Salivary Diagnostics

Saliva and other oral fluids (e.g., gingival crevicular fluid, combined secretions of minor salivary glands) support the health of soft and hard tissues in the oral cavity.^1-7 The protective functions of saliva include maintaining a neutral oral pH, cleaning and remineralizing teeth, facilitating swallowing and digestion, and protecting oral tissue against desiccation and invasion by microorganisms.^{2, 7} Adequate saliva is essential for maintaining oral health, and reduced salivary secretion (i.e., hyposalivation) or xerostomia (i.e., dry mouth) can contribute to oral problems such as dental caries, mucositis, fungal infections, and periodontal diseases.^8-11

Potential advantages of using saliva for disease diagnostics include ease of access, noninvasive sample collection, increased acceptance by patients, and reduced risks of infectious disease transmission.^12-14 Readily accessible fluids include whole saliva, secretions from specific glands, mucosal transudate or gingival crevicular fluid.¹³ However, the various techniques and durations of collecting saliva and other oral fluids (e.g., spitting, drooling, dribbling, and collection with or without coughing) can affect the precision and determination of biomarkers of interest. 

Saliva is a heterogeneous biofluid that can also include epithelial cells and leukocytes.¹⁵ Composition of saliva is variable as specimens will be affected by the time of day they are collected, the location where oral fluid is collected, and the sample storage conditions, including the time to analysis.^16-19 To date, there are no established uniform criteria for the collection of human saliva.² 

There continues to be interest in oral fluid as a non-invasive diagnostic medium for rapid, point-of-care testing.^12-14 Interest in saliva tests expanded significantly with emergence of the coronavirus disease 2019 (COVID-19) pandemic, which fueled development of rapid diagnostic tests using alternative sample specimens, such as saliva, for improved detection of individuals with SARS CoV-2 infection.²⁰ During early stages of the pandemic, initial studies showed detectable viral loads of SARS CoV-2 in saliva^21-24 and in individuals showing no symptoms of COVID-19 (asymptomatic).²⁵ Subsequently, studies that evaluated saliva specimens using polymerase chain reaction (PCR) testing for COVID-19 showed that such tests performed relatively well.^26-30 One meta-analysis³¹ reported 91% sensitivity for saliva tests of previously confirmed COVID-19 patients compared with 98% sensitivity for nasopharyngeal swabs (a commonly used specimen type for COVID-19 diagnosis). 

Sampling oral fluids, instead of blood, provides an accessible medium for detecting a range of candidate biomarkers, such as proteins, electrolytes, hormones, antibodies and DNA/RNA, as well as other substances such as therapeutic or recreational drugs.¹ While oral fluid assessment has been suggested to be useful in screening for or diagnosing oral or systemic diseases, monitoring viral or fungal infections, detecting drug exposure, and evaluating endocrine disorders and cancer risk, evidence of utility for most of these purposes is limited, and further clinical validation research is required. 

Currently, oral fluid testing by clinical laboratories is regulated under the Clinical Laboratory Improvement Amendments of 1988 (CLIA). The federal CLIA regulatory standards apply to clinical laboratories and any facility examining human specimens for diagnosis, prevention, treatment of a disease or for assessment of health. CLIA regulations are designed to help ensure test results are accurate and reliable.³² However, the CLIA program does not address the clinical validity of any test—that is, the accuracy with which the test identifies, measures, or predicts the presence or absence of a clinical condition or predisposition in a patient.³² 

To make a claim of clinical validity, a test must be submitted to the U.S. Food and Drug Administration (FDA), which regulates tests under the Federal Food Drug, and Cosmetic Act. The clinical validity of a test is evaluated by FDA in its premarket clearance and approval processes.³² 

As of mid-2021, several tests have received FDA emergency use authorization for detection of SARS CoV-2 infection using saliva collected from individuals with COVID-19 symptoms. Some of the tests allow saliva specimens to be self-collected at home (and/or in special collection devices), or to be collected in a health care setting (when determined to be appropriate by a health care provider).³³ In June 2021, the FDA granted emergency use authorization to the first point-of-care antibody assay that uses oral fluid samples (i.e., gingival crevicular fluid) for qualitative detection of total antibodies (e.g., IgG, IgA, IgM) to SARS CoV-2 (the antibody test is intended for use in diagnosing recent or prior infection with SARS CoV-2).³⁴

In addition, there are two direct-to-consumer (DTC), oral fluid tests that have received marketing authorization from FDA, which test for gene alleles associated with increased cancer risk. The FDA includes more information on DTC genomic tests at the following website: https://www.fda.gov/medical-devices/in-vitro-diagnostics/direct-consumer-tests.

