A microscopic view of saliva with a blue background

The COVID-19 pandemic and the need for rapid, reliable testing has heightened interest in saliva as a convenient and reliable testing medium for infectious diseases. The Food and Drug Administration (FDA) has issued more than 30 emergency use authorizations (EUA) for saliva-based SARS-CoV-2 tests, recently including high throughput instruments such as Thermo Fisher Scientific’s Amplitude Solution system. Recent studies also have shown that saliva is just as effective as a nasopharyngeal swab for traditional SARS-CoV-2 PCR tests and useful in at-home rapid antigen tests.

But saliva’s use as a health indicator did not start with SARS-CoV-2, nor will it end with this virus. Researchers and laboratorians are investigating oral fluid’s potential in detecting heart disease, human papilloma virus (HPV)-related head and neck cancers, breast cancers, lung cancers, as well as monitoring treatment efficacy, detecting disease reoccurrence, and stratifying patient risk. Israeli company Salignostics has even submitted a saliva-based pregnancy test for FDA approval in the U.S.

As a testing medium, saliva, unlike blood and other body fluids, is easy to collect, handle, and store. Add that to increasingly sensitive lab instruments, and it seems like there should be a lot of opportunities to exploit saliva in the clinic.

COVID-19 Testing on a Community Scale

As using saliva becomes more mainstream, in vitro diagnostic companies are showing its utility in high-throughput instruments. In October 2021, Thermo Fisher Scientific received an FDA EUA to run SARS-CoV-2 tests from saliva samples obtained with their Spectrum Solutions SpectrumDNA SDNA-1000 collection device on their Amplitude Solution system. The company rolled out the Amplitude Solution system in 2020 for SARS-CoV-2 testing of nasopharyngeal and anterior nasal swab specimens. The 2021 EUA expanded Amplitude’s use with a saliva-specific workflow.

“Amplitude is a high-throughput automated molecular diagnostic testing system that can help labs quickly scale COVID-19 PCR testing to changes in demand volume and offers maximum throughput—up to 8,000 test results in 24 hours—with minimal hands-on time, equipment, and staffing,” explained Manoj Gandhi, MD, PhD, senior medical director in the Genetic Testing Solutions Business at Thermo Fisher. The saliva collection system is self-contained and provides sample consistency and long-term stability while protecting the genetic material needed for accurate PCR test results.

Thermo Fisher got to demonstrate such an instrument’s value when it was used during the 2021 Summer Olympics in Tokyo to test athletes, officials, and staff in the Olympic Village. “About one million tests from saliva samples were performed on the Amplitude system over 50 days,” Gandhi said.

With the new saliva protocol, the company hopes that Amplitude will help “provide a means to address the routine testing needed for companies, schools, universities, and communities to curb infection rates and help us return to pre-pandemic life,” Gandhi said.

Using Saliva to Screen for HPV-Related Cancers

In 2010, Chamindie Punyadeera, PhD, left a career in industry, where she was working on point-of-care saliva diagnostics to detect drugs of abuse, to start a new path in academia, where she could pursue more blue-sky research on using saliva for disease detection.

Early in her research career she worked with Johnson & Johnson to develop a companion diagnostic to their HPV vaccine Gardasil. This companion diagnostic was a saliva-based screen for persistent oral HPV infection—a condition that has been linked to increased risk for head and neck cancers. “DNA from HPV-related head and neck tumors sheds directly into the saliva and could potentially be used as a biomarker for early cancer diagnosis,” said Punyadeera. “Saliva testing is a more effective way to detect lower levels of oral HPV than through a blood draw,” she added, “because we are going directly to the source of the viral infection—the mouth and throat.”

Punyadeera has also partnered with the lifestyle company Viome on an mRNA test to detect oral squamous cell carcinoma and oropharyngeal cancer from saliva. In April 2021, Viome received an FDA breakthrough device designation for using saliva as a liquid biopsy for mouth and throat cancers.

Now a professor at Griffith University in Brisbane, Australia, Punyadeera is championing the validation and use of saliva HPV testing as a standard clinical tool for screening and early detection of HPV-related head and neck cancers for at-risk populations. “I am persistent in pursuing support and funding to bring saliva-based HPV screening to clinical practice. These simple, non-invasive tests have the potential to save people’s lives,” she said.

Using Sound to Sift for Pathogens

The quest to deliver more diagnostic information from saliva is also accelerating because of its special properties that interest researchers in both diagnostic technology and molecular biology. For example, the National Institute of Health (NIH) established the Extracellular RNA Communication Consortium to explore how extracellular RNA (exRNA) can traverse from organs and blood to saliva and other body fluids. The consortium aims to develop exRNA’s potential for diagnostic and therapeutic value. So far, the initiative has catalogued exRNA biomarkers—including categories unique to saliva—for nearly 30 diseases.

Researcher David T.W. Wong, DMD, DMSc, has been a key player in the consortium, focusing on new technologies to identify and sort different types of exRNA from saliva samples. A professor, associate dean for research, director of the University of California, Los Angeles Center for Oral/Head & Neck Oncology Research, and Felix and Mildred Yip Endowed Chair in Dentistry, Wong has been collaborating with researchers at Duke University to develop what they call an acoustofluidic chip. The small device uses sound waves to sort molecular-scale particles, including exRNA, in saliva by size and density.

