1Senior Lecturer, Department of Oral Medicine and Radiology, Thai Moogambigai Dental College and Hospital
2Professor and Head of the Department, Department of Oral Medicine and Radiology, Thai Moogambigai Dental College and Hospital
3,4,5Junior resident, Thai Moogambigai Dental College and Hospital
Saliva, a readily accessible biofluid, has emerged as a promising diagnostic tool for various oral diseases and systemic conditions. Its non-invasive nature and the presence of numerous biomarkers make it ideal for early detection, monitoring disease progression, and informing treatment decisions. This review gives an overview into the potential of salivary diagnostics in oral cancer, dental caries, periodontal diseases, Sjogren's syndrome, oral lichen planus, oral leukoplakia, and systemic diseases like cardiovascular diseases, diabetes, and viral infections. The article discusses the factors influencing saliva composition, the role of salivaomics in diagnostic applications, and the challenges and future directions in this field. By harnessing the power of salivary biomarkers, researchers aim to develop innovative diagnostic tools that can improve patient care and revolutionize healthcare. This article gives an overview about saliva as a potential diagnostic tool for oral diseases and oral cancer.
Saliva is emerging as a promising diagnostic tool for oral cancer and various oral diseases due to its non-invasive nature and the presence of numerous biomarkers. Recent studies highlight the potential of salivary diagnostics to enhance early detection, monitor disease progression, and inform treatment decisions, making it a valuable asset in clinical practice. Saliva is a clinically valuable bio-fluid that serves various roles in prognosis, diagnosis, patient monitoring, and management of both oral and systemic diseases.1 Due to its easy collection and storage, saliva is ideal for early disease detection, as it contains specific soluble biomarkers. These biomarkers make saliva suitable for multiplexed assays, which are being developed for point-of-care devices, rapid tests, or centralized laboratory formats. Research has shown that saliva contains a wide array of molecular and microbial analytes. This makes salivary diagnostics a rapidly advancing field that is becoming integral to disease diagnosis, clinical monitoring, and informed clinical decision-making.2 Human saliva is a clear, slightly acidic (pH 6.0 to 7.0) bio-fluid, composed primarily of water (99%), with small amounts of proteins (0.3%) and inorganic substances (0.2%). Average saliva production ranges from 0.3 to 0.7 ml per minute, yielding about 1 to 1.5 litres daily. Three major glands (parotid, submandibular, and sublingual) and numerous minor glands around the mouth and throat produce saliva, with the major glands accounting for 90% of its volume.3 The salivary glands, surrounded by capillaries, allow easy transfer of blood-based molecules into saliva-producing cells, suggesting that blood-derived molecules might affect saliva's molecular makeup. Saliva sampling offers advantages: it is quick, cost-effective, non-invasive, and well-tolerated by children and individuals with disabilities, and it poses minimal risk to healthcare providers. Serum substances can enter saliva via passive diffusion, active transport, or ultrafiltration, supporting the idea that saliva can act as a “window to health.” Using saliva as a diagnostic tool aligns with the need for non-invasive, accessible, and efficient testing methods.4 Its composition, which includes water, electrolytes, enzymes, mucus, proteins, and various biomolecules, reflects systemic health and provides disease-specific biomarkers, making it a valuable resource for disease detection, particularly for oral health conditions.5
Saliva versus blood
Blood is a complex bodily fluid known to contain a wide range of molecular components, including enzymes, hormones, antibodies, and growth factors. Although life-saving, the procedures to collect and eventually analyse blood samples can often be expensive, problematic, and physically intrusive. Employing salivary fluids for biomarker evaluation alleviates subject/ patient discomfort through the provision of a non-invasive method of disease detection. Saliva includes hormones, antibodies, growth factors, enzymes, bacteria and their by-products, just like serum.6