Although a variety of biocomponents (Table 1) are found in oral fluid including proteins and related molecules, nucleic acid components (e.g., human and microbial DNA, messenger RNA [mRNA] and microRNA), and endogenous and exogenous metabolites,^{12, 13} a number of factors, including collection methods used to obtain the sample (e.g., stimulated or unstimulated), affect their concentration.³⁵

Factors affecting the ability to accurately assess biomarkers in samples obtained from the oral cavity depend not only on marker biochemistry, the source and type of the sample, and the mechanism by which the marker enters the oral cavity,¹³ but marker stability.^{36, 37} Proteomic technologies significantly improved the ability to identify candidate biomarkers at the molecular level. These technologies have advanced cataloging the human salivary biocomponents and evaluating their diagnostic value.^{19, 38}

Table 1. Examples of Biocomponents Detectable in Oral Fluid^{2, 6, 12-14, 39-49}

Biocomponent Class and Examples

Hormones
— Cortisol, androgens, estriol, estrogen, progesterone, aldosterone, melatonin, insulin

Cytokines
— Interleukins (IL-1beta, IL-6, IL-8), tumor necrosis factor, troponin

Antibodies
— IgG, IgA

Proteins/Enzymes
— Amylase, pepsin, matrix metalloproteinases, C-reactive protein (CRP), mucins, lactoferrin, antimicrobial peptides (cystatin, hystatin, statherin, calprotectin)

Growth Factors
— Epidermal growth factor (EGF), vascular endothelial growth factor (VEGF) insulin-like growth factor

Nucleic Acids
— Human and microbial DNA, mRNA, microRNA, tRNA-derived small RNA (sRNA)

Viruses
— HIV, HSV-1, HSV-2, EBV, HPV, CMV, VZV, HCV, Anelloviridae

Bacteria
— P. gingivalis, S. mutans, Lactobacillus spp, T. forsythia, E. coli, H. pylori, M. tuberculosis

Fungi
— Candida, Aspergillus

Drugs
— Anticonvulsants, chemotherapeutic agents (including antibiotics and antineoplastic agents), analgesics, drugs of abuse, ethanol

Metabolites/Electrolytes
— Phosphate, calcium, sodium, potassium, glucose, chloride, nitrate, uric acid, amino acids, lipids, carbohydrates

Tumor Markers
— CA 15-3, HER2/neu, CA 19-9, p53, leptin, CA 125, alpha fetoprotein, CEA, somatic mutations in tumor suppressor genes, loss of heterozygosity, promoter hypermethylation of genes, microsatellite DNA alterations

The salivary genome consists of both human and microbial DNA.¹³ Nearly 70% of the salivary genome is of human origin, whereas the remaining 30% is from the oral microbiota.¹

The ability to determine the presence or absence or even to quantify the amount of a biomarker in oral fluids is not synonymous with its being of clinical significance. As of mid-2021, there are no FDA-approved salivary diagnostic tests for evaluating risk of periodontal disease, dental caries, or head and neck cancer.

Oral fluid contains secretions from the major and minor salivary glands, oro-nasopharyngeal secretions, gingival crevicular fluid, and cellular debris. The mixture of fluids obtained varies depending on the collection method used (e.g., spitting, suctioning, drooling, draining, or collection on some type of absorbent material).² In addition, drug concentrations can be affected by the collection method, as well as by whether saliva stimulation methods were used. Several collection devices are commercially available in the United States and they generally involve collection on absorbent material (e.g., foam pad). Drug concentrations may also vary depending on how the oral fluid is recovered from the collection device (e.g., by centrifugation or by applying pressure). Another issue is that drug concentrations may not reflect blood levels because of residual amounts of drug (specifically those ingested or smoked) remaining in the oral cavity after recent use.

Currently available oral fluid tests have been developed for the detection of specific infectious agents (such as SARS CoV-2, HIV, HPV, HSV, Candida albicans, etc.),^{50, 51} drug metabolizer status,⁵⁰ and detection of illicit drugs^52-55. Oral fluid tests are capable of screening over 20 drugs of abuse (e.g., cocaine, amphetamines, ethanol).^53-55 However, the clinical insight gained from an oral fluid test that measures presence or absence of oral HPV may be of limited value for predicting oropharyngeal squamous cell carcinoma risk⁵⁶ or post-treatment prognosis⁵⁷ because the test does not account for virus clearance.

