Article

The emergence of the SARS-CoV-2 Delta variant highlights the importance of widespread vaccination and the ongoing need for accurate testing.

New variants can quickly change the public health situation

Positive developments related to the pandemic situation between the end of 2020 and early 2021 generated a great deal of hope. Multiple vaccines were starting to become available and by March 8, 2021, the CDC declared that fully vaccinated people could gather indoors without wearing masks.¹ At that time, many believed that the worst of the pandemic was in the past. However, the emergence of the SARS-CoV-2 Delta variant significantly impacted the public health situation across much of the United States, with the CDC issuing new masking guidance and urging more people to become vaccinated in a July 27, 2021, announcement.²

As we now know, the Delta variant is ~2x more contagious than previous variants,^2-5 which has led to very rapid spread in areas with low vaccination rates.⁵ In the United States, the 7-day moving average of reported cases increased from ~12,000 in late June to ~60,000 by July 27.² One of the main lessons the medical community can learn from the rapid spread of the Delta variant is how quickly a new variant can change the local public health situation from in to out of control. So how can we stop the emergence of new variants, one or more of which may be worse than Delta?

Stopping the emergence of new variants requires controlling disease spread

As with many viruses, the emergence of SARS-CoV-2 variants is the result of random errors introduced into the viral genome during replication.⁶ The best way to reduce the emergence of new variants is to limit virus replication by limiting transmission.⁷

Variants are expected. The best way to slow the emergence of new variants is to reduce the spread of infection by taking measures to protect yourself, including getting a COVID-19 vaccine when available.⁷

Until vaccination rates increase sufficiently to limit transmission, SARS-CoV-2 testing will continue to be needed

Despite the effectiveness of vaccines in reducing the risk of death from SARS-CoV-2 and limiting disease spread, vaccine hesitancy remains a challenge.^8,9 Given a vaccination level of ~55% of the total population in the United States,¹⁰ it seems plausible that we may see additional outbreaks from new variants, although Delta is the only variant of concern currently in the United States.¹¹

In addition, many scientists believe that COVID-19 is here to stay, becoming an endemic disease.^12,13 Whether COVID-19, during its transition to endemicity, becomes a milder disease and/or a disease of the young,^12,13 diagnostic testing will most likely continue to be an important tool for ensuring public health.

References

1. CDC. CDC Museum COVID-19 Timeline. Centers for Disease Control and Prevention. Published August 4, 2021. Accessed August 23, 2021. https://www.cdc.gov/museum/timeline/covid19.html
2. CDC. Coronavirus Disease 2019 (COVID-19). Delta Variant: What We Know About the Science. Centers for Disease Control and Prevention. Published February 11, 2020. Accessed September 24, 2021. https://www.cdc.gov/coronavirus/2019-ncov/variants/delta-variant.html
3. Li B, Deng A, Li K, et al. Viral Infection and Transmission in a Large, Well-Traced Outbreak Caused by the SARS-CoV-2 Delta Variant.; 2021:2021.07.07.21260122. doi:10.1101/2021.07.07.21260122
4. Wang Y, Chen R, Hu F, et al. Transmission, viral kinetics and clinical characteristics of the emergent SARS-CoV-2 Delta VOC in Guangzhou, China. EClinicalMedicine. 2021;40:101129. doi:10.1016/j.eclinm.2021.101129
5. Bian L, Gao Q, Gao F, et al. Impact of the Delta variant on vaccine efficacy and response strategies. Expert Rev Vaccines.:1-9. doi:10.1080/14760584.2021.1976153
6. Chen J, Wang R, Wei G-W. Review of the mechanisms of SARS-CoV-2 evolution and transmission. ArXiv. Published online September 15, 2021:arXiv:2109.08148v1.
7. CDC. Coronavirus Disease 2019 (COVID-19). What you need to know about variants. Centers for Disease Control and Prevention. Published February 11, 2020. Accessed September 24, 2021. https://www.cdc.gov/coronavirus/2019-ncov/variants/variant.html
8. Tram KH, Saeed S, Bradley C, et al. Deliberation, Dissent, and Distrust: Understanding Distinct Drivers of Coronavirus Disease 2019 Vaccine Hesitancy in the United States. Clin Infect Dis Off Publ Infect Dis Soc Am. Published online July 16, 2021:ciab633. doi:10.1093/cid/ciab633
9. Siegler AJ, Luisi N, Hall EW, et al. Trajectory of COVID-19 Vaccine Hesitancy Over Time and Association of Initial Vaccine Hesitancy With Subsequent Vaccination. JAMA Netw Open. 2021;4(9):e2126882. doi:10.1001/jamanetworkopen.2021.26882
10. CDC. COVID Data Tracker. Centers for Disease Control and Prevention. Published March 28, 2020. Accessed September 24, 2021. https://covid.cdc.gov/covid-data-tracker
11. CDC. Coronavirus Disease 2019 (COVID-19). SARS-CoV-2 Variant Classifications and Definitions. Centers for Disease Control and Prevention. Published February 11, 2020. Accessed September 24, 2021. https://www.cdc.gov/coronavirus/2019-ncov/variants/variant-info.html
12. Torjesen I. Covid-19 will become endemic but with decreased potency over time, scientists believe. BMJ. 2021;372:n494. doi:10.1136/bmj.n494
13. Phillips N. The coronavirus is here to stay — here’s what that means. Nature. 2021;590(7846):382-384. doi:10.1038/d41586-021-00396-2

