Chairside Salivary Diagnostics for Oral Diseases

Saliva testing products gain in momentum

by Susan Vogell, RDH, BS, MBA

Saliva plays many important roles in the mouth. Saliva helps us breathe and swallow by lubricating the oral tissues. Enzymes found in saliva begin the digestive process. Saliva also helps protect the teeth from cariogenic bacteria. At present, saliva is rapidly gaining popularity as an important diagnostic tool, thereby offering the possibility to detect diseases earlier than before.¹ Saliva is now being used to detect a growing number of oral diseases including caries, periodontal disease, and oral cancer, as well as systemic disorders.² Point-of-care technologies offer the ability to help facilitate chairside saliva testing.² Some tests are readily available on the market and more are emerging. These exciting new medical advances will allow dental hygienists to expand their role in offering this valuable service to patients. In addition to facilitating early detection of a disease, salivary tests also offer an important educational aid for oral hygiene counseling and patient motivation.

The National Institute of Dental and Craniofacial Research (NIDCR) is the primary funding agency for oral health research in the United States and has provided substantial research funding to develop saliva as a diagnostic medium.³ With funding support from NIDCR, several research centers in the United States have successfully identified 1,166 proteins in human saliva and have classified the specific proteins associated with certain diseases.³

The benefits of using saliva as a diagnostic fluid

Saliva is a clear, slightly acidic (pH = 6.0-7.0) watery fluid which is secreted from the major salivary glands including the parotid, submandibular, and sublingual glands in addition to other minor glands.⁴ It contains a variety of enzymes, hormones, antibodies, antimicrobial constituents, and cytokines. Many of these compounds enter the saliva through the blood, which is why most compounds found in blood are also present in saliva.^2,4 As a result, saliva acts as a source to monitor both oral and systemic health of an individual.^2,4 Saliva is often described as a mirror of oral and systemic health because it contains biomarkers.⁵ According to the National Institutes of Health, a biomarker is a substance that is measured objectively and evaluated as an indicator of normal biologic processes, pathogenic processes, and pharmacologic responses to a therapeutic intervention.⁶ The utilization of biomarkers serves as an early predictor for disease, thereby contributing to the effective prevention and treatment of the disease.⁷ Furthermore, biomarkers can aid in the assessment of potential health risks for the individual.^7,8

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Consider reading:

UCLA Dentistry discovers cellular signals between pancreatic cancer tumors and saliva

Oxidative Stress and the Use of Antioxidants in Dentistry [Part I]

Oral cancer and the human papillomavirus: Part 2

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Saliva is an ideal alternative to blood.^2,4 , 7 It contains sufficient quantities of disease biomarkers.^4,5 Saliva collection is noninvasive and easy because it is readily available. Furthermore, the utilization of saliva instead of blood alleviates the common fear of needle sticking. Saliva is easier to handle during diagnostic procedures than blood, because it does not clot.² Collecting saliva has a reduced potential for accidental transmission of infectious diseases such as HIV and hepatitis compared to blood samples.⁴

By testing saliva, clinicians can help find the cause of a disease, monitor the activity of the disease, and evaluate the success of the therapy.⁹ Salivary diagnostics offer dentists and dental hygienists the ability to provide a higher standard of care for patients. Incorporating the appropriate salivary diagnostic test into practice is easy, because administering the tests take very little time. Salivary test results can help the clinician formulate an individualized therapy plan for both professional care and oral hygiene home care.

Point-of-care testing

The overall goal of point-of-care (POC) testing is to move salivary diagnostics out of the laboratory and into clinical practice to allow for more timely diagnosis of the disease.² POC testing is testing that can be rapidly performed directly at the dental clinic, without the need for steps such as sending samples to a laboratory.² This process reduces turnaround time, thereby allowing therapy to begin immediately and thus improving the quality of care delivered. Moreover, by offering immediate results, problems such as patient follow-up can be averted. In addition, these types of tests can lower overall costs, because they will eliminate the need to draw blood and avoid the cost associated with sample shipping and handling to a centralized laboratory.¹⁰

A newer generation of POC technology is under development.¹⁰ The technology, called lab-on-a-chip, seeks to integrate and automate all the complexities of a laboratory procedure into a device the size of a computer chip.¹³ This sophisticated technology will be able to measure the amounts of multiple biomarkers in a small saliva sample.^10,13