A search of MEDLINE via PubMed Clinical Queries for systematic reviews was conducted in 2018 (plus a supplemental search in mid-2021) for pertinent systematic reviews published within the last 8 years using the following search string: systematic[sb] AND ((saliva* OR oral fluid*) AND (diagnosis OR diagnostic* OR biomarker*)). The following section summarizes the results of the identified systematic reviews with respect to saliva/oral fluid testing and oral disease(s) affecting the oral cavity.

Cancer (including Oral and Oropharyngeal Cancer)

A 2017 guideline and systematic review^{58, 59} from the ADA Center for Evidence-Based Dentistry on the use of adjuncts in the evaluation of potentially malignant disorders in the oral cavity made a conditional statement based on low quality evidence recommending against the use of commercially available salivary adjuncts for the evaluation of potentially malignant disorders among adult patients with or without clinically evident, seemingly innocuous, or suspicious oral lesions, and that "their use should be considered only in the context of research."

Recent reviews that have examined the potential of human salivary microRNAs, cell-free nucleic acids, mRNA, cytokines and peptide markers find the data on their utility for cancer diagnosis, progression prediction, or determining susceptibility for development of mucositis following radiotherapy to be preliminary and to require further clinical validation.^60-67

Periodontal Disease 

Recent studies and systematic reviews demonstrate the continued research interest in investigating panels of oral biomarkers in saliva and gingival crevicular fluid to assist in screening and diagnosis of periodontal disease.^68-78 Using the revised classification system for periodontal diseases (proposed by the 2017 World Workshop on Periodontal and Peri-implant Diseases and Conditions),⁷⁹ researchers have continued to evaluate potential candidate biomarkers, expression patterns of microRNAs, point-of-care tests and diagnostic models for predicting and identifying periodontal disease.^80-83

Research on diagnostic models and the predictive accuracy of salivary biomarker panels for diagnosing periodontal disease has steadily advanced but requires further clinical validation in larger cohorts.^{71, 73} Such research will entail highly accurate measurement studies and statistical analyses, which are further complicated because analytes are only present in trace amounts in saliva, and saliva contains bacterial proteases that can degrade protein biomarkers.^{71, 73, 76, 84}

A 2017 literature review⁸⁵ attempted to determine whether measurement of the inflammatory cytokines IL-1beta or TNF-alpha in saliva could be used as markers associated with clinical evidence of periodontal disease. Fifteen papers meeting selection criteria were included in the qualitative review; the authors determined that meta-analysis could not be performed because of between-study heterogeneity. The review concluded that although salivary IL-1beta and TNF-alpha are associated with early periodontal disease and increase with disease progression, they were characterized as “promising tools” in the early diagnosis of periodontal diseases. The results of this review are hypothesis generating about the potential clinical utility of these salivary markers.

The association between chronic periodontal disease and IL-8 levels in oral fluids (gingival crevicular fluid [17 studies] and saliva [4 studies]), as well as gingival tissue biopsies (10 studies), was explored in a 2017 systematic review and meta-analysis.⁸⁶ The systematic review showed conflicting associations for both saliva and gingival crevicular fluid IL-8 concentrations with chronic periodontitis, while higher IL-8 concentrations in gingival tissue were found to be more consistently associated with chronic periodontitis. Separate meta-analyses were performed for the associations with salivary IL-8 (2 studies) and gingival crevicular fluid IL-8 (7 studies), finding mixed results for salivary IL-8 and lower concentrations of IL-8 in gingival crevicular fluid of patients with chronic periodontitis.

