March 2021 Share
The COVID-19 pandemic continues to cause illness across the world, but there is hope on the horizon as improved testing, treatments, and vaccines make their way to the global population. With the emergence of new SARS-CoV-2 variants and mutations, however, it is more important than ever that testing is widely available as early diagnosis of infected individuals plays a critical role in stopping further escalation of this deadly disease.
Early in the pandemic, nasopharyngeal swabs (NPS) were the standard method to test for viral infection. Today, the demand for alternative testing methods has increased due to the inherent limitations and challenges associated with NPS testing. One appealing option is saliva testing, which is quickly gaining traction as an alternate testing source with a growing body of research suggesting that saliva-based tests could simplify, accelerate, and increase the availability of COVID-19 testing – and potentially lead to better patient outcomes.
The “gold standard” for COVID-19 testing to date has been to collect a sample of nasal mucus using a nasopharyngeal swab (NPS). The swab is inserted through the nostril into the pharynx region where it is rotated to collect secretions. The swab is then removed and sent to a certified lab for analysis. This method is routinely used for detecting respiratory pathogens, including SARS-CoV-2. There are, however, several drawbacks to using NPS as the primary form of COVID-19 testing.
Administration of this diagnostic test requires trained medical personnel, and mass testing necessitates an increase in the number of trained personnel at testing facilities. Because the professional performing the test needs to be in close physical proximity to the patient, specimen collection inevitably increases exposure risk for medical personnel. Ongoing shortages of swabs, reagents, and personal protective equipment (PPE) are frequently reported, making significant expansion of testing difficult to scale.
From the patient’s perspective, NPS testing can be physically uncomfortable. It is also not recommended for those with certain medical conditions like coagulopathy, who are presently undergoing anticoagulant therapy, or those with a significantly deviated septum. As a result, the reliance on NPS testing is contributing to an economic and logistical burden on healthcare systems, without an easy path for expanding and scaling the number of tests being performed.
It is more important than ever that the healthcare community has access to options when it comes to COVID-19 testing, especially with shortages in available supplies (and patients who actively avoid the discomfort of testing by swab). Additional sample types – like saliva – are stepping in to provide options that could help to scale up testing effectively and affordably.
One of the primary benefits of saliva testing as compared to NPS testing is that it is non-invasive and can be self-administered, and the collected samples are reliable when kept at room temperature. They also demonstrate less variability in results, even when samples are self-collected. Not only is saliva collection more comfortable for people being tested, it also decreases exposure risk to healthcare personnel and minimizes the need for PPE, which is especially critical when supplies are scarce.
Saliva collected from the mouth contains more than just salivary gland secretions – among all the things it contains are expectorated airway surface liquid and mucus, epithelial and immune cells from the oral mucosa and upper airways, and oral microbes and viruses. In fact, saliva has been widely used for a number of diagnostic tests for other infections and diseases, including other coronavirus-caused diseases like severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS).
As testing demand for SARS-CoV-2 continues to increase, a growing body of research is finding that not only are saliva samples useful for detecting infection, but they could be preferable to the NPS swabs that have been predominantly used. Research demonstrates that saliva can provide greater detection, sensitivity, and consistency throughout the course of infection – meaning that saliva could be an incredibly reliable sample source when it comes to testing for active SARS-CoV-2 infection. Saliva is not only a viable and sensitive alternative to NPS testing, picking up even asymptomatic cases that NPS testing misses, but it can potentially be administered at home and allow for accurate, wide-scale testing.
More sensitive and consistent detection of SARS-CoV-2, especially in mild and asymptomatic cases, could be a critical factor in assessing whether and when individuals are able to return to work, as well as the safety of reopening local economies.
For both NPS and saliva testing methods, the laboratory processing the sample commonly uses a form of polymerase chain reaction tests, also called PCR tests, to detect and identify SARS-CoV-2 viral RNA. Early in the pandemic, the predominant focus of this testing was on obtaining qualitative results that delivered a yes or no result as to whether viral RNA was detected. As we continue to learn more about this virus and the disease it causes, laboratories are more frequently looking to quantify the number of viral particles that are detected per ml of the sample, and these quantitative results are being researched to discover whether there is a correlation between viral load and the development of the more severe forms of COVID-19 that can result in hospitalization or death.
Early research seemed to indicate a correlation between viral load and severe COVID-19, but these results were hard to replicate. It is important to note that early studies like this were using data almost exclusively from NPS testing. As the body of work regarding viral load in NPS samples has been expanded upon, there have been conflicting reports about how strongly disease severity correlates to viral load. Some studies showed strong correlations, while others delivered inconsistent findings. New research, however, could hold the answer – and the answer could very well be found in saliva.
A study from Yale looked at patients hospitalized with COVID-19, with confirmation of the diagnosis obtained via NPS testing at admittance. Later, the team had the patients collect their own saliva samples – and, in conjunction with this, healthcare workers also collected another NPS sample from these patients. The results of the two tests were compared with one another, and the researchers confirmed that more copies of SARS-CoV-2 RNA were detected in the saliva samples than in the NPS samples. Furthermore, saliva samples tested positive for as many as 10 days post-diagnosis, indicting that saliva may very well be a more accurate sample source when testing for active infection.