By using saliva as the diagnostic medium, POC technologies will be able to provide rapid, simple, inexpensive, and accurate measurements directly from saliva.² Incorporating POC salivary tests into the dental office will substantially change the dentist's and dental hygienist's roles in prevention, risk assessment, and disease management.¹¹ In addition, due to its simplicity and low cost, POC salivary tests may be advantageous from a public-health perspective.² Overall, chairside salivary tests will translate into improved access and health-care outcomes for patients.¹²

Currently, biologists and engineers are developing chairside saliva diagnostic technologies for oral diseases such as dental caries, periodontal diseases, and oral cancer.²

Saliva testing for caries

Dental caries is still highly prevalent among all age groups.¹⁵ Dental caries is caused by bacterial plaque that in combination with fermentable carbohydrates produces acids. These acids lower the pH at the surface of the tooth, compromising the enamel, dentin, and cementum, and ultimately affecting the structural integrity of the tooth. The disease is multifactorial, involving a combination of biological and environmental factors.¹⁶ An individual caries-risk assessment can aid in the identification of etiological factors responsible for the disease.17 Salivary tests are included in the caries-risk assessment and are important in the identification of high-caries-risk individuals.17 There is no known single salivary factor sufficient to lead to a reliable diagnosis of dental caries to date.¹⁵ As salivary diagnostic technology continues to develop, the likelihood of predicting dental caries before tooth demineralization occurs is promising.¹⁷ Currently, there are salivary tests that when used in combination with caries risk assessment tools can help the dentist to make a diagnosis.¹⁵ The salivary tests most commonly used for this purpose are salivary bacteria count, salivary flow rate (resting and stimulated), salivary pH and buffer capacity, and finally biofilm activity level.¹⁸

Salivary bacteria count

Although caries is a multifactorial disease, it has been shown that specific oral bacteria play a significant role in caries development.¹⁵ A great deal of evidence supports the association of Streptococcus mutans (SM) and lactobacilli (LB) with dental caries; however, Actinomyces and Candida have also been implicated in the disease.¹⁵ SM are highly cariogenic because they produce copious amounts of acid, they are very adherent, and they are capable of rapid uptake of sucrose in competition with other plaque bacteria.¹⁵ LB are also acid-producing and prefer to live in low-pH niches that are difficult to clean.¹⁵

Two commercially available salivary bacterial cultural tests are Dentocult Strip Mutans (Orion Diagnostica, Espoo, Finland) and Ivoclar CRT (Ivoclar Vivadent, Amherst, N.Y.). Both tests can detect levels of SM and LB, but each test requires a 48-hour incubation period and a follow-up appointment for discussing the results with the patients. Another test, Saliva-Check Mutans (GC America), resolves the recall issue as the test can detect salivary SM levels chairside in 15 minutes (see chart).

The findings of salivary microbial tests for detection of cariogenic bacteria can be used for motivation of patients on an individual level. For example, it has been observed that mothers' saliva is the major source of SM transmission to their infants.19 When mothers are informed of preventive measures that interrupt SM transmission, there is a decrease in caries occurrence in children 3 years of age and younger.¹⁹ Salivary tests that detect SM are also justified for use for preschool children as it has been observed that a delay in colonization of SM is associated with a reduced level of decay.¹⁹ Testing also aids in determining recall appointment interval length and helps evaluate the efficacy of oral hygiene efforts at home.

In addition to saliva testing for bacteria counts, saliva should be tested for quality, quantity, and buffering capacity.

Saliva flow

Saliva offers an important protective role in maintaining oral health.²⁰ Insufficient salivary flow may lead to prolonged demineralization and consequently dental caries.20 Testing salivary flow rate can aid in predicting future caries susceptibility, diagnosing salivary gland hypofunction, and medical compromise from illness and/or medication.^18,20 Saliva can be categorized as resting (unstimulated) or stimulated. Type, intensity, and duration of stimulation and the time of day, diet, age, sex, certain diseases, and a number of medications all affect the salivary flow rate.¹⁷ The strongest evidence linking decreased salivary flow with caries is in cases of Sjögren's syndrome, xerostomia, and radiation therapy.¹⁹

The stimulated flow rate can be easily tested in the dental office or clinic by having the patient chew on paraffin wax and expectorate into a graduated cup, measuring mls. Normal values are 8.6-mL/5 min for women and 10.1-mL/5 min for men.¹⁷ Inadequate saliva is measured as less than 0.7 ml of stimulated saliva per minute.¹⁷ If the flow rate remains low over time, this can place a patient at high or extreme risk for caries.¹⁷

While it can be concluded that the absence of salivary flow will most likely lead to dental caries, there are a number of other factors that can enhance saliva's protective effect.