This sort-of lab-on-a-chip is also tunable, allowing the same device to be used for different particles Wong said. Aside from ease of collection, one of the benefits of using saliva with the acoustofluidic chip is that the samples do not need to be processed before entering the device.

Testing for SARS-CoV-2 infection is a near-term application for the acoustofluidic chip technology. However, Wong is also enthusiastic about the chip’s downstream capabilities. “From a single droplet of saliva, we could identify in minutes an individual’s complete COVID-19 infection status—including viral load and antibody levels—simultaneously,” Wong said. “This pathogen isn’t going away. We need to employ technologies like the acoustofluidic chip that can quickly tell you if your antibodies are low, making you vulnerable to a breakthrough infection.”

Validation Concerns

With most assays developed specifically for blood, clinical laboratories must use caution before using tests off-label for saliva or other body fluids. “From a regulatory perspective, manufacturers will indicate testing parameters only for the body fluids that their diagnostics have been approved for,” said Darci Block, PhD, DABCC, FADLM, co-medical director of the central clinical lab at the Mayo Clinic. “And most body fluid diagnostics have not been approved for use with saliva.” Block is well-versed in body fluid testing and the issues surrounding the validation of such tests.

Proper clinical validation of alternate sample types is essential to ensure a test’s accuracy and precision. “It’s a major logistical challenge for us,” said Block. “Modifying the use of a test without validation is against the law.”

One of the main concerns clinical laboratories have in using saliva is matrix interference. “The way the assay is designed, the concentration of reagents, the amount of sample dilution that happens during testing, is optimized to the indicated testing liquid,” Block noted. “So, when you test a sample that doesn't have the exact properties of the indicated testing liquid, you have to wonder what the test results really mean.”

With saliva, specifically, Block noted that viscosity is a potential problem. If the sample is too thick to aspirate into the testing instrument, it is imperative to find a way to increase fluidity without affecting the analytes.

Block stressed that when considering adding any new assay to a laboratory’s repertoire, laboratorians should always involve the people who order the tests in the conversation—and create a policy for how the lab handles testing fluids outside manufacturers’ parameters.

A Drool-Worthy Future?

Subject matter experts offer many reasons to champion the future use of saliva as a testing medium, despite the important barriers to clinical integration.

“Saliva provides the opportunity for minimally invasive specimen collection and is a convenient alternative to other common respiratory samples, like nasal and nasopharyngeal swabs,” Gandhi said. “It’s readily available from most patients and can be collected with minimal discomfort or inconvenience.”

Saliva might even find a place as a matrix of choice for important new biomarkers like tau protein for Alzheimer’s disease, Wong noted. “The day will come when we can detect the tau protein in saliva instead of through a spinal tap procedure,” he said. “However, without credentialing these new methods for clinical deployment, they will remain fairytales.”

Punyadeera sees saliva diagnostics as potentially having a transformative effect on medical care by “increasing access in low resource settings, reducing healthcare costs, driving precision healthcare, and providing more equitable healthcare solutions for remote and rural communities.”

While a decade of advances has brought us closer to realizing the usefulness of saliva as a diagnostic medium, the last paragraph of the Clinical Chemistry paper that Punyadeera co-authored still rings true (2011; doi: 10.1373/clinchem.2010.153767): “The development of specific and standardized analytical tools, establishment of defined reference intervals, and interlaboratory tests will make saliva diagnostics a clinical reality.”

Sarah Michaud is a freelance writer who lives in London. +Email: [email protected] 

Finding Asymptomatic Cancer With Saliva Screening

While developing the saliva-based HPV screening protocol for Johnson & Johnson, Punyadeera and her colleagues identified three individuals with persistent oral HPV infections from the 700 study participants they screened from their university lab in Brisbane, Australia. “There is a strong association between persistent HPV infection and cervical cancer, so we theorized the same could be true for persistent oral HPV infection and throat cancer,” she said. “I didn’t feel comfortable not sharing this information with these three study participants.”

Punyadeera and her clinical team obtained permission from their ethics committee to contact the three participants with high levels of HPV. Of the three, one individual appeared healthy but presented with an abnormal tonsil. Even though an MRI scan of the tonsil was clear, “this person’s salivary HPV DNA levels were sky high,” said Punyadeera. “We knew something was going on.”

The patient opted for a voluntary tonsillectomy in which a pathologist found a 2mm cancerous tumor. Per Punyadeera, this was the first time a cancer was detected with a saliva test before the patient showed symptoms of illness. Results from her study were published in Frontiers in Oncology (2020; doi: 10.3389/fonc.2020.00408).

David T.W. Wong, DMD, DMSc, and his colleagues at Duke University developed an acoustofluidic chip that uses sound waves generated by gold interdigital transducers to isolate molecules in saliva according to size and density. The microfluidic channel for the saliva sample is shown in red, and the coin is for scale.