Open questions remain as to whether the quantifiable viral load in saliva samples do, in fact, correlate to patient outcomes – are patients with high viral loads in early stages of infection more likely to develop severe forms of COVID-19? New research is increasingly finding a correlation between the two, and it appears that the viral load detected in saliva could more accurately reflect the respective viral load deep in the lungs, which is where much of the damage associated with COVID-19 occurs.
A recent study from Yale, published as a preprint and not yet peer-reviewed, followed 154 people with active SARS-CoV-2 infection throughout the course of disease progression, testing regularly with both NPS and saliva samples to compare the two. They found that viral load detected in saliva correlated strongly with the severity of COVID-19, while NPS samples did not. Higher viral load in saliva samples was positively associated with increased presence of inflammatory markers that are common with COVID-19, including many interleukins and cytokines, as well as progressive depletion of platelets, lymphocytes, and certain effector T cell subsets. Overall, the study shows that viral load measured in saliva samples (but not NPS samples) is a correlate of disease presentation, severity, and mortality over time.
Saliva is argued to be a better potential predictor of disease outcomes than nasal mucus, as nasal swabs are testing viral presence in the upper respiratory tract while the more harmful damage comes from deeper in the lungs. Saliva can better measure what is happening in the lower respiratory tract, as mucus from deep in the lungs gets moved to the throat and mixes with saliva (much like what happens when coughing). As a result, saliva could very well be a more accurate measure of how the virus may be replicating in the lower respiratory tract.
If these results are confirmed in subsequent research, quantification of viral load in saliva samples could be used to identify patients in the early stages of the disease who would be at-risk for developing severe COVID-19. With this information in-hand early on, doctors could use these results to identify those patients who could most benefit from early intervention with known therapeutics like antibodies (to help reduce the viral load) or steroids (to help suppress overactive, nonspecific immune responses) in the hopes of preventing severe disease progression.
Research to date has not yet provided enough insight to confidently determine likelihood of developing severe forms of the disease, which makes further research critical for expanding upon recent findings. But with further validation, widespread adoption of saliva sampling could transform public health efforts by resolving many of the resource and safety issues associated with current NPS testing methods. At the same time, saliva sampling could also empower doctors to identify and treat at-risk patients early in the disease and potentially prevent hospitalizations and deaths, which could lead to better patient outcomes and help to lessen the burden on overwhelmed medical institutions.
The widespread adoption of saliva testing faces technical hurdles when it comes to scaling testing by an order of magnitude in an efficient and cost-effective way. One approach is to perform pooled saliva tests, in which samples from several unique individuals are combined, or pooled, and the pool is then tested. There are a number of different methods being used for a pooled approach as laboratories look to develop efficient methods for testing the greatest number of people in the fewest number of individual tests in order to both speed up the testing process and to keep costs contained. The traditional approach to pooling involves combining samples from multiple people and testing them all at once, and if the pool comes back positive, retesting the individual samples from that specific pool. This is normally an efficient approach, but when there are high rates of community infection like we are seeing with SARS-CoV-2, this can lead to the need for a lot of retesting.
To make more efficient use of resources, institutions are getting creative in how they pool and test among larger populations. Colorado State University, as an example, is using what they call “paired pooling” that allows for 64 individuals to be screened using 16 tests. The 64 individuals are arrayed into an 8×8 grid, with each individual saliva sample split in half. Each row is then pooled and screened, and each column is pooled and screened. Using this method, each sample is being tested twice (once for its row, and once for its column), and a total of 16 tests are used to screen all 64 samples in duplicate. If only one row and one column test positive, it becomes straightforward to identify the individual – they are the sample sitting at the intersection of that given row and column. No retesting of individual samples is required. If a larger number of pools come back positive (four or more), limited retesting is then performed.
Pooling samples allows for faster and cheaper mass testing, which could be a game-changer in an ongoing pandemic. Still, there are technical hurdles to overcome in pooled saliva testing before widespread adoption becomes practical.
A major challenge with pooled saliva testing is that pooling of samples leads to dilution of the signal analyte. Without a method for enriching the target analyte, even ultrasensitive diagnostic testing may be insufficient for providing accurate results. This is particularly concerning with a disease like COVID-19, where asymptomatic patients with early-stage disease or low viral loads are difficult to identify but are still able to spread the disease to others. Therefore, it is critical that saliva-based testing has the sensitivity required for detecting these cases, even in pooled samples.
While traditional sample preparation relies on magnetic bead-based enrichment of the analyte, this technology comes with inherent limitations and has failed to evolve over the past 40 years in a way that meets current needs. For saliva testing in particular, magnetic technology is limited in the volume of the sample it is able to interrogate. Since magnetic bead-based methods are only able to process a small portion of the overall sample, the majority of the sample is wasted along with any RNA contained within it. This is an unnecessary constraint that can severely impact the ability to detect active infection and is especially concerning when processing pooled samples.
