 test is based on CRISPR (clustered regularly interspaced short palindromic repeats technology), and others that can test an exhaled breath for the “chemical signature” of patients with COVID-19. Companies are now mobilized to help clinicians diagnose COVID-19 quickly and more accurately.

References

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2. Ra SH, Lim JS, Kim GU, Kim MJ, Jung J, Kim SH. Upper respiratory viral load in asymptomatic individuals and mildly symptomatic patients with SARS-CoV-2 infection. Thorax. 2021;76(1):61-63. Published correction appears in Thorax. 2021 May;76(5):e3. doi:10.1136/thoraxnjnl-2020-215042

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4. Wölfel R, Corman VM, Guggemos W, et al. Virological assessment of hospitalized patients with COVID-2019. Nature. 2020;581(7809):465-469. Published correction appears in Nature. 2020 Dec;588(7839):E35. doi:10.1038/s41586-020-2196-x

5. Larremore DB, Wilder B, Lester E, et al. Test sensitivity is secondary to frequency and turnaround time for COVID-19 surveillance. medRxiv. Published online September 8, 2020. doi:10.1101/2020.06.22.20136309

6. Guidance for SARS-CoV-2 point-of-care and rapid testing. Centers for Disease Control and Prevention. Updated July 8, 2021. Accessed June 30, 2021. https://www.cdc.gov/coronavirus/2019-ncov/lab/point-of-care-testing.html#updates-previous-content

7. Dinnes J, Deeks JJ, Berhane S, et al. Rapid, point-of-care antigen and molecular-based tests for diagnosis of SARS-CoV-2 infection. Cochrane Database Syst Rev. 2021; 3(3):CD013705. Published March 24, 2021. doi:10.1002/14651858.CD013705.pub2

8.Nguyen Van JC, Gerlier C, Pilmis B, et al. Prospective evaluation of ID NOW COVID-19 assay used as point-of-care test in an emergency department. medRxiv. Published April 4, 2021. doi:10.1101/2021.03.2921253909

9. Mahmoud SA, Ganesan S, Ibrahim E, et al. Evaluation of six different rapid methods for nucleic acid detection of SARS-COV-2 virus. J Med Virol. 2021;93(9):5538-5543. doi:10.1002/jmv.27090.

10. Hansen G, Marino J, Wang ZX, et al. Clinical performance of the point-of care cobas Liat for detection of SARS-CoV-2 in 20 minutes: a multicenter study. J Clin Microbiol. 2021; 59(2):e02811-20. Published January 21, 2021. doi:10.1128/JCM.02811-20

11. Renzoni A, Perez F, Ngo Nsoga MT, et al. Analytical evaluation of Visby Medical RT-PCR portable device for rapid detection of SARS-CoV-2. Diagnostics (Basel). 2021;11(5):813. Published April 29, 2021. doi:10.3390/diagnostics11050813

12. Creager HM, Cabrera B, Schnaubelt A, et al. Clinical evaluation of the BioFire Respiratory Panel 2.1 and detection of SARS-CoV-2. J Clin Virol. 2020;129:104538. doi:10.1016/j.jcv.2020.104538