Healthcare disparities in the US have become strikingly apparent throughout the COVID-19 pandemic, affecting historically underserved populations (e.g., lower-income households, Black and Minority Americans, immigrants, people with disabilities, rural populations, etc.). Point-of-care (POC) testing can help. To reduce the spread of infections and bring health equity forward, we need to take a closer look at POC testing and how to get it to communities that need it most.

Healthcare disparities in the US at the point of care

The rapidity of spread and mortality associated with the COVID-19 pandemic has shined a light on health disparities in the US. COVID-19 infection is higher among groups already affected by health disparities across age, race, ethnicity, language, income, and living conditions.¹ Since the beginning of the pandemic, the top-third of vulnerable counties (defined by the COVID-19 vulnerability Index) have seen 21% more cases and 47% more deaths than the bottom-third of vulnerable counties, despite receiving 27% fewer tests.^{2 3}

One study found that transportation barriers are associated with significantly higher odds of a positive COVID-19 test, which suggests a greater risk of disease exposure associated with reliance on public transit and/or shared rides, or it may suggest inadequate access to health care, including timely COVID-19 testing.¹ What’s more, COVID-19 has shifted the utilization of the American healthcare system where individuals are delaying or forgoing care—which can have serious and life-threatening health consequences. By June 2020, over 40% of U.S. adults reported that they delayed health care due to COVID-19 concerns.⁴

Adding to these challenges is the threat of false negative COVID-19 test results, which could potentially lead to positive case clusters.^{5 6} When false negatives are suspected, individuals have to leave the facility and wait for their reflex testing results to become available. Ideally, individuals awaiting reflex testing results (as well as those who receive a positive COVID-19 test result) will quarantine, but this is not always possible, especially for those relying on public transportation and living in overcrowded housing facilities.

Minimizing the time it takes to get accurate results and being able to communicate those results to patients quickly is paramount for minimizing community spread and delivering proper treatment. This supports the need for rapid and reliable testing, as well as in-visit test-to-answer POC workflows—especially for communities that have been shown to have less access to medical care and are less inclined to use telehealth service.

What Point-of-Care Diagnostic Tests Should Look Like

To drive health equity forward, we need diagnostic testing to be more accessible and reliable for underserved populations.

Point-of-care diagnostics should enable rapid and effective test-to-treat workflows all in one visit

Because underserved populations are more likely to miss appointments due to transportation and are more likely to have limited access to telehealth,^7-10 it is imperative that these individuals get the information and treatment they need while they are in the testing facility. To confidently make treatment decisions during the same visit, point of care diagnostic tests should be sensitive without compromising speed and operational efficiency.

Not only will test-to-treat workflows at the point of care help detect disease and deliver proper care earlier, but it can help minimize health care expenses due to missed appointments and hospitalization. This approach is stressed in the Sexually Transmitted Infections National Strategic Plan (2021-2025), where several objectives are discussed to ensure continuity of care. The plan touches on expanding personnel training to enhance screening, testing, and treatment capabilities within a single facility, specialized programs, and integrated and collaborative approaches in settings that serve communities disproportionately affected by STIs, HIV, and viral hepatitis.¹¹

Point-of-care diagnostics should be flexible enough to adapt to different environments and different communities

Point-of-care locations can range from large pop-up centers in rural areas and places of work to metropolitan mobile clinics. Each community is unique, and POC tests need to be adaptable to the specific needs of the community. The instruments being used to detect SARS-CoV-2 should be space efficient for multiple types of facilities and, ideally, flexible enough to test for multiple infectious diseases and disease variants that are prevalent in the community being tested. This can save time for both the individual and the testing facility.