Akadeum Life Sciences is pursuing an alternative approach: buoyant, functionalized microbubbles. The microbubbles are functionalized to capture nucleic acid, including SARS-CoV-2 viral RNA. Unlike with magnetic bead-based separation, microbubbles do not have the same volume and equipment restrictions. They can simply be mixed into the sample, where they grab onto the target (in this case, the viral RNA) and float it to the top for further processing and analysis. Microbubbles are able to interrogate the full sample volume, which in a pooled saliva application would allow for capture of RNA from the entire sample. This is of critical importance when it comes to developing accurate pooled saliva testing and enables key advances in widespread community testing that can be done at scale.
Akadeum is actively developing nucleic acid extraction microbubbles, with results to date demonstrating that Akadeum’s microbubbles bring 10x to 25x more SARS-CoV-2 RNA to the downstream PCR. This has far-reaching implications when it comes to developing the ability to accurately detect and diagnose viral infection in real-world individuals. In a head-to-head comparison of microbubbles vs magnetic in a small set of real-world patient saliva samples, Akadeum’s microbubble protocol was able to confirm positive results in all four samples, whereas the magnetic-based isolation only confirmed one of the four. Greater concordance with NPS test results is incredibly important for ensuring that patients are getting accurately diagnosed, versus falsely coming up negative, as false negative test results have been an ongoing issue with current testing capabilities and one that Akadeum is hoping to help alleviate. This also means that Akadeum’s microbubble approach is successfully pushing the limit of detection lower, increasing our capacity to detect and diagnose COVID-19 positive patients.
Akadeum Life Sciences is actively seeking partnerships to explore opportunities in functionalizing its novel microbubble platform to solve the problems of tomorrow in areas like research, diagnostics, cell separation, cell therapy, and bioprocessing. If you are interested in leveraging the power of microbubbles to overcome hurdles in your workflow, we want to hear from you! Learn more about partnership opportunities by contacting Matt Potter, head of Global Partnerships, at firstname.lastname@example.org.
Saliva viral load is a dynamic unifying correlate of COVID-19 severity and mortality. Julio Silva, Carolina Lucas, Maria Sundaram, Benjamin Israelow, Patrick Wong, Jon Klein, Maria Tokuyama, Peiwen Lu, Arvind Venkataraman, Feimei Liu, Tianyang Mao, Ji Eun Oh, Annsea Park, Arnau Casanovas-Massana, Chantal B. F. Vogels, M. Catherine Muenker, Joseph Zell, John B. Fournier, Melissa Campbell, Michael Chiorazzi, Edwin Ruiz Fuentes, Yale IMPACT Team, Nathan D. Grubaugh, Shelli Farhadian, Charles Dela Cruz, Albert I. Ko, Wade L. Schulz, Aaron Ring, Shuangge Ma, Saad Omer, Anne L Wyllie, Akiko Iwasaki. medRxiv 2021.01.04.21249236; doi: https://doi.org/10.1101/2021.01.04.21249236
Saliva could hold clues to how sick you will get from COVID-19. Robert F. Service. 2021.01.13. American Association for the Advancement of Science. https://www.sciencemag.org/news/2021/01/saliva-could-hold-clues-how-sick-you-will-get-covid-19
Viral Load Can Predict COVID-19 Severity Only in Saliva Specimens, New Study Shows. Caleb Williams. 2021.01.18. The Dark Intelligence Group. http://covid19briefings.com/2021/01/18/viral-load-can-predict-covid-19-severity-only-in-saliva-specimens-new-study-shows/
Saliva samples preferable to deep nasal swabs for testing COVID-19. Michael Greenwood. 2020.04.24. Yale News. https://news.yale.edu/2020/04/24/saliva-samples-preferable-deep-nasal-swabs-testing-covid-19
Saliva as a Candidate for COVID-19 Diagnostic Testing: A Meta-Analysis. László Márk Czumbel, Szabolcs Kiss, Nelli Farkas, Iván Mandel, Anita Hegyi, Ákos Nagy, Zsolt Lohinai, Zsolt Szakács, Péter Hegyi, Martin C. Steward and Gábor Varga. Front. Med., 04 August 2020; doi: https://doi.org/10.3389/fmed.2020.00465
COVID-19 Saliva Tests Are Gaining Popularity for Ease-Of-Use and Reliability. Jennifer Chesak. 2020.12.18. verywellhealth. https://www.verywellhealth.com/covid-19-saliva-tests-easy-to-use-and-reliable-5092897
The Best COVID Warning System? Poop and Pooled Spit, Says One Colorado School. Rae Ellen Bichell. 2020.11.03. KAISER FAMILY FOUNDATION. https://khn.org/news/the-best-covid-warning-system-poop-and-pooled-spit-says-one-colorado-school/
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