A systematic review⁷⁵ of measurement of matrix metalloproteinase-8 (MMP-8) in gingival crevicular fluid and saliva as a potential marker of periodontal disease included six studies meeting selection criteria. Although the studies found significantly higher levels of MMP-8 in the oral fluid of patients with periodontal disease compared with healthy controls as well as increased levels in patients presenting with more advanced periodontal disease, the authors found that the findings only “imply the potential adjunctive use of MMP-8 in the diagnosis of periodontal disease.” Another systematic review on MMP-8 levels found significantly higher levels of salivary MMP-8 in patients with periodontal disease, but concluded that “further high-quality studies are still needed to verify [this] conclusion.”⁸⁷

Other Conditions 

Oral Lichen Planus
Two systematic reviews evaluated the evidence for the relationship between salivary and serum levels of IL-6 or TNFalpha and oral lichen planus.^88-89 The meta-analyses by Liu et al.⁸⁸ (salivary and serum IL-6 analyzed separately; 5 studies each) found significant differences in serum and salivary IL-6 levels between patients with disease and healthy controls, but called for additional research to confirm their meta-analytic findings. The meta-analyses by Mozaffari et al.⁸⁹ (salivary and serum TNFalpha analyzed separately; 7 studies each) found higher levels of TNFalpha in saliva compared with serum in patients with oral lichen planus, suggesting that the salivary marker may be more useful than serum measurement for diagnosis and monitoring treatment; however, the authors cautioned that confounding factors such as age, stress, smoking and genetics should be taken into consideration when interpreting results.

A 2019 systematic review reported elevated serum and salivary IL-4 levels in patients with oral lichen planus, but noted that secondary infections could influence its concentration.⁹⁰ Two other systematic reviews indicated that salivary cytokine and nitric oxide measurements and elevated levels of salivary IL-8 may have potential applications in monitoring disease activity in patients with oral lichen planus.^{91, 92}

Laryngopharyngeal Reflux
Two systematic reviews^{93, 94} evaluated the utility of salivary pepsin assessment as a diagnostic biomarker of laryngopharyngeal reflux, a condition in which stomach acid and/or gastric content flow back up the esophagus and into the larynx or throat. The review by Calvo-Henriquez et al.⁹³ included 12 studies meeting study selection criteria; eight of these evaluated salivary pepsin as a biomarker for laryngopharyngeal reflux. The review concluded that although pepsin might be a reliable marker of disease, “questions remain about optimal timing, location, nature, and threshold values for pepsin testing,” and that future research is required. The review and meta-analysis by Wang et al.⁹⁴ was limited to salivary pepsin measurement; 11 studies met study selection criteria. This review concluded that although pepsin in saliva has moderate value in the diagnosis of laryngopharyngeal reflux, because the cutoff value used could affect its diagnostic value, further research is required “to find the optimal method to detect salivary pepsin.” 

General

Studies of the saliva metabolome have identified 853 metabolites and metabolic species in human saliva.⁹⁵ A saliva ontology-based database and a Human Salivary Proteome Wiki have also been established to support data accessibility for expanded transcriptomic and proteomic research.^{96. 97} The field of salivary diagnostics drew extensive research interest and public support during the COVID-19 pandemic, in the U.S. and internationally, as communities searched for simple, non-invasive and accurate testing options for improved public health surveillance and protective measures.

The use of saliva for disease diagnostics and surveillance has considerable potential for future diagnostic tests for oral and systemic diseases, including biosensors that could potentially provide continuous monitoring of salivary analytes associated with oral or systemic health.⁹⁸ Extensive research, however, is still required to identify and assess oral fluid biomarkers and to validate saliva-based testing modalities for future clinical application.^{98, 99} Similar recommendations are presented in a 2014 “critical review”¹⁰⁰ of the literature for salivary and biofilm diagnostics found that the majority of studies published at that time were primarily concerned with validating metrics and identifying biomarkers with diagnostic potential. A 2015 review¹⁰¹ similarly concluded that “[m]ost reports on discovered candidate [salivary] biomarkers have been only preliminary and require extensive validation in large patient or subject cohorts before they can be translated into real world diagnostic and screening applications.”

Large-scale, multicenter clinical trials and independent validation studies are required to establish evidence of clinical utility of salivary and oral fluid diagnostics in the early diagnosis and/or monitoring of oral cancer and other diseases or conditions. Current challenges include identification of disease-specific markers, establishing sensitivity and specificity of home-use and point-of-care tests using saliva specimens, and standardization of collection/storage of salivary samples.⁴³ Refinement of oral fluid screening and diagnostic tests may further elucidate our understanding of the relationship between oral health and overall health. Lastly, as of mid-2021, there are oral fluid-based diagnostic tests available for the detection of HIV infection, drugs of abuse and SARS CoV-2 (under the FDA’s Emergency Use Authorization authority). However, no FDA-approved salivary diagnostic tests for evaluating risk of periodontal disease, dental caries, or head and neck cancer.

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