Systemic diseases encompass a wide range of conditions that affect multiple organs and biological systems, often leading to significant morbidity and mortality. These diseases, including diabetes mellitus, cardiovascular diseases, autoimmune disorders, and metabolic syndromes, are frequently characterized by a complex interplay of genetic, environmental, and immunological factors. One of the most challenging aspects of systemic diseases is their early diagnosis, as they often present with nonspecific symptoms that can be easily mistaken for other health conditions. This delay in diagnosis can lead to poor patient outcomes due to the progression of the disease before adequate interventions are initiated. Timely and accurate diagnosis is, therefore, crucial in managing these diseases effectively.

Traditionally, diagnosing systemic diseases relies on a combination of clinical evaluation, imaging techniques, and laboratory tests. However, these diagnostic methods are not without limitations. Blood tests, for instance, are invasive, requiring venipuncture, and may cause discomfort or anxiety for the patient. Additionally, imaging studies can be expensive, time-consuming, and sometimes offer limited sensitivity in detecting early disease stages. As a result, there is a growing need for alternative diagnostic approaches that are non-invasive, cost-effective, and capable of providing early and accurate detection of systemic diseases.

One such promising approach is the use of salivary biomarkers for diagnostic purposes. Saliva is an easily obtainable, non-invasive biofluid that offers a wide range of potential biomarkers for various diseases. Unlike blood or urine, which often require specialized techniques for collection and processing, saliva collection is straightforward, painless, and can be performed repeatedly without significant patient burden. Furthermore, saliva contains a wealth of biological markers that reflect systemic physiological conditions, including enzymes, cytokines, hormones, and antibodies. This makes saliva a valuable diagnostic medium for not only oral diseases but also for systemic conditions that affect distant organs.

Saliva is composed of various molecules, such as proteins, nucleic acids, metabolites, and lipids, which can serve as potential biomarkers for a range of diseases. These biomarkers can provide insights into the underlying pathophysiology of systemic diseases by reflecting the biochemical changes occurring in the body. For example, elevated levels of C-reactive protein (CRP), a well-known marker of systemic inflammation, have been associated with conditions like cardiovascular disease and autoimmune disorders. Interleukin-6 (IL-6), a pro-inflammatory cytokine, is another biomarker that has been shown to correlate with diseases involving chronic inflammation, such as rheumatoid arthritis and diabetes. Other biomarkers like cortisol (a hormone involved in the body’s stress response) and glucose (a marker of metabolic disorders) can provide critical insights into endocrine and metabolic dysfunctions.

In recent years, significant advancements in diagnostic technologies have made it possible to detect and quantify these biomarkers in saliva with high sensitivity and specificity. Techniques such as enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), and mass spectrometry have been employed to measure the concentrations of various biomarkers in salivary samples. These innovations have paved the way for the development of salivary diagnostics that can be used alongside or even as alternatives to traditional diagnostic tools.

Despite the growing body of research supporting the role of salivary biomarkers in diagnosing diseases, there remains a need for more extensive clinical studies to validate their efficacy in diagnosing systemic diseases. Many of the studies conducted thus far have been limited by small sample sizes or have focused on specific disease categories, without addressing the broader applicability of salivary biomarkers in diverse systemic diseases. Furthermore, the variability in salivary composition due to factors like age, gender, diet, medications, and individual health conditions can pose challenges to the standardization of salivary diagnostics. Therefore, it is essential to assess the reliability and diagnostic accuracy of salivary biomarkers in larger, more varied populations, to ensure that these tools can be implemented effectively in clinical settings.

This study aims to evaluate the role of salivary biomarkers in diagnosing systemic diseases, particularly diabetes mellitus, cardiovascular diseases, and autoimmune disorders, and to compare their diagnostic performance with conventional diagnostic methods. By analyzing specific salivary biomarkers—CRP, IL-6, cortisol, and glucose—this research seeks to establish a correlation between biomarker levels in saliva and the clinical parameters of systemic diseases. The findings from this study will contribute to the growing evidence supporting the potential of salivary biomarkers as an alternative or supplementary diagnostic tool, with the ultimate goal of improving patient care through early detection and more efficient disease management.

In summary, the use of salivary biomarkers for diagnosing systemic diseases represents a promising advancement in clinical diagnostics. This non-invasive, cost-effective approach could revolutionize the way we detect and monitor systemic diseases, making it easier for healthcare providers to identify health conditions at an earlier stage, reduce patient discomfort, and ultimately improve outcomes. The current study aims to further investigate this potential and provide valuable insights into the clinical application of salivary diagnostics.

Materials and Methods

Study Design and Setting: This cross-sectional study was conducted at the GCRG Institute of Medical Sciences, Lucknow, Uttar Pradesh, over a one-year period from January 2018 to December 2018.

Participants: A total of 200 patients, aged 18-70 years, diagnosed with systemic diseases such as diabetes mellitus, cardiovascular diseases, and autoimmune disorders, were included. Healthy controls (n=50) were also recruited for comparative analysis.

Inclusion Criteria:

1. Patients diagnosed with systemic diseases confirmed by clinical and laboratory parameters.
2. Participants willing to provide informed consent.

Exclusion Criteria:

1. Individuals with local oral infections or recent dental procedures.
2. Patients on medications known to affect salivary composition.

Sample Collection and Analysis: Unstimulated saliva samples were collected in sterile containers and processed within two hours. Salivary biomarkers, including:

• C-reactive protein (CRP)
• Interleukin-6 (IL-6)
• Cortisol
• Glucose levels were quantified using enzyme-linked immunosorbent assay (ELISA) kits. Clinical and laboratory data were simultaneously recorded.

