August 2008: Volume 34, Number 8
Saliva Tests: Ready to Spit?
Oral-based Tests Poised for Wider Array of Analytes
By Gina Rollins
In Western culture, saliva has a distinctly unsavory connotation, but a wave of research involving cutting-edge technologies is changing people’s opinions on the heretofore undervalued biofluid. In fact, some experts believe that analysis of saliva may be equivalent, and in some instances superior, to assessment of serum, urine, or other biofluids in diagnosing oral as well as systemic diseases. With none of the anxiety and discomfort associated with needlesticks, coupled with the micro- and nanotechnologies employed to amplify its diverse but comparatively low concentration of analytes, saliva has the potential to offer a revolutionary change in medicine.
“It’s been the poor step-child of serum, yet the vast majority of proteins in serum are also present in saliva, but in minute amounts. You simply have to look harder to reveal information that’s contained there,” explained John McDevitt, PhD, professor of chemistry at the University of Texas at Austin.
Two forces are driving saliva’s rising currency as a diagnostic tool. One is the development of mass spectrometry and other technological advances that enable high throughput analysis and detection of concentrations of analytes that are 1,000 to 10,000 times lower than in serum. The many biological roles of saliva are well documented, such as starting the digestive process through the release of enzymes and the cleansing and diluting of detritus in the mouth. Pioneering researchers like Irwin Mandel, DDS, professor emeritus of dentistry at Columbia University, N.Y. have long maintained that saliva is a window to the body. But until recently, analysis techniques were not sensitive enough to detect nano-concentrations of biomarkers in saliva.
The other factor contributing to saliva’s rising star is crucial funding for two lines of investigation by the National Institute of Dental and Craniofacial Research (NIDCR) in Bethesda, Md. In 2002, NIDCR awarded grants to seven research teams to develop salivary diagnostic platforms. All focused on using microfluidics and microeletromechanical systems to detect and analyze the components of saliva, including proteins, electrolytes, DNA, and mRNA, with the goal of commercializing the products. Four of these labs received a second round of funding in 2005, and all are well on their way to developing “lab on a chip” oral-based detection systems using different platforms. Since the initiative commenced, NIDCR has funded $43.4 million, according to a spokesperson.
On the heels of the salivary diagnostic initiative, in 2003 NIDCR funded a complementary undertaking to identify and catalogue the entire salivary proteome. It has subsequently awarded $24.1 million to three labs. Efforts from both NIDCR initiatives are bearing fruit in 2008.
A Mouth Full
Saliva comes primarily from three pairs of glands in the craniofacial structure: the parotid glands, located between the back of the jaw and each ear; the sublingual glands, under the floor of the mouth; and the submandibular glands, just inside the back of the jaw in the floor of the mouth. About 10% comes from minor glands clustered in the oral mucosa. Each gland, encased in a capsule of connective tissue, is composed of individual lobules supplied by a fine network of capillaries. The lobules, with serous and/or mucous cells, depending on the gland, actively take up substances from serum, such as water and salt, passively diffuse and filter others, blend them with salivary proteins, then secrete the resulting concoction—ranging from 500mL to 1.5L per day—into ducts that transport saliva to the mouth. As a result of this process of diffusion, absorption, and secretion, saliva reflects natural and artificial substances in the body, albeit at micro levels.
Researchers distinguish between ductal saliva, which comes directly from the salivary glands, and whole saliva, which includes the entire oral fluid mix, including saliva, bacterial waste products, and serum leaked from crevicular junctures around the teeth. Ductal saliva is considered more valuable as a biofluid, so collection techniques typically use something like a Lashley cup that adheres via mild suction to the parotid duct orifices. The composition of saliva is affected by numerous factors, so some researchers also follow strict specimen protocols, such as collecting fasting early morning samples.