Major advantages that saliva carries over blood
1. Collection procedure is undemanding.
2. Procedure is non-invasive.
3. Samples are easier to ship and store.
4. The procedure is economical.
Factors Influencing Saliva Composition and Salivaomics in Diagnostic Applications
Saliva composition and total volume are highly variable, influenced by numerous factors such as the time of day, hydration levels, body posture, medications, smoking, psychological states, dietary intake, and other systemic conditions. These variables can significantly alter saliva characteristics within the same individual. Saliva sampling may occur at rest ("unstimulated saliva") or following stimulation with agents like chewing gum or citric acid, which increases volume and alters composition.7For instance, parasympathetic stimulation raises flow rates, while sympathetic activation yields a lower flow with a higher concentration of proteins and peptides. In clinical trials, unstimulated saliva collection generally follows fasting for at least one hour, during which the subject remains seated and avoids oro-facial movements, rinsing the mouth with deionized water before sampling. Saliva can be obtained from whole saliva or a single gland, although single-gland collection is less common due to patient discomfort. Whole saliva includes additional materials like food particles, bacteria, epithelial cells, and leukocytes.8 Standard collection methods employ specialized devices (e.g., Salivette®, Quantisal®, Orapette®, and SCS®) to ensure sample integrity. Literature highlights some debate regarding centrifugation protocols, speed, use of protease inhibitors, and storage conditions; protease inhibitors and cold storage (?80 °C) are widely recommended to prevent bacterial growth and preserve salivary proteins.9The emerging field of "salivaomics," coined in 2008, encompasses various “omics” within saliva—genome, transcriptome, proteome, metabolome, and microbiome. Approximately 70% of the DNA in saliva is human, while the remaining 30% originates from the oral microbiota. Despite containing less DNA than blood, saliva can still be effective for genotyping and epigenetic analysis, with techniques like PCR and sequencing arrays enabling detection of epigenetic changes such as aberrant DNA methylation, an early marker for neoplastic processes.10
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Transcriptomes |
mRNA and microRNA in saliva can be detected through reverse transcriptase PCR and microarray. Stabilization techniques for salivary RNA, pioneered enable processing for cancer and disease diagnostics. Noncoding RNAs, especially microRNAs (miRNAs), have shown significant potential due to their role in oncogenesis and high stability in saliva. MicroRNAs are linked to cell differentiation, proliferation, and survival, with dysregulated levels observed in cancer tissues. Salivary miRNAs are particularly stable, making saliva suitable for transcriptome analysis.11 |
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Metabolome |
Saliva contains metabolites like nucleic acids, vitamins, lipids, and amino acids. Salivary metabolomics can offer insights into general health or disease states. Nuclear Magnetic Resonance (NMR) spectroscopy and Liquid Chromatography–Mass Spectrometry (LC-MS) are key methods, with LC-MS being highly sensitive and capable of analyzing a broad range of analytes through molecular fragmentation patterns.12 |
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Proteome |
The salivary proteome consists of over 2000 proteins, some detectable in plasma, offering numerous biological insights. Rapid degradation of saliva proteins is a challenge; thus, protease inhibitor cocktails (PIC) are recommended. Techniques like NMR, gas and liquid chromatography (GC-MS and LC-MS), and two-dimensional gel electrophoresis (2D-PAGE and 2D-DIGE) are widely used. Immunoassays, particularly enzyme-linked immunosorbent assay (ELISA), detect antibody or antigen presence and are widely applied in diagnostics for their sensitivity and specificity.8,9 |
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Microbiome |
Saliva microbiome research, enabled by next-generation sequencing, has identified approximately 19,000 microorganisms. Oral dysbiosis is linked to conditions like periodontal disease, caries, and possibly cancer. Techniques like 16S rRNA gene sequencing and PCR are commonly used, though cost limits routine diagnostic use. Electromigration techniques and mass spectrometry methods, such as MALDI-TOF MS (matrix-Assisted Laser Desorption-Time of flight Mass Spectometry), provide quick and accurate microorganism profiling, while histopathology remains essential for conditions requiring direct immunofluorescence (DIF) or biopsies.11 |
