Salivary Diagnostics

For dental professionals, saliva can be a hindrance during examinations and procedures. Consequently, it is suctioned out of the oral cavity. Recent advances in salivary diagnostic research, however, indicate that saliva holds promise for the screening of oral and systemic health problems. Saliva is said to be a “mirror of the body” because it is an indicator of health not just in the oral cavity but throughout the body.¹ The molecular composition of saliva includes therapeutic, hormonal, immunologic, and toxicological molecules, which can provide vital clues to systemic health.²

Saliva has been used informally as a clinical diagnostic medium for more than 2,000 years. Historically, the viscosity and odor of saliva were used as diagnostic symptoms for certain diseases. In diagnostics, saliva is an excellent alternative to serum or urine since it contains sufficient quantities of disease biomarkers, ribonucleic acid (RNA), and deoxyribonucleic acid (DNA), and the collection method is noninvasive, safe, and easy. Collecting saliva also has a reduced potential for accidental transmission of infectious diseases compared to blood samples. In the past, the intrinsic value of saliva as a disease indicator was not fully recognized due to the lack of highly sensitive and specific analytic techniques. However, a high level of sensitivity is now achievable through the use of different techniques that amplify the magnitude of disease biomarker signals.

RECENT ADVANCES

Through the work of the Human Salivary Proteome Project, more than 1,000 proteins in saliva have been identified. Advancements in analytical techniques have enabled scientists to discover the specific proteins associated with human diseases. These proteins are referred to as biomarkers.

Ever since the catalog of human salivary proteome was made available, there has been an increased effort to examine and compare the proteomic signatures in diverse diseases. Specific biomarkers are associated with dental caries, periodontal diseases, and oral cancer.² Currently, biologists, engineers, and dental practitioners are developing “lab-on-a-chip” platforms that use oral fluids in rapid tests to accelerate clinical decision making. Ultimately, these types of tests will lower the cost of health care because they eliminate the need for trained phlebotomists to draw blood.³

The vision of the technology is to provide the simultaneous assessment of multiple aspects of oral health. The assessment will then help clinicians determine the most appropriate preventive and therapeutic approaches, all during the same office visit.^2,3 Lab-on-a-chip platforms will be able to perform multiple operations in nonlaboratory settings such as satellite clinics, field sites, or at home.⁴ Furthermore, these testing methods use smaller amounts of sample and reagents (substances used in chemical reactions), further decreasing costs. Current diagnostic approaches that take days or weeks to obtain results are less effective than this new realm of point-of-care diagnostics that provide results within minutes or hours so the treatment plan can begin immediately (Figure 1).

DIAGNOSTICS FOR CARIES

Saliva-based diagnostic technologies have the potential to improve dental care by detecting oral infections early. Dental caries is a significant health problem that affects millions of Americans. The ultimate demineralization of teeth seen in dental caries is caused by acids produced by bacteria, particularly Streptococcus mutans and Lactobacilli.

Prediction of caries risk is often difficult because of the complex interactions of the host and its environment, as well as behavioral risk factors. Changes in buffer capacity, salivary composition, rate of salivary flow, and pH are all risk factors for caries. One of the most significant predictors of caries is the relative level of S. mutans and Lactobacilli colonization, which can be determined through saliva or plaque samples. S. mutans are normally transferred from mother to child. If the transmission occurs in very young children, the resulting dental caries may be more severe.⁵ Clearly, early detection has the potential to lessen the burden of this disease.

A commercially available kit developed in Finland called Dentocult® SM (Orion Diagnostica) tests for the adherence and growth of S. mutans. The bacteria appear as dark- to light-blue raised colonies on the surface of the test strip. Clinical studies show that the kits’ results are sensitive,⁶ however, the test requires a 48-hour incubation period. A recently developed S. mutans detection system, Saliva-Check SM (GC America), eliminates the need for an incubation period. It can detect salivary S. mutans levels in 30 minutes compared to the 48 hours needed for bacterial culture tests.⁶ One study found that results produced by Saliva-check SM were equal to those gained from traditional culture methods.⁶ Saliva tests are practical for use in dental clinics because they do not require expensive equipment.

Researchers at the University of Southern California in Los Angeles are developing the Caries Assessment and Risk Evaluation (CARE) test to help determine which children are most at risk of developing cavities. The CARE test detects specific glycoproteins in saliva that are associated with bacterial attachment to teeth.⁷ The goal of CARE is to help dental professionals pinpoint children at risk of developing cavities, and then prophylactically apply protective sealants to high-risk patients.