“Protein levels in saliva change due to medications, systemic diseases, the time of day, and whether the patient has eaten or drunk anything. There are so many variables that can impact the results,” explained Brian Schmidt, DDS, MD, PhD, associate professor and an oral and maxillofacial surgeon at the University of California at San Francisco. While these types of collection techniques are important as scientists perfect their knowledge about saliva, any commercial oral diagnostic devices likely will feature simpler means of gathering samples, such as oral swabs or spitting into a tube, he said.
Saliva’s Utility as a Diagnostic Fluid
Some researchers view oral-based tests as the Holy Grail of diagnostics, mainly because samples can be obtained non-invasively. Although this would appeal to squeamish people, it would be of particular importance to children and elderly patients, who typically experience the most discomfort in providing blood samples. “If the consumer is presented a choice of obtaining a sample through blood, urine, saliva, or spinal fluid and the results are equally accurate, that will drive adoption,” said David Wong, DMD, DMSc, professor and associate dean of research at the UCLA School of Dentistry.
In addition, collecting saliva poses less risk to healthcare workers. Saliva samples are also easier to handle in that saliva doesn’t clot like blood. In combination with micro- and nano-technologies, oral-based diagnostics offer the possibility of condensing the full processing power of flow cytometers into low-cost, hand-held devices—an ideal solution for developing countries that lack resources and infrastructure.
But there are also advantages for industrialized countries. Oral-based diagnostics could enable speedy, low-cost POC devices that do not depend on trained phlebotomists for sample collection. And saliva-based tests could provide rapid, perhaps even field-based, results. “You can’t be slower and more expensive with new diagnostics and expect economies. That’s where electronics has a powerful impact for our country,” said McDevitt. “We have taken advantage of the microfabrication methodologies popularized by the electronics industry to make en masse our new nano-bio-chip sensors. The electronics industry has provided our society with so many examples of powerful yet affordable tools, from computers, to televisions, to cell phones. The marriage of electronics and in vitro diagnostics has the potential to change the practice of medicine by creating powerful yet affordable diagnostic aids. These devices may be mini-sized, but [could] exceed performance of refrigerator-sized instruments.”
A few oral-based diagnostics are already available commercially. Most notably, OraQuick (OraSure Technologies, Bethlehem, Pa.) was approved by the FDA in 2004 as an oral-based qualitative immunoassay for HIV-1 and HIV-2. It is a screening test only and requires a confirmatory reactive test. The same test using blood samples was approved in November 2002. There are a number of breath analyzers to detect blood alcohol concentrations using infrared spectrophotometer or electrochemical fuel cell technologies. Specimens for DNA testing used in forensics, paternity, and genealogical investigations are now commonly collected via buccal swabs. Steroid hormones, hepatitis A, B and C, and substances of abuse such as marijuana and methamphetamine also can be detected through salivary analysis.
On the Cutting Edge
Recent research seems likely to expand this relatively modest group of oral diagnostics to encompass a host of systemic and oral diseases. In addition, three NIDCR-funded research teams from five universities are working to categorize the salivary proteome. In April 2008, the group reported that they had used different separation and fractionation technologies, along with mass spectrometry, to identify 1,166 proteins in saliva collected from 23 adults of several races and both sexes. Researchers matched their results against the plasma and tear proteomes and found that 650 salivary proteins also are in plasma and 259 are in tears. The proteome is significant in that researchers are now “empowered with a more extensive parts list than they’ve ever had before,” according to Lawrence Tabak, DDS, PhD, director of NIDCR. This initial chart of the salivary proteome will open wide the doors for further discovery, he predicts. “The challenge and exciting part is figuring out what all the parts do. I think, as with other similar efforts, how proteins work in concert will be more important than what they do individually.”
Oral-based diagnostics are being investigated actively for a number of high-impact diseases, including oral, breast, pancreatic, and lung cancer; HIV (as a combined screening and confirmatory test); as well as tuberculosis, malaria, heart disease, Sjögren’s syndrome, asthma, type 2 diabetes, COPD, and pre-eclampsia, among others.