I.Saliva in oral disease:
Dental Caries
Dental caries, a common and multifactorial oral disease, progresses through demineralization of tooth structures such as enamel, dentin, and cementum, potentially leading to tooth loss and pain. As it is irreversible in advanced stages, early detection and prevention through good oral hygiene are essential.11 Saliva serves as a valuable diagnostic tool for caries risk, as it contains structures like remineralizing molecules and bacteria. Key bacterial markers include Lactobacilli and Streptococcus mutans, with other species like Streptococcus sobrinus and Prevotella varying between healthy and caries-affected individuals. Salivary proteins such as statherin, cystatin, histatins, and proline-rich proteins contribute to enamel health and are found in higher concentrations in those with no caries history.11 Increased levels of immunoglobulin A (IgA) have also been associated with caries. Additionally, oxidative stress in saliva can exacerbate carious lesions by affecting dentin’s response to bacterial acids, which, combined with the presence of enzymes like matrix metalloproteinases (MMPs), may lead to dentin destruction and tissue damage.13 Mucins in saliva, particularly MUC7 (mucin7), play a protective role by aiding bacterial agglutination and preventing demineralization; lower levels of mucins are associated with a higher risk of caries. The quantity, flow rate, and viscosity of saliva are also crucial, as insufficient salivary flow can hinder acid clearance, shifting the pH balance towards acidity and increasing caries risk.13 Reduced saliva flow rates and altered chloride levels have been linked to tooth erosion, especially in children. Commercial tests (e.g., CRT bacteria®) indicate that high salivary S. mutans and Lactobacilli levels are correlated with caries presence, though specificity and sensitivity of caries risk assessments vary due to the complex interplay of local and systemic factors in caries development.15
Periodontal and Peri-Implant Diseases
Periodontal diseases, which cause inflammation of the gingiva, periodontal ligament, and alveolar bone around teeth, are a major factor in tooth loss. Similarly, peri-implantitis affects tissues around dental implants and leads to rapid bone resorption. Although both conditions share similar etiologies, peri-implantitis advances more quickly.16 Early diagnosis efforts for these diseases focus on identifying biomarkers in gingival crevicular fluid (GCF) and saliva. Key bacterial markers in these diseases include gram-negative bacteria like Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis. Elevated levels of salivary immunoglobulins (IgA, IgG, IgM) indicate an immune response to these pathogens, with radial immunodiffusion and nephelometer commonly used for immunoglobulin detection.17 Enzyme-linked immunosorbent assay (ELISA) also identifies various biomarkers in saliva, with levels decreasing post-treatment. Interleukins, particularly IL-1 and IL-6, play a role in triggering bone and tissue degradation in periodontal disease. These interleukins are produced by multiple cell types and interact with matrix metalloproteinases (MMPs) like MMP-8 and MMP-9, key enzymes in tissue destruction. Point-of-care tests like Perio Safe and Implant Safe detect active MMP-8 in oral fluids, correlating positively with periodontitis and peri-implantitis activity and turning negative after effective treatment. Oxidative stress markers, such as total antioxidant capacity and nitric oxide levels, also help indicate periodontal health.18 Antioxidant levels decrease in those with periodontal disease, while nitric oxide levels rise in cases of gingivitis and aggressive periodontitis. Further, specific salivary markers, including hormones, epithelial keratins, ions, and immunoglobulins, are examined in both saliva and GCF to monitor disease progression. Salivary genomics represents a promising area of study for periodontal disease diagnosis, as variations in over 70 genes have been associated with these conditions. In diabetic patients, elevated salivary IL-6 levels correlate with periodontal infection, showing the systemic impact of diabetes on periodontal health and offering a future biomarker for assessing periodontitis risk and severity in these individuals.19