Salivary pH

Although resting and stimulated salivary pH is easily and accurately measured chairside with the use of pH sensitive test strips, there is not a direct correlation between salivary pH and caries susceptibility.¹⁸ Salivary pH always follows the rate of secretion; therefore, it is lowest at night and in the morning.¹⁷ The normal range is between 5 and 8.¹⁷ Salivary pH assessment should not be used to predict caries susceptibility, but rather as a teaching tool to modify behavior in choosing products that will help neutralize acid.

Salivary buffer capacity

Salivary buffer capacity is defined as the quantitative measure of resistance to pH changes.^17,18 The buffering capacity of saliva is one of the best indicators of caries susceptibility, because it is indicative of the individual's response to acid challenge within the oral cavity.¹⁸ According to Larmas (1992), "Low salivary buffer capacity is indicative of reduced host response to cariogenic bacteria, reduced salivary flow rate, possible malnutrition, or pregnancy" (p. 205).²¹

The Dentobuff Strip (Orion Diagnostica, Espoo, Finland) is a chairside salivary buffer capacity test that consists of pH indicator paper that has been impregnated with acid. Results of the test are then compared to a chart. Other chairside salivary buffer capacity tests are available (Fig. 1).

Measuring biofilm activity

Another type of testing involves biofilm activity level measured by adenosine triphosphate (ATP) bioluminescence. ATP bioluminescence is a technology that has been used for years to test bacterial activity in places such as food manufacturing facilities and wastewater treatment plants.¹⁸ ATP bioluminescence can assess an individual's risk for caries by measuring the overall level and activity of cariogenic bacteria.¹⁸ ATP bioluminescence is a simple chairside test that involves swabbing a specific site on teeth and then a 15-second measurement with a meter. An example of an ATP bioluminescence test is the CariScreen caries susceptibility test (Oral BioTech, Albany, Ore.)

Salivary tests for caries susceptibility can aid in educating patients, motivating patient compliance, determining effectiveness of anti-caries therapy or furthering caries-control measures, and setting frequency of dental checkups.

Saliva testing for periodontal disease

One of the leading causes of tooth loss in adults is periodontal disease, affecting more than 50% of the U.S. population.22 The traditional method to diagnose periodontal disease relies on measuring pocket depth and clinical attachment loss, and evaluating radiographs for bone loss. These assessments do not predict periodontal disease in its earliest state.⁷ Since periodontal disease is an irreversible disease, early diagnosis is imperative. Furthermore, it has been shown that untreated periodontal disease can lead to systemic disorders such as cardiovascular disease and diabetes.²²

Researchers have been investigating ways to detect periodontal disease in its preclinical phase using genetic, microbial, and protein biomarkers.²² Since the early 1990s, much research was generated to learn about biomarkers of periodontal disease.⁵ Gingival crevicular fluid (GCF) became an early medium to examine for biomarkers due to its location within the sulcus and easy accessibility. Chapple (2009) states the advantages of using GCF: "The biomarkers found in GCF indicate the presence or absence of periodontal pathogens, gingival and periodontal inflammation, the host inflammatory-immune response to certain pathogenic species, and host tissue destruction" (p. 9-10). The disadvantages of using GCF is that it is expensive, time-consuming, requires multiple samples of individual tooth sites, and requires laboratory processing.⁵ Saliva is more readily available and easier to collect than is GCF. Saliva contains a plethora of biomarkers for periodontal disease, including GCF, and has emerged as the medium of choice to test for periodontal disease.⁵

OralDNA Labs (Brentwood, Tenn.) offer two salivary tests that evaluate for periodontal disease. MyPerioPath is a DNA test that uses saliva to determine an individual's risk for periodontal disease by identifying the specific bacterial pathogens (microbial biomarkers) associated with the disease. MyPerioID uses saliva to determine a patient's genetic susceptibility for periodontal disease by testing for a genetic biomarker. It is believed that 30% of the population carries this genetic variation²³ (see chart). While both tests provide useful information regarding an individual's risk for periodontal disease, they require the use of a laboratory. However, POC tests are under development.