Spotlight on Oral Cancer
Wong’s laboratory used oral cancer as a first proof-of-principle disease for salivary transcriptome diagnostics. His team found that four genes—IL-8, ornithine decarboxylase, spermidine acetyltransferase, and IL-1ß—could discriminate and predict whether a saliva sample was from a patient with cancer or a healthy individual, with both sensitivity and specificity of 91% (ROC 0.95). The biomarkers were validated in approximately 200 subjects, and found to be consistently higher in people with oral cancer than in matched control subjects. Wong and colleagues went a step further and compared the accuracy of the salivary biomarkers with four serum RNA biomarkers for oral cancer detection and found the serum biomarkers had a sensitivity and specificity of 91% and 71%, respectively (ROC 0.88). “This demonstrates clearly that for oral cancer detection, salivary transcriptome diagnostics have a slight edge over serum,” Wong noted. “We can’t just say how good saliva is; we have to benchmark it against the gold standard—serum.”
Concurrent with its analysis of salivary biomarkers and participation in the salivary proteome project, Wong’s lab is developing an Oral Fluid NanoSensor Test (OFNASET), a handheld, automated, integrated microelectromechanical system that will enable simultaneous and rapid detection of multiple salivary protein and nucleic acid targets. The team is investigating the potential of using OFNASET in several diseases, including Sjögren’s syndrome and pancreatic and lung cancers.
Schmidt is pursuing another avenue of investigation: saliva as an indicator of oral cancer. His early work testing levels of salivary endothelin-1 (ET-1), a vasoactive peptide normally synthesized by human keratinocytes but over-produced by several cancers, found that the concentration of ET-1 in saliva from oral cancer patients was significantly higher than in that of healthy controls. He also discovered that ET-1 mRNA was overexpressed in 80% of oral squamous cell carcinoma (OSCC) specimens. The study shows the potential of ET-1 as a discriminate marker for OSCC. In follow-up research, Schmidt is comparing pre- and post-operative ET-1 levels in patients with OSCC. “It’s very promising. We hope it might turn out to be like PSA level in prostate cancer,” he said.
Another line of investigation related to oral cancer that Schmidt is pursuing involves analyzing saliva to detect hypermethylation in the promoter region of five genes as an early indicator of the disease.
The Oral Fluid NanoSensor Test is a handheld, automated, integrated microelectromechanical system that will enable simultaneous and rapid detection of multiple salivary protein and nucleic acid targets. The device was developed by a team of researchers at the UCLA School of Dentistry led by David Wong, DMD, DMSc.
Infectious Diseases and More
Saliva-based diagnostics also have applications in infectious diseases. HIV and the viral and bacterial diseases that create opportunistic infections in HIV/AIDS patients are the research focus of a team led by Daniel Malamud, PhD, professor of basic sciences and craniofacial biology at the New York University College of Dentistry. Malamud and colleagues are developing a single microfluidic chip that will detect HIV, malaria, and tuberculosis using a detection system based on up-converting phosphor technology. In the case of HIV, the research team already has developed a chip that tests for antibodies, antigens, or nucleic acid as independent pathways on the chip, making it both a screening and confirmatory diagnostic tool. Plans are for the TB assay to detect antibodies in saliva, while the malaria test will identify nucleic acid as well as antigens.
Malamud’s team is working with other labs that have already developed reagents for TB and malaria to accelerate the process of converting benchtop tests to microfluidic systems. Eventually the separate pathways will be combined into a single analytical system. “The potential is incredible,” said Malamud. “No one dies of HIV; but it suppresses the immune system and makes it possible for TB and malaria to develop. If clinicians knew an HIV patient also had TB, they could start treatment right away and improve the patient’s survival and quality of life.”