Salivary Biomarkers in Sjögren’s Syndrome
Sjogren’s syndrome (SS) is a systemic autoimmune disorder primarily affecting the exocrine glands, especially the salivary and lacrimal glands, resulting in inflammation, glandular destruction, and often severe dry mouth (xerostomia). Primarily affecting menopausal women, SS can lead to systemic manifestations, including joint, gastrointestinal, and central nervous system involvement, and an increased risk of lymphoma. Traditionally, SS diagnosis involves clinical assessment and salivary gland biopsy.20 However, advancements in salivary biomarker research allow for non-invasive diagnostic alternatives, helping distinguish SS from other autoimmune diseases like systemic lupus erythematosus (SLE). Key autoantibodies, including Anti-Ro/SSA and Anti-La/SSB, have been identified in both serum and saliva, effectively differentiating SS from SLE. Salivary analyses also reveal elevated cytokine levels, including Th1 (T-Helper cells 1), Th2, and Th17, similar to those observed in serum. Proteomic studies in SS have identified elevated inflammatory proteins and immune-related molecules in saliva. Notably, a recent study highlighted soluble siglec-5 as a promising marker, with its elevated levels reflecting hyposalivation severity in SS patients. Moreover, novel miRNAs, such as miR-146a, miR-768-3p, and miR-574, are linked to SS-related glandular inflammation, suggesting their utility in early diagnosis.21 Epigenetic modifications, such as reduced DNA methylation and alterations in specific gene expressions [e.g., DNMT1 (DNA methyltransferase 1) and Gadd45a], further contribute to SS pathogenesis. One of the most critical areas of SS research involves the early detection of MALT(mucosa associated lymphoid tissue)-type lymphoma, a potential SS complication. Specific markers, including anti-cofillin-1, anti-alpha-enolase, and anti-Rho GDP-dissociation inhibitor 2, have been identified as elevated in SS patients who develop this lymphoma, aiding in early intervention. Additionally, research into the SS-associated microbiome has identified differences in microbial diversity compared to healthy individuals, with specific bacterial genera (e.g., Bifidobacterium, Dialister, Lactobacillus, and Leptotrichia) being more prevalent in SS patients. Such microbial shifts, influenced by steroid treatment, reveal the potential of microbiome analysis in assessing therapeutic responses in SS.22
II. Saliva in the diagnosis of cancer and potentially malignant oral disorders:
Saliva in Oral leukoplakia (OL)
Oral leukoplakia (OL) is a commonly encountered precancerous lesion with a 10% risk of developing into cancer. It is defined as a “white plaque of questionable risk” after excluding other known conditions without an increased cancer risk, with an estimated prevalence rate of 1.7–2.7% in the general population. The WHO categorizes OL as an oral potentially malignant disorder (OPMD), with a reported 1% average annual malignant transformation rate. Despite advancements in molecular biology, there is no definitive biomarker to predict malignant transformation in OL, although cytokines are widely studied as potential indicators.25 Studies using ELISA techniques have documented elevated levels of interleukin-1? (IL-1?), interleukin-6 (IL-6), cystatin, and apolipoprotein A-1 in unstimulated saliva from OL patients, suggesting inflammation's role in this condition. Proteomic research on saliva in OL remains limited; however, certain biomarkers have been found useful in distinguishing between OL and oral squamous cell carcinoma, including proteins such as complement component C4D, malondialdehyde (MDA), endothelin-1, and lactate dehydrogenase (LDH. These findings indicate that while specific biomarkers in OL remain under investigation, cytokines and particular proteins may hold promise for early detection and monitoring of malignant transformation risk.25
Oral Squamous Cell Carcinoma
