SARS-CoV-2 Salivary Tests

The benefits of saliva sampling for frequent and massive COVID-19 testing

Saliva tests for detection of SARS-CoV-2 RNA and antigens are becoming widely available lately. What are the advantages and disadvantages of sampling saliva over the nasal swabs? Saliva sampling simply involves spitting into a collection container. Recently, the U.S. Food and Drug Administration (FDA) also authorized the first diagnostic test with the option of home-collected saliva samples by using the Spectrum Solutions LLC SDNA-1000 Saliva Collection Device. The collected samples are then sent to a lab for further processing and analysis. Therefore, saliva sampling is much simpler and less uncomfortable than nasal swab sampling. This makes taking a sample at home or point of care much easier and more practical. It does not require collection by trained and protected medical personnel wearing personal protective equipment, thus reducing a considerable risk to healthcare workers. In contrast to nasal swab sampling, this approach is not affected by global shortages of swabs and personal protective equipment. In general, saliva sampling should permit more widespread and frequent testing. It is, of course, important that the test be as reliable and sensitive as the nasal swab test, but these appear to be reasonably similar [(1-3).

The current gold standard for COVID-19 diagnosis is real-time reverse transcription polymerase chain reaction (RT-PCR) detection of SARS-CoV-2 from collected samples. Concomitant with the advent of saliva sampling, techniques to simplify the detection of viral RNA have been introduced by eliminating the need for specific equipment, thermal cyclers. This makes it far more adaptable in resource poor settings, which often don’t have the relatively expensive PCR thermal cyclers. One such technique is reverse transcription loop-mediated isothermal amplification (RT-LAMP), which has been previously used to detect other viruses, including Zika and Ebola. A typical RT-LAMP assay takes place at a constant 63°C and the presence of viral RNA generates a color change in as little as a half an hour. A recent modification of the technique that uses inhibitors of salivary RNAs has been claimed to detect a single copy of viral RNA. These methods are obviously not specific to saliva tests, but use of saliva samples should facilitate a mass testing of SARS-CoV-2.

Several new developments in testing for viral RNA combine LAMP isothermal amplification with a technique called lateral flow, in which amplified samples are applied to a strip and allowed to flow along the strip. Amplified viral cDNA is detected by application of a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-CAS12 complex designed to bind to a specific viral sequence. When the CRISPR-CAS12 complex finds and binds its target, it releases a chromophore, which is visualized directly or fluorescently as a specific band on the strip. The CRISPR-CAS12 DETECTR is claimed to have a sensitivity of 95% and specificity of 100% compared to the CDC RT-PCR test (4) despite a concern of false negative results. In addition, STOP (SHERLOCK Testing in One Pot) uses similar technology. STOP has a detection limit of 100 viral RNA copies. Results from both tests are obtained in an hour or less, and analyses of the strips are obviously simple. Positive results are indicated by an exhibition of specific band on the strip.

Of course, all these tests, even though they can use point of care collection, require analyses in laboratories. A true point of care test would be one that could be used without need for laboratory involvement, similar to home pregnancy tests. Several of these kinds of tests are in development. For example, a test would involve placing a drop of saliva onto a device, the size of a quarter, and plugging it in to a smart phone. DNA aptamers on the device bind to viral proteins and then are detected by voltage generated by room temperature electron tunneling. Another potential test uses a microfluidic chip in a cartridge and isothermal amplification. Results can be read and uploaded to a smart phone.

The actual impact of these new technologies still need to be ascertained, yet, they provide a snapshot of innovative testing, not only for SARS-CoV-2, but  for other pathogens. Rapid point of care tests could ultimately be used for screening a large group of people (i.e., airline passengers, concert attendees) especially if they can be linked to smart phones. This could be an important interventional strategy in preventing transmission of the virus and in preparing for future pandemics.

 

  1. L. Azzi et al., Saliva is a reliable tool to detect SARS-CoV-2. J Infect 81, e45-e50 (2020).
  2. M. Baghizadeh Fini, Oral saliva and COVID-19. Oral Oncol 108, 104821 (2020).
  3. K. K. To et al., Consistent detection of 2019 novel coronavirus in saliva. Clin Infect Dis, (2020).
  4. J. P. Broughton et al., CRISPR-Cas12-based detection of SARS-CoV-2. Nat Biotechnol 38, 870-874 (2020).

 

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