As suggested by the Office of the Assistant Secretary of Health (OASH) and Office of Minority Health (OMH), to expand access to communities that are unable to access traditional testing sites, the federal government should partner with test kit manufacturers to develop SARS-CoV-2 test kits that are flexible enough to be used at the point of care or at-home and should establish and enforce policies that require public and private health insurance to cover SARS-CoV-2 testing to minimize financial barriers.¹²

Point-of-care diagnostics should be easy-to-implement and easy-to-use

Adopting new healthcare technologies is resource intensive—requiring a substantial amount of time for implementation and training. With healthcare personnel shortages increasing in underserved areas,¹³ it can be difficult to maintain an efficient operational workflow, or worse, patients cannot get the tests they need because there aren’t enough people to perform them. POC testing needs to be easy to implement and intuitive so that facilities can get up and running as quickly as possible and maintain efficiency through personnel changes.

Point-of-care diagnostics should have features that streamline reporting and communication to patients, labs and governing bodies

Timely reporting is essential for sharing results, monitoring where outbreaks occur, tracking the incidence and prevalence of infection, and evaluating the impact of interventions. Not only can it help determine if a newly placed mobile clinic is effective and quickly get test results to patients, but it also enables proper allocation of critical resources to those in greatest need.

Moving Towards Health Equity with Point-of-Care Testing

With the CDC planning to invest $2.25 Billion to address COVID-19-related health disparities and advance health equity among populations that are at high-risk and underserved,¹⁴ there is no better time than now to evaluate and adopt a new POC testing solution.

To ensure there is timely and equitable access to testing with rapid return of results in communities disproportionately affected by COVID-19, we need to consider the recommendations outlined by the CDC:^{1 5}

• Use a social vulnerability index¹⁶ to help select testing sites
• Review the groups to prioritize for screening testing
• Carefully consider the different types of SARS-CoV-2 tests when planning for diagnostic or screening use

By providing easy access to actionable results at the point-of-care in public health settings, the Talis One molecular testing solution empowers you to quickly and confidently diagnose infections, promote safety and peace of mind for your patients, and ultimately combat healthcare disparities in the US.