Significant advances are in development for the screening of periodontal disease. Researchers have reported that high levels of the inflammatory biomarker C-reactive protein (CRP) have been associated with chronic and aggressive periodontal disease.¹⁰ Researchers at Rice University in Houston, Tex., are developing a lab-on-a-chip system, which will help quantify the difference in CRP levels between healthy individuals and patients with periodontal disease.¹⁰ This detection system is called an electronic taste chip (ETC).10 In addition to the device's ability to measure CRP, the ETC will simultaneously monitor several additional biomarkers for periodontal disease.¹⁰

The University of Michigan in collaboration with the NIDCR has developed a rapid POC device, known as an integrated microfluidic platform for oral diagnostics (IMPOD).^10,12 This handheld, pocket-sized test determines the amount of the enzyme matrix metalloproteinase-8 (MMP-8) in saliva, in less than 10 minutes.^10,12 Herr states (2007), "MMP-8 has been identified as a major tissue destructive enzyme in periodontal disease. Consequently, MMP-8 is a promising candidate for diagnosing and possibly more importantly, assessing the progression of periodontal disease"¹² (p. 5268).

It is clear that individual susceptibility, along with a variety of local and systemic conditions can influence the initiation and progression of periodontal disease.²⁴ Therefore, it is important that advances in diagnostic testing are made to help identify early periodontal risk. The use of saliva-based diagnostics appears promising for future application to diagnose periodontal disease and to predict periodontal treatment outcomes. In the near future, clinicians will be able to assess periodontal disease with a rapid chairside saliva test.²⁵

Saliva testing for oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is the most common malignancy of the oral cavity among oral cancers, accounting for more than 90% of clinical cases and ranking among the top 10 types of cancers worldwide.⁹ The occurrence of OSCC has been increasing in frequency among individuals.²⁶ The survival rate of oral cancer is 60-80% when detected during its early stages; however, this number drops to 30-40% when the cancer is diagnosed during the advanced stages.⁸ Due to the lack of a reliable early-stage diagnostic marker for OSCC, most OSCC cases are diagnosed when the cancer has developed well into the advanced stages.⁸ Moreover, because OSCC has a very high recurrence rate, early identification and detection is critical for patient survival.⁸ Saliva is now being considered as a diagnostic medium for OSCC.

One benefit of using saliva as a diagnostic tool for OSCC is that it contains the exfoliated cells in the oral cavity, which allow for the screening and identification of potential biomarkers for oral cancer.⁸ In addition, multiple salivary proteins and DNA have been used to detect OSCC.^4,26 For patients, the noninvasive saliva collection procedure can reduce anxiety and discomfort and is easily accessible as compared with tissue biopsies. Furthermore, whereas biopsies can take up to seven days for results, saliva-based diagnostic technology has the potential to decrease the wait time to less than one hour.⁸

The University of California, Los Angeles (UCLA) Collaborative Oral Fluid Diagnostic Research Laboratory, led by Dr. David Wong, developed a POC device used to detect oral cancer in saliva.^2,10 The test, known as the oral fluid nanosensor test (OFNASET), is a POC, automated, and easy-to-use integrated system that uses electrochemical detection of salivary proteins and nucleic acids and can measure up to eight different biomarkers in a single test in less than 15 minutes^2,10 (see chart). The OFNASET will screen for the risk of oral cancer to allow for only test-positive patients to be referred for biopsies.² The test is expected to detect oral cancer at an earlier stage, when treatment is more effective and less costly. In addition to detecting oral cancer, the OFNASET will be able to assess for pancreatic, breast, and lung cancers, Sjögren's syndrome, Alzheimer's disease, and Type II diabetes. According to Dr. Wong, the OFNASET is currently a fully functioning academic prototype. He estimates that in approximately two to three years' time, we will begin to see the FDA approve salivary biomarkers for diseases. It is at that point that the OFNASET will become available on the market (personal communication, May 19, 2013).

Early detection of a disease can provide patients with timely treatment and, therefore, yield better treatment outcomes. It can minimize patient discomfort, save money, and most importantly, save lives. Saliva is on its way to becoming the medium of choice in the diagnostics field. New POC testing will allow for the diagnosis of oral diseases right at the patient's chairside. Salivary diagnostics will expand the role of both dentists and dental hygienists and will allow for substantial involvement of the patient in decision-making and self-care. Salivary diagnostic testing not only presents the clinician with the ability to provide a higher standard of care for patients; it also can help increase the patient's understanding of the overall value of comprehensive care and subsequently facilitate positive behavioral changes. RDH

Susan L. Vogell, RDH, BS, MBA, is a clinical assistant professor of dental hygiene at New York University College of Dentistry and a clinical instructor at Farmingdale State University, where she provides dental hygiene clinical education. She has more than 15 years of private practice experience.

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