DIAGNOSTIC TESTING FOR EARLY PERIODONTAL DISEASE

Significant advances are in development for the screening of periodontal diseases.⁸ The current method of diagnosing periodontitis is through assessment of clinical parameters and radiographs, however, this is not necessarily the most efficient method for early diagnosis. If periodontal diseases are detected early, treatment can be easier and less painful for the patient. Left untreated, periodontal diseases may lead to systemic problems such as cardiovascular disease and diabetes. Therefore, early screening for periodontal diseases is essential during dental examinations. The only commercially available techniques for screening oral fluids for periodontitis is by testing gingival crevicular fluid (GCF) for inflammatory markers⁸ or DNA detection tests for specific periodontal pathogens.

The Rapid Periodontal Test™ (GeneEx™ Inc) uses GCF, an inflammatory exudate, collected from each tooth and placed onto filter paper strips in its anaylsis. This method is advantageous because the contributing inflammatory mediators and tissue destructive molecules associated with periodontitis are found in this fluid. It is time consuming, however, because each periodontal site must be individually tested and analyzed. Developments in oral biology research are discovering the specific biomarkers associated with periodontal diseases and several salivary markers haven been identified (Table 1).⁸

Two DNA-based saliva tests are available from OralDNA® Labs: MyPerioPath® and MyPerioID® PST®. My PerioPath uses a saliva sample to identify the type and concentration of the specific bacteria that cause periodontal diseases. MyPerioID PST test also uses saliva to determine a patient’s genetic susceptibility to periodontal diseases and which patients are at higher risk of more serious periodontal infections. Both tests require the shipping of saliva samples to a laboratory for results.

The future of salivary diagnostics is a testing platform that is able to analyze oral fluids/saliva for a variety of biomarkers. Several study groups have reported elevated serum C-reactive protein (CRP) levels in periodontitis patients. The higher the levels of CRP in periodontitis patients, the more severe the disease, even with adjustments for external factors.⁹ There is a kit to test serum levels of CRP, however, there is no commercially available kit for measuring CRP in saliva samples.

Currently, researchers at Rice University in Houston, Tex, are developing a lab-on-a-chip system using a new detection system for measuring analytes in saliva based on an electronic taste chip (ETC). The ETC methodology was compared with the standard laboratory technology (ELISA) for measuring CRP in saliva, and displayed a 20-fold lower limit of detection than the ELISA. With this technique it was possible to quantitate the difference in CRP levels between healthy individuals and patients with periodontal diseases.⁹ Additional studies confirmed the ability of the ETC platform to simultaneously monitor several additional biomarkers.⁹ The prospect of a commercially available ETC lab-on-a-chip platform that can detect multiple biomarkers for early diagnosis of periodontal disease is promising.

DIAGNOSTIC TESTS FOR ORAL SQUAMOUS CELL CARCINOMA

Currently, the most definitive procedure for identifying oral squamous cell carcinoma (OSCC) is a biopsy of suspected lesions, followed by histopathological identification by an oral pathologist. It requires a biopsy and a waiting period of approximately 7 days, however, to obtain the pathology report. Saliva-based diagnostic technology for screening OSCC has the potential to decrease the wait time to less than 1 hour at the dental clinic.

Research on more sensitive salivary methods of diagnosis is crucial for oral cancer screenings. The laboratory of David T. Wong, DMD, DMSc, at the University of California, Los Angeles (UCLA), is using a microfluidic electrochemical detection system for the identification of OSCC “signatures” in saliva. The signature is composed of two salivary proteins—IL8 and thioredoxin—that can discriminate saliva obtained from oral cancer subjects as compared to control subjects. IL8 is important because both protein expression and specific messenger RNAs were found to be elevated in saliva of oral cancer patients. The UCLA group identified seven salivary messenger RNAs that were consistently elevated in saliva from oral cancer patients, and of the seven RNAs, four of these in combination are capable of distinguishing oral cancer patients from controls with a sensitivity and specificity of 91%.¹⁰ Other studies on oral cancer biomarkers using salivary proteomic methods have discovered additional candidate markers including M2BP, MRP14, CD14, catalase, and profilin.¹¹ Collectively, these biomarkers have demonstrated 90% sensitivity and 83% specificity for OSCC detection.¹²

In addition to protein and mRNA markers for OSCC, John McDevitt, PhD, from Rice University has demonstrated 93% specificity and 97% sensitivity in detecting OSCC by combining the over-expression of epidermal growth factor receptor (EGFR) in OSCC with a morphological analysis of the nuclear/cytoplasmic size ratio of the cells. This technology analyzes a brush biopsy of a lesion in 8 minutes to 10 minutes.¹³

Oral cancer affects 37,000 Americans yearly and kills more than 8,000.¹⁴ Survival rates have not improved over the past 50 years, largely because most cases are diagnosed too late. Implementation of these new discoveries should improve early detection rates, with the hope of reducing patient morbidity.