References

1. Rozenfeld, Yelena, et al. “A model of disparities: risk factors associated with COVID-19 infection.” International journal for equity in health 19.1 (2020): 1-10.
2. Bringing Greater Precision to the COVID-19 Response. (2020, December). Retrieved July 01, 2021, from https://precisionforcovid.org/ccvi
3. Smittenaar, P., Stewart, N., Sutermaster, S., Coome, L., Dibner-Dunlap, A., Jain, M., … & Sgaier, S. K. (2021). A COVID-19 Community Vulnerability Index to drive precision policy in the US. medRxiv.
4. Czeisler, Mark É., et al. “Delay or avoidance of medical care because of COVID-19–related concerns—United States, June 2020.” Morbidity and mortality weekly report 69.36 (2020): 1250.
5. False negative rate of COVID-19 PCR testing: A discordant testing analysis. (2021, January 9). Retrieved July 01, 2021, from https://virologyj.biomedcentral.com/articles/10.1186/s12985-021-01489-0
6. Cao G, Tang S, Yang D, Shi W, Wang X, Wang H, et al. The potential transmission of SARS-CoV-2 from patients with negative RT-PCR swab tests to others: two related clusters of COVID-19 outbreak. Jpn J Infect Dis. 2020. https://doi.org/10.7883/yoken.JJID.2020.165.
7. Samuels, R. C., Ward, V. L., Melvin, P., Macht-Greenberg, M., Wenren, L. M., Yi, J., … & Cox, J. E. (2015). Missed appointments: factors contributing to high no-show rates in an urban pediatrics primary care clinic. Clinical pediatrics, 54(10), 976-982.
8. Fischer, S. H., Ray, K. N., Mehrotra, A., Bloom, E. L., & Uscher-Pines, L. (2020). Prevalence and characteristics of Telehealth utilization in the United States. JAMA network open, 3(10), e2022302-e2022302.
9. Pierce, R. P., & Stevermer, J. J. (2020). Disparities in use of telehealth at the onset of the COVID-19 public health emergency. Journal of telemedicine and telecare, 1357633X20963893.
10. Vogels, E. (2021, June 22). Digital divide persists even as Americans with lower incomes make gains in tech adoption. Retrieved July 01, 2021, from https://www.pewresearch.org/fact-tank/2021/06/22/digital-divide-persists-even-as-americans-with-lower-incomes-make-gains-in-tech-adoption/
11. U.S. Department of Health and Human Services. 2020. Sexually Transmitted Infections National Strategic Plan for the United States: 2021–2025. Washington, DC.
12. COVID-19 Health Equity Task Force: Long COVID, PPE, Testing and Therapeutics Subcommittee Interim Recommendations. (2021, June 25). Retrieved July 01, 2021, from https://www.minorityhealth.hhs.gov/Assets/PDF/June-COVID19HETFSubcommitteesRecommendations-062521-508.pdf
13. Malayala, Srikrishna Varun, et al. “Primary care shortage in medically underserved and health provider shortage areas: Lessons from Delaware, USA.” Journal of Primary Care & Community Health 12 (2021): 2150132721994018.
14. CDC announces $2.25 billion to address Covid-19 health disparities in communities that are at high-risk and underserved. (2021, March 17). Retrieved July 01, 2021, from https://www.cdc.gov/media/releases/2021/p0317-COVID-19-Health-Disparities.html
15. Overview of Testing for SARS-CoV-2 (COVID-19). (2021, March 17). Retrieved July 01, 2021, from https://www.cdc.gov/coronavirus/2019-ncov/hcp/testing-overview.html
16. CDC/ATSDR Social Vulnerability Index. (2021, April 28). Retrieved July 01, 2021, from https://www.atsdr.cdc.gov/placeandhealth/svi/index.html

In the antigen vs molecular test debate, understanding which test is best is a matter of timing and application—the answer could change depending on how you’re using the test and how quickly you need the test results. In addition, the emergence of newer testing technologies may change the parameters of the debate, tipping the balance towards molecular testing for a number of situations.

To understand which test technology will work best for your application, let’s take a closer look at the pros and cons of each technology (note that for the sake of brevity, we are leaving the discussion of sample type for another article).

Molecular tests performed in a central lab are sensitive and specific, but take time to deliver results

What a molecular test detects

In general, molecular tests detect nucleic acid—either RNA or DNA.

Why this matters for diagnosing COVID-19

Molecular tests can detect the presence of SARS-CoV-2 even during the early stages of infection because they detect viral RNA, which can accumulate to high levels within a cell before virus particles are formed.

Benefits of molecular tests

Molecular tests are ideal for detecting emerging infectious diseases such as the SARS-CoV-2 virus because they are quick to develop and deploy.

Most molecular tests that are conducted in a central lab setting are very sensitive and specific, with commercially available SARS-CoV-2 molecular tests exhibiting >95% sensitivity and >99% specificity, with the potential to detect as little as a few dozen to a few hundred copies/mL of a nucleic acid sequence from the pathogen (i.e., they have low limits of detection (LOD)).¹

Thus, molecular tests can detect disease in the very early stages of infection, when the viral load is fairly low.

Challenges of molecular tests

While the sensitivity and specificity of lab-conducted molecular tests is high and the LOD is low, the complexity of these tests typically restricts their use to the central lab setting. This adds time between when the sample is taken and when results are delivered. At best, the turnaround time can be as little as 24-48 hours, but at the height of the pandemic, some labs were experiencing turnaround times of as much as two weeks due to reagent shortages and insufficient testing throughput.

For highly infectious diseases like SARS-CoV-2, the delay in receiving test results can negatively impact both the patient—by potentially forcing them to quarantine while waiting for results—and public health, if an infectious patient does not quarantine while waiting for results.