Oral cancers, particularly oral squamous cell carcinoma (OSCC), are among the most prevalent cancers in the head and neck region, with a high mortality rate globally. OSCC is linked to environmental and genetic factors, with tobacco and alcohol consumption being the most modifiable risk factors. Human papillomavirus (HPV) has also gained recognition as a contributing factor in recent studies. Traditionally, biopsies have been the standard diagnostic tool for oral carcinomas, but due to their invasive nature, there is a growing demand for non-invasive diagnostic methods, such as salivary biomarkers. Over 100 biomarkers have been identified for oral cancer diagnosis, including proteins, RNA, and DNA found in saliva. MicroRNAs (miRNAs) such as miRNA-9, miRNA-191, miRNA-125a, and miRNA-200a, have shown potential as biomarkers for head and neck cancers, with specific patterns linked to OSCC progression. Additionally, the tumor suppressor p53 gene and its associated anti-p53 proteins, along with other markers like CA15-3 (cancer antigen 15-3) and CA125, have been implicated in the diagnosis of head and neck cancers as well as those in other regions like breast, ovarian, and lung cancers. Matrix metalloproteinases (MMPs) also play a critical role in tumor progression, from cell proliferation to invasion and metastasis, and studies have shown elevated levels of MMP-1, MMP-2, MMP-9, MMP-10, and MMP-12 in OSCC patients. Salivary biomarkers have proven useful in distinguishing OSCC from other oral conditions and predicting the malignant transformation of oral potentially malignant disorders (OPMDs) such as leukoplakia. Proteins such as IL-6, IL-8, IL-1, TNF-? (tumor necrotic factor – ?), and cell-surface glycoproteins like CD44, CD59, and CEA (carcino-embryonic antigen) have been extensively studied as potential OSCC biomarkers. Non-targeted proteomic studies have identified additional proteins, such as M2BP(membrane attack cell-2 binding protein), MRP14(myeloid related protein-14), CD59, catalase, and profilin, that show promise in diagnosing OSCC with high sensitivity and specificity. Furthermore, recent studies have highlighted the potential of metabolic markers and glycoproteins, such as fucose and sialic acid, in diagnosing and monitoring OSCC. The role of the microbiome in oral cancer is also being explored, with some studies suggesting that microbial communities could modulate cancer metabolism and contribute to carcinogenesis. The presence of certain microorganisms, such as Neisseria and Candida species, has been linked to the production of carcinogenic agents like acetaldehyde and N-nitrosamines in the oral cavity, further supporting the multifactorial nature of OSCC development. Salivary diagnostic tools, utilizing biomarkers like miRNAs, MMPs, and microbial analysis, are emerging as valuable non-invasive alternatives to traditional biopsy methods, offering the potential for early detection and better prognosis for patients with OSCC.4,17,20,24,25,26 Saliva in Oral lichen planus (OLP)
Oral lichen planus (OLP) is a chronic inflammatory condition of the oral mucosa, prevalent in about 0.5%–2% of adults, with a slight female predominance. It manifests in several clinical forms, including reticular, erythematous, and ulcerative types, impacting patients' quality of life by causing symptoms from mild discomfort to severe pain. Although the exact cause is not fully understood, autoreactive T cells are implicated in its pathogenesis, and potential triggers include stress, hepatitis C infection, and certain medications. The premalignant nature of OLP, with an estimated annual transformation rate into oral squamous cell carcinoma (OSCC) of 0.04–1.74%, has led to substantial research into salivary biomarkers for early detection, disease monitoring, and assessing malignancy risk. Key biomarkers include cortisol, immunoglobulins (IgA and IgG), cytokines, and oxidative stress-related molecules. Cortisol, often elevated in OLP patients, reflects a complex relationship between stress and disease onset, while immunoglobulins, specifically IgA and IgG, highlight immune responses in OLP. Cytokines like interleukin-6 (IL-6) and interleukin-8 (IL-8) play essential roles in inflammation; IL-6 levels correlate with heightened cancer risk by impacting p53 function, while IL-8, elevated particularly in the erosive form of OLP, is linked to keratinocyte damage and shows therapeutic response by decreasing after dexamethasone treatment. Oxidative stress is marked by reduced antioxidant capacity and elevated levels of thiobarbituric acid-reactive substances (TBARS), indicating increased cellular damage and lipid peroxidation, which contribute to keratinocyte apoptosis in OLP. Proteomic studies reveal specific proteins associated with inflammation and immune modulation in OLP, including elevated complement C3c and fibrinogen fragment D, as well as decreased cystatin SA, an antimicrobial cysteine protease inhibitor. Additionally, proteins like S100A8 and S100A9 support the role of T-cell responses, while zinc-binding AZGP1 (alpha-2-glycoprotien 1) is associated with T-cell proliferation and differentiation. Together, these findings offer a detailed profile of OLP’s biomolecular landscape, supporting the use of salivary biomarkers in diagnosing, monitoring, and managing OLP, potentially advancing early detection of malignant transformation and enabling personalized therapeutic strategies.23,24