SUMMARY

Saliva is becoming accepted as an excellent diagnostic medium and an indispensable tool in the field of diagnostics. It is likely that the developments of saliva-based methods will impact and expand the role of dental hygienists. Thousands of individuals visit dental clinics on an annual basis to receive dental hygiene services, and many do not regularly see a physician. Dental hygienists are the guardians of oral health, responsible for detecting abnormalities related to oral disease. They have unique access to large at-risk populations and are potentially able to rapidly screen many individuals for oral and systemic health risks and diseases, allowing for efficient preventive methods and early treatment.

Integrating these new salivary diagnostics methods into clinical practice is important to aid dental professionals in making essential health-related decisions for patients. In the near future, taking a saliva sampling in a dental clinic will become as routine as obtaining a urine or serum sample at a physician’s office. With tests for cardiac markers, infectious disease, and unique cancer “signatures,” more timely treatment and referrals for oral and systemic diseases can be made.

ACKNOWLEDGEMENTS

Supported by National Institutes of Health, National Institute of Dental and Craniofacial Research (NIDCR), and National Institute of Allergy and Infectious Diseases Grants U01DE017855 and U19DE018385, in addition to a New York State Foundation for Science, Technology and Innovation award.

The authors would like to thank Lillian Shum, PhD, and Isaac Rodriguez-Chavez, PhD, MS, MHS, at NIDCR, as well as William Abrams, PhD, for help in preparing this manuscript.

---

REFERENCES

1. Mandel ID. Salivary diagnosis: promises, promises. Ann N Y Acad Sci. 1993;694:1-10.
2. Zhang L, Xiao H, Wong DT. Salivary biomarkers for clinical applications. Mol Diagn Ther. 2009;13:245-259.
3. Tabak LA. Point-of-care diagnostics enter the mouth. Ann NY Acad Sci. 2007;1098:7-14.
4. Lee JM, Garon E, Wong DT. Salivary diagnostics. Orthod Craniofacial Res. 2009;12:206-211.
5. Li Y, Caufield PW, Dasanayake AP, Wiener HA, Vermud SH. Mode of delivery and other maternal factors influence the acquisition of Streptoccus mutans in infants. J Dent Res.2005;84:806-8011.
6. Matsumoto Y, Sugihara N, Koseki M, Maki Y. A rapid and quantitative detection system for Streptococcus mutans in saliva using monoclonal antibodies. Caries Res. 2006;40;15-19.
7. Denny PC, Denny PA, Takashima J, Galligan J, Navazesh M. A novel caries risk test. Ann NY Sci. 2007;1098:204-215.
8. Millar CS, Foley JD, Bailey AL, et al. Current developments in salivary diagnostics. Biomarkers Med. 2010;4;171-189.
9. Christodoulides N, Mohanty S, Millar CS, et al. Application of microchip assay system for the measurement of C-reactive protein in human saliva. Lab Chip. 2005;5;261-269.
10. Wong DT. Salivary diagnostics for oral cancer. J Calif Dent Assoc. 2006;34;303-308.
11. Hu S, Arellano M, Boontheung P, et al. Salivary proteomics for oral cancer biomarker discovery. Clin Cancer Res. 2008;14:6246-6252.
12. Shpitzer T, Bahar G, Feinmesser R, Nagler RM. A comprehensive salivary analysis for oral cancer diagnosis. J Cancer Res Clin Oncol. 2007;133;613-617.
13. Weigum SE, Floriano PE, Redding SW, et al. Nano-Bio_chip sensor platform for examination of oral exfoliative cytology. Cancer Prev Res. 2010;3:518-528.
14. Oral Cancer Facts. Available at: www.oralcancerfoundation.org/facts/index.htm. Accessed January 18, 2011.

From Dimensions of Dental Hygiene. February 2011; 9(2): 56-59.