Best applications for SARS-CoV-2 molecular tests

With their high sensitivity and specificity, SARS-CoV-2 molecular tests are often considered the gold standard for diagnosis. The Infectious Disease Society of America (ISDA) recommends molecular tests for symptomatic patients even if the suspicion of COVID-19 is low, and prefers that results be available within 48 hours.²

For asymptomatic patients, the ISDA recommends testing only in certain situations², such as:

• Upon known or suspected exposure to COVID-19
• Upon admission to a hospital in an area with a high prevalence of COVID-19
• Before certain medical procedures

Antigen tests can be performed at the point-of-care, but are not as sensitive as molecular tests leading to higher false negative rates

What an antigen test detects

In general, an antigen test detects a protein that’s unique to the pathogen. For SARS-CoV-2 detection, the antigen tests detect a protein of the virus particle.

Why this matters for diagnosing COVID-19

Because antigen tests rely on production of virus particles, they can miss newly-infected patients that have virus replicating within their cells but have not yet produced measurable amounts of virus particles.

Benefits of antigen tests

Antigen tests are easy to conduct, inexpensive, and deliver results in minutes. They are also highly specific and can be done at the point-of-care.³

Challenges of antigen tests

The benefits of antigen testing are balanced by a lower sensitivity than molecular tests, with sensitivity dependent on whether or not symptoms are present and how long after symptom onset the test is given. For tests given within seven days of symptom onset, pooled antigen test sensitivity is 84%, dropping to 49% in asymptomatic individuals.³

The lower sensitivity can lead to a higher false negative rate, i.e., more missed infections, which restricts their usefulness to certain situations when molecular tests are widely available.

Best applications for SARS-CoV-2 antigen tests

While the ISDA recommends molecular tests over antigen tests for symptomatic individuals unless test results will not be delivered within three days, their recommendations for asymptomatic individuals are neither for nor against antigen testing due to a lack of sufficient evidence either way.³

That said, a more recent simulation study based on real-world data suggests that although RT-PCR tests perform better than antigen tests in detecting infected individuals and preventing transmission, more frequent antigen testing, e.g., every day or every 3 days, is comparable to less frequent RT-PCR tests, at the expense of many more false-negative tests. This indicates that frequent antigen tests, potentially self-administered at home could be an important tool in combating spread of infection.⁴

The Talis One delivers all the benefits of molecular tests with similar speed and convenience of antigen tests

As can be seen from the above discussion, the antigen vs. molecular test debate has primarily been one of test quality versus speed, with the molecular test being used as the gold standard, although it can take days to get results, and the antigen test being used when speed is more important than sensitivity.

However, the Talis One is set to change the cost/benefit balance in this debate. It’s a molecular test that can be done quickly and easily at the point-of-care, delivering results in under 30 minutes. And unlike other point-of-care tests, the Talis One’s unique cartridge technology includes solid-phase nucleic acid extraction, which gives it a sensitivity and LOD that’s comparable to molecular tests performed in a lab, enabling excellence at the point of care.

Conclusion

In the antigen vs. molecular test debate, the best test is the one that gives you the highest sensitivity and specificity in the shortest amount of time. Molecular testing is the gold standard, although antigen testing can be effective when speed is necessary. Now, with the Talis One, you can get similar speed and point-of-care convenience as an antigen test with the quality you could previously only get from a central lab molecular test.

References

1. Antigen and Molecular Tests for COVID-19. COVID-19 Testing Toolkit website. Accessed July 9, 2021. https://www.centerforhealthsecurity.org/covid-19TestingToolkit/molecular-based-tests/current-molecular-and-antigen-tests.html#Current-Molecular-Tests
2. Hanson KE, et al. IDSA Guidelines on the Diagnosis of COVID-19: Molecular Diagnostic Testing. Clin Infect Dis. 2020 Jun 16;ciaa760. doi: 10.1093/cid/ciaa760.
3. Hanson KE, et al. IDSA Guidelines on the Diagnosis of COVID-19: Antigen Testing. Clin Infect Dis. 2021 Jun 23;ciab557. doi: 10.1093/cid/ciab557.
4. Ke R, et al. In vivo kinetics of SARS-CoV-2 infection and its relationship with a person’s infectiousness. medRxiv. Preprint. 2021 Jun 30. doi: 10.1101/2021.06.26.21259581. Accessed July 10, 2021.

As new variants of SARS-CoV-2 arise, they can impact diagnostic testing, potentially resulting in false negative results. To confidently make treatment decisions, achieve best-possible patient outcomes, and drive operational and clinical efficiency in a point-of-care setting, you need highly sensitive, rapid molecular tests capable of detecting all existing and emerging SARS-CoV-2 variants.