III. saliva in diagnosis of systemic disease:
Systemic Diseases
Cardiovascular diseases (CVDs), including hypertension, atherosclerosis, and myocardial infarction, remain among the leading causes of death worldwide. Traditional diagnostic tools such as clinical findings, electrocardiograms, and blood tests are used to identify these conditions, but emerging research is exploring the potential of saliva as a non-invasive diagnostic tool. Several biomarkers in saliva can reflect changes associated with CVDs, making it a promising medium for early detection and monitoring. For instance, C-reactive protein (CRP), a marker of inflammation, can be detected in saliva using nano-biochip-based systems, providing a quick, chair-side diagnostic tool. In patients with acute myocardial infarction, biomarkers such as CRP, MMP-9, IL-1?, adiponectin, E-selectin, and IL-18 were found to be elevated in saliva more than in serum, suggesting that saliva may offer more sensitive markers for cardiovascular events. Additionally, the levels of MMP-8 and lysozyme in saliva have been found to increase in hypertensive individuals, further supporting the role of saliva in cardiovascular disease monitoring. In the case of atherosclerosis, antioxidants like vitamin C, lutein, lycopene, ?-tocopherol, and ?-carotene play a crucial role in protecting against oxidative stress. Lower levels of these antioxidants in the saliva of patients with ischemic heart disease and periodontitis indicate potential biomarkers for cardiovascular health. Saliva’s nitric oxide levels, which are linked to carotid endothelial damage, could also serve as an indicator of atherosclerosis progression. Diabetes, traditionally monitored through blood tests, has also shown correlations with saliva biomarkers. Salivary glucose and urea levels align with blood concentrations, offering a potential non-invasive method for diabetes management. In diabetic patients, increases in amylase, total protein, and electrolytes like potassium, calcium, and chloride in saliva have been noted. salivary biomarkers could aid in diabetes diagnosis and monitoring. Saliva is also a key diagnostic tool for viral infections. It is commonly used for the detection of HIV, hepatitis, rubella, and other viral infections. Saliva-based diagnostic methods have been particularly useful in detecting COVID-19. Studies comparing saliva and nasopharyngeal samples for SARS-CoV-2 detection have shown that saliva offers promising results, with high sensitivity (84.2%) and specificity (98.9%) for detecting the virus. In renal disease diagnosis, salivary biomarkers have also shown promise. Increased creatinine levels in saliva correlate with renal disease, and selenium levels decrease in the presence of renal stones, making it a potential biomarker for kidney health. Furthermore, salivary pH levels tend to rise in dialysis patients due to high urea concentrations, providing additional diagnostic insights.28,29,30
CONCLUSION
Saliva-based diagnostics are advancing rapidly, driven by innovations in molecular analysis and device miniaturization, improving sensitivity and specificity for disease detection and monitoring. Technologies such as PCR-based assays for DNA and RNA biomarkers, mass spectrometry and ELISA for protein quantification, and lab-on-a-chip devices for real-time testing are transforming salivary diagnostics. Additionally, biosensors enhance detection sensitivity, enabling personalized medicine, while liquid biopsy methods are proving effective in detecting oral squamous cell carcinoma and other cancers. Despite these advancements, challenges like saliva composition variability and the need for standardization remain. Further research is essential to validate these technologies and integrate them into routine clinical practice, offering a non-invasive, accessible tool for diagnosing and monitoring various diseases.
REFERENCES
Dr. K. Pazhanivel, Dr. Priya Ramani, Dr. Illakhiya V.*, Dr. K. Jelin Lilly, Dr. A. Joshua, Saliva – A Diagnostic Tool for Oral Cancer and Oral Diseases- A Review, Int. J. of Pharm. Sci., 2024, Vol 2, Issue 11, 1334-1343. https://doi.org/10.5281/zenodo.14221670
10.5281/zenodo.14221670
