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Saliva testing Blog: your spit can save your life
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Saliva testing Blog: your spit can save your life

Controlling the spread of COVID-19 through rapid and scalable direct saliva testing

Date: January 4, 2021

Author: Bhavna Chaudhuri, PhD

Categories: Current events

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You've heard it all before: long lines in front of testing facilities, trained technicians in full personal protective equipment (PPE), and a notoriously uncomfortable nasal swab test. This scenario—combined with supply chain issues, high testing costs, and a long wait for results—makes the current testing procedure inadequate for servicing a pandemic of such enormity. Nasopharyngeal swabs have been the gold standard for testing during this pandemic. Still, they are challenging to use for regular and routine screening because of the shortage of swab kits, a long workflow, and the test's uncomfortable nature. More importantly, since the test does not provide results the same day, presymptomatic or asymptomatic patients could potentially infect many others in the 2–5 days while they wait to get their results. With over 20 million COVID-19 cases in the US alone, finding a sensitive, quick, easy, and inexpensive workflow for testing is the key to controlling the spread of the virus. As we move towards reopening schools, colleges, and workplaces, the demand for such rapid and scalable testing will increase significantly. 

What makes an ideal testing workflow? 

To provide better infectious disease management and improve clinical decision-making, the WHO established ASSURED criteria for diagnostic testing in resource-limited point-of-care settings. Tests should be Affordable, Sensitive, Specific, User-friendly, Rapid (and robust), Equipment-free, and Deliverable to end users (Blacksell 2012). The ideal testing method for any infectious disease should require minimal diversion of resources (including personnel and expensive reagents from clinical diagnostic testing facilities), be affordable and scalable, and allow for rapid and reliable identification of viral presence for asymptomatic or subclinical infections (Ott et al. 2020). Current testing methods fail to meet many of these criteria or could be significantly improved. Efforts to simplify the sample collection and testing workflow would accelerate time to results.

What is the best sample for COVID-19 testing? 

The standard COVID-19 diagnostic test detects SARS-CoV-2 using RT-qPCR on viral RNA isolated from nasopharyngeal swab samples. However, the sample collection and testing procedure suffers from several drawbacks: exposure risks to healthcare workers, global shortages of testing and safety supplies, and long turnaround times, which can limit the ability to expand testing (Figure 1). Validation of new diagnostic approaches that bypass these issues is critical to expanding testing efforts. Saliva testing offers several promising advantages over nasopharyngeal swab testing. Saliva tests are comparable in sensitivity to nasal swab tests for SARS-CoV-2 detection and more conducive for widespread adoption (Wyllie et al. 2020): sample collection is noninvasive, can be reliably done at home without exposing trained health professionals, and does not rely on a limited supply of swabs and specialized collection tubes. Saliva testing is most effective during the disease's infectious phase, which spans 1–3 days before and about 7 days after symptoms start. In contrast, nasal swab testing is most reliable after the patient develops symptoms (Wyllie et al. 2020; Ott et al. 2020). 

 SARS-CoV-2 Nasal-swab test

Figure 1. Nasal swab testing for SARS-CoV-2 is lengthy and suffers from supply-chain issues that are preventing expansion efforts. 

 

How can testing be improved and scaled up?

To get ahead of the pandemic and open the economy, massive expansion of testing is needed using inexpensive, quick, noninvasive tests that do not require specialized swabs and tubes. Such a diagnostic test would allow testing people several times per week until we can administer a reliable vaccine at scale. Recently, several protocols were developed that have made saliva testing extraction-free and significantly faster. The sample is collected in any sterile tube, treated with a reagent to isolate viral RNA, and processed directly for RT-qPCR, without the lengthy RNA extraction steps. This rapid and simple protocol, combined with noninvasive sample collection that does not require specialized equipment, is significantly speeding up testing efforts. Indeed, the development of extraction-free saliva tests that bypass the nucleic acid isolation step has made the tests much faster, cheaper, and independent of supply chain issues (SalivaDirect and I-COVID, Vogels et al. 2020, and Ranoa et al. 2020, respectively). These extraction-free saliva tests are comparable in sensitivity to the nasal swab tests and are being used in various universities across the US to regularly screen students, staff, and sports teams—as often as twice per week. 

Supporting the development of extraction-free SARS-CoV-2 detection methods

Seeing the great need for rapid, accurate, and extractionless SARS-CoV-2 detection from crude saliva, we adapted and optimized our gold-standard PrimeScript III technology for this challenging sample type, resulting in the Direct One-Step RT-qPCR Mix for SARS-CoV-2. Researchers developing new extractionless detection methods can start with our simple protocol: mix the saliva sample and pretreatment reagent and heat treat, then combine an aliquot of this mixture with the RT-qPCR master mix and perform RT-qPCR. The entire workflow from pretreatment to detection takes less than one hour (~5 minutes for pretreatment and ~50 minutes for real-time RT-qPCR), enabling fast time to results. This reliable method has a high sensitivity for detecting low copy numbers of the SARS-CoV-2 genome (as low as 10 SARS-CoV-2 RNA copies; Figure 2).

 SARS-CoV-2 Saliva-swab test

Figure 2. Direct One-Step PrimeScript RT-qPCR Mix uses a simple 3-step protocol to detect SARS-CoV-2 in saliva samples across a range of target RNA copy numbers. Panel A. Our simple protocol takes less than an hour to complete. Panel B. Unpurified saliva samples spiked with synthetic RNA of the N1 and N2 regions of SARS-CoV-2 were processed according to the Direct One-Step PrimeScript RT-qPCR Mix for SARS-CoV-2 protocol, and positive detection was achieved for these targets as well as human RNase P (RP), the positive control.


Light at the end of the tunnel

Just like there was never a night that could defeat sunrise, we will overcome this pandemic and come out stronger. Several safe and effective vaccines have recently been approved for release, and in the near future, enough of the population will be immunized to significantly reduce the spread of the virus. In the meantime, testing people often and at scale is critical to control the spread.

It has been a year of tremendous hardship and suffering for people worldwide, and we hope that a scalable, rapid, and affordable detection method, such as extraction-free saliva testing, will help contain the spread of SARS-CoV-2.

References

Blacksell, S. D. Commercial dengue rapid diagnostic tests for point-of-care application: recent evaluations and future needs. J. Biomed and Biotechnol, 12, Article ID 151967 (2012).

Ott, I. M., et al. Simply saliva: stability of SARS-CoV-2 detection negates the need for expensive collection devices. MedRXiv. doi: 10.1101/2020.08.03.20165233 (2020).

Ranoa, D. R. E. et al. Saliva-Based Molecular Testing for SARS-CoV-2 that Bypasses RNA Extraction. BioRxiv. Doi: 10.1101/2020.06.18.159434 (2020).

Wu, G. and Zaman, M. H. Low-cost tools for diagnosing and monitoring HIV infection in low resource settings, Bulletin of the World Health Organization, 90, 914920 (2012).

Wyllie, A. L. et al. Saliva is more sensitive for SARS-CoV-2 detection in COVID-19 patients than nasopharyngeal swabs. MedRxiv. doi: https://doi.org/10.1101/2020.04.16.20067835 (2020).

 

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