A saliva-based test for heart disease is one of several areas of focus in McDevitt’s lab at the University of Texas. The lab is adapting a miniaturized sensor based on a micro-bead array for several analyte classes. Chemically sensitized bead “micro-reactors” are populated in micro-etched pits inside a silicon wafer. When new molecular reagents are attached to the micro-reactors, they can detect different array platforms.
The lab’s early efforts related to heart disease involved establishing the lowest limit of detection of CRP reported thus far, defining the physiological range of CRP in healthy, edentulous and periodontitis patients, and developing a dual platform to detect CRP and total white blood cell count. More recently it has developed a system to detect in saliva a panel of four important cardiac biomarkers: BNP, troponin I, CK-MB, and myoglobin. In a study comparing levels of the biomarkers in control subjects to those of patients with acute coronary syndrome, researchers found for the first time that all were detectable in both unstimulated and stimulated saliva samples, but the rate of detection varied for each enzyme based on sample stimulation status. The findings suggest that salivary samples could be used to detect a standard battery of cardiac biomarkers. McDevitt envisions that the panel will help clinicians identify patients with non-ST-elevated myocardial infarction (non-STEMI) earlier and fast-track treatment for them, given that non-STEMI cardiac events often are not detected via EKG.
McDevitt’s lab also has adapted a lab-on-a-chip platform to detect, from whole saliva,12 biomarkers associated with cardiovascular disease. He hopes that one day it may be used to track patients at risk of recurrent heart attack and death after an initial coronary event. “By looking at early indicators, we think we can capture [a second event] before it happens. We project there is a unique signature here,” explained McDevitt. “It’s not practical or convenient for patients to give blood every day, but they can spit. That’s where saliva has such a strong potential to make the transition to wellness and health promotion rather than reactive medicine.”
Another area of focus for the McDevitt lab is oral-based detection systems for breast, ovarian, and cervical cancer. In addition, it has already developed a serum-based lab-on-a-chip platform for measuring CD4 lymphocyte counts (licensed to LabNow in Austin, Texas), which is being tested clinically in Africa, India, and the U.S.
Oral-based microarrays for diagnosing pulmonary diseases such as asthma, chronic obstructive pulmonary disease, and acute pneumonia are the focus of a consortium led by David Walt, PhD, professor of chemistry at Tufts University in Medford, Mass. Walt’s multi-institution team is developing a complete diagnostic system including a microfluidic chip coupled to an optical fiber microarray for detecting multiple analytes. The results will be read optically, processed using data mining and pattern recognition techniques, and reported both through a liquid crystal display and over a wireless network. In the case of asthma and COPD, “our hope is that within half an hour clinicians will be able to identify both the nature of the inflammation and the causative agents,” Walt noted. For instance, patterns of protein-based inflammatory response could pinpoint whether an asthmatic exacerbation was caused by a bacterial infection or exposure to pet dander. The Walt laboratory also is exploring the use of saliva-based biomarkers to detect the onset of end-stage renal disease.
At Walt’s lab, like others developing oral-based micro-diagnostics, part of the challenge in transitioning from the bench to the bedside is harnessing the passion and intelligence of a diverse team of scientists, from oral biologists and analytical chemists to engineers and computer scientists. “We have an energetic group of people with a common goal, but getting them all speaking the same language and figuring out how to work together has been challenging,” he said.
How Far Off?
Just how long it will take for oral-based microfluidic diagnostic systems to be used in patient care remains to be seen. The NIDCR-funded consortia are working diligently and have had success in demonstrating proof of concept and in developing first-generation prototypes. But as they add analytes to the platforms, integrate them into a single process, and move into larger trials, challenges are no doubt in the offing. Many of the teams are aiming for commercialization within 3 years, but whether that goal will be realized remains to be seen. “It’s hard to predict, because even if the technology works, it needs to be validated in a clinical setting,” said Walt. What is clear is that the emerging science of saliva-based testing is an area that laboratorians will want to keep an eye on.
Gina Rollins is a freelance writer in Silver Spring, Md.