Which SARS-CoV-2 Tests Are Susceptible to False Negatives?

Molecular tests are straightforward to design for pathogen detection because they are largely based on an RNA sequence. However, viruses that rapidly evolve, such as SARS-CoV-2, can influence test sensitivity based on the sequence of the variant, the design of the test and the prevalence of the variant in the population.

Earlier this year, the FDA identified four EUA-authorized molecular tests that could be impacted by SARS-CoV-2 genetic variants, such as the B.1.1.7 variant that has been associated with an increased risk of transmission.^1-2

It is important to be aware that genetic variants are expected to continue to emerge in SARS-CoV-2, which can threaten the sensitivity of virtually any molecular test. This is due to the possibility that one of the targets is driving the sensitivity, so that even if the others still match perfectly the sensitivity can be reduced based on a mutation in a single target. By choosing a test that uses multiple genetic targets, a test will be less likely to be impacted by increased prevalence of genetic variants.³

How To Know If SARS-CoV-2 Variants Impact Your Molecular Test?

Consider the patterns of detection for your specific test. By choosing a test that uses multiple genetic targets, unfamiliar patterns may reveal the presence of new variants without impacting the final result of your test.

Stay up-to-date on emerging SARS-CoV-2 variants⁴, and their ability to evade detection by specific viral diagnostic tests.

Perform a repeat test. Per FDA recommendations⁵, consider negative results in combination with clinical observations, patient history and epidemiological information and repeat testing with a different test targeting different genetic regions if COVID-19 is still suspected.

How Do SARS-CoV-2 Molecular Tests Perform at the Point-of-Care?

All molecular tests used at the point-of-care are significantly faster than central-lab molecular tests 

Point-of-care molecular tests integrated with nucleic acid extraction are more sensitive than those without this crucial sample preparation step

Talis One provides rapid and accurate SARS-CoV-2 detection—quickly delivering central lab-quality results at the point of care

Confidently determine who needs to quarantine and be treated to achieve better patient outcomes with the Talis One COVID-19 Test. 

References

1. SARS-CoV-2 Viral Mutations: Impact on COVID-19 Tests. U.S. Food and Drug Administration website. Updated June 3, 2021. Accessed June 25, 2021. https://www.fda.gov/medical-devices/coronavirus-covid-19-and-medical-devices/sars-cov-2-viral-mutations-impact-covid-19-tests
2. Emergence of SARS-CoV-2 B.1.1.7 Lineage — United States. Centers for Disease Control and Prevention website. Updated January 22, 2021. Accessed June 25, 2021.https://www.cdc.gov/mmwr/volumes/70/wr/mm7003e2.htm
3. SARS-CoV-2 E Gene Variant Alters Analytical Sensitivity Characteristics of Viral Detection Using a Commercial Reverse Transcription-PCR Assay https://journals.asm.org/doi/full/10.1128/JCM.00075-21 Accessed August 12, 2021
4. Genetic Variants of SARS-CoV-2 May Lead to False Negative. U.S. Food and Drug Administration website. Updated March 30, 2021. Accessed June 25, 2021. https://www.fda.gov/medical-devices/letters-health-care-providers/genetic-variants-sars-cov-2-may-lead-false-negative-results-molecular-tests-detection-sars-cov-2
5. Science Brief: Emerging SARS-CoV-2 Variants. Centers for Disease Control and Prevention website. Updated January 28, 2021. Accessed June 25, 2021. https://www.cdc.gov/coronavirus/2019-ncov/science/science-briefs/scientific-brief-emerging-variants.html
6. Manufacturers’ instructions for use accessed August 12, 2021 at https://www.fda.gov/medical-devices/coronavirus-disease-2019-covid-19-emergency-use-authorizations-medical-devices/in-vitro-diagnostics-euas-molecular-diagnostic-tests-sars-cov-2#individual-molecular
7. Subsoontorn, P., Lohitnavy, M. & Kongkaew, C. The diagnostic accuracy of isothermal nucleic acid point-of-care tests for human coronaviruses: A systematic review and meta-analysis. Sci Rep 10, 22349 (2020). https://doi.org/10.1038/s41598-020-79237-7 Accessed August 12, 2021