Statistical Analysis: Descriptive and inferential statistics were performed using SPSS software. Correlations between salivary biomarkers and systemic disease indicators were evaluated using Pearson’s correlation coefficient. A p-value < 0.05 was considered statistically significant.

Results

A total of 200 patients (120 males and 80 females) diagnosed with systemic diseases, including diabetes mellitus, cardiovascular diseases, and autoimmune disorders, participated in the study. Additionally, 50 healthy controls (30 males and 20 females) were recruited for comparative analysis.

The demographic characteristics of the participants, including age and gender distribution, are summarized in Table 1.

Table 1- Demographics:

The levels of salivary biomarkers, including C-reactive protein (CRP), Interleukin-6 (IL-6), cortisol, and glucose, were significantly higher in patients with systemic diseases compared to healthy controls. The biomarker levels and their statistical comparison are presented in Table 2.

Table 2-Biomarker Analysis:

Receiver Operating Characteristic (ROC) curve analysis was performed to evaluate the diagnostic performance of the biomarkers. The following results were observed:

Table 3- ROC Curve Analysis:

Further analysis revealed that salivary biomarkers were elevated across all disease categories studied, with the following disease-specific levels:

Table4- Disease-Specific Analysis:

The potential of salivary biomarkers in the diagnosis of systemic diseases has garnered significant interest in recent years, owing to the ease of collection and non-invasive nature of saliva. Traditional diagnostic methods for systemic diseases are often invasive, expensive, and time-consuming, which can create barriers, especially in resource-constrained settings. Saliva, being rich in various biological markers, offers an alternative diagnostic medium. This study's findings corroborate earlier research suggesting that salivary biomarkers can serve as a reliable diagnostic tool for detecting systemic diseases, including diabetes mellitus, cardiovascular diseases, and autoimmune disorders.

The study primarily focused on four biomarkers: C-reactive protein (CRP), Interleukin-6 (IL-6), Cortisol, and Glucose. These biomarkers were selected for their well-established roles in systemic inflammation, metabolic dysfunctions, and endocrine regulation. The findings highlight that elevated salivary levels of these biomarkers were significantly correlated with the presence of systemic diseases, reinforcing their diagnostic potential.

CRP and IL-6 as Inflammatory Markers

CRP and IL-6 are both widely recognized markers of systemic inflammation. Elevated CRP levels in saliva have been linked to various systemic inflammatory conditions, including cardiovascular diseases and autoimmune disorders. Our study showed significantly higher CRP levels in patients compared to controls (mean levels of 15.8 µg/mL vs. 3.2 µg/mL), with a strong correlation to systemic disease indicators (Pearson’s correlation coefficient, r = 0.85). This finding is consistent with previous studies that have demonstrated the association between elevated CRP and chronic inflammatory conditions, such as atherosclerosis and rheumatoid arthritis.

Similarly, IL-6 is a pro-inflammatory cytokine that plays a critical role in immune responses and has been implicated in a variety of systemic diseases. The mean level of IL-6 in the patient group (12.4 pg/mL) was significantly higher compared to the control group (2.8 pg/mL). The correlation coefficient (r = 0.81) indicates a strong association between IL-6 and systemic disease, corroborating findings from studies on its role in conditions like sepsis, cardiovascular diseases, and autoimmune disorders (such as lupus and rheumatoid arthritis). The diagnostic performance of IL-6, with an AUC of 0.89, sensitivity of 85%, and specificity of 84%, emphasizes its potential utility in clinical settings for early disease detection.

Cortisol as an Endocrine Marker

Cortisol, a hormone produced by the adrenal glands, serves as an essential biomarker for stress response and endocrine dysfunction. Elevated cortisol levels are frequently observed in individuals with chronic stress, metabolic disorders, and certain autoimmune diseases. In our study, salivary cortisol levels were significantly elevated in patients compared to controls (mean levels of 15.4 µg/dL vs. 7.8 µg/dL), which is in line with literature highlighting its role in the pathophysiology of systemic diseases.

The correlation coefficient of 0.75 between salivary cortisol levels and systemic disease underscores the relationship between endocrine dysfunction and disease presence. The diagnostic accuracy, reflected by an AUC of 0.87 and sensitivity of 83%, suggests that cortisol could be a valuable tool in diagnosing conditions that involve dysregulated stress responses or adrenal axis abnormalities, such as in chronic fatigue syndrome and Addison’s disease.

Glucose as a Metabolic Marker

Glucose, a central metabolic marker, is elevated in diseases like diabetes mellitus, which is characterized by chronic hyperglycemia. Elevated salivary glucose levels can serve as a diagnostic indicator for metabolic dysfunctions, particularly in diabetes. Our study found that mean salivary glucose levels in patients with diabetes (28.6 mg/dL) were significantly higher compared to controls (5.4 mg/dL), with a strong correlation coefficient of 0.78. The AUC of 0.92, sensitivity of 90%, and specificity of 86% for glucose underscore its diagnostic value. This aligns with previous studies that have demonstrated the presence of elevated glucose levels in saliva in diabetic patients. The ease of measuring glucose from saliva provides an attractive option for regular monitoring, particularly for individuals with diabetes or those at high risk.

Comparison with Traditional Diagnostic Methods

Conventional diagnostic techniques, such as blood tests, imaging, and tissue biopsies, are often invasive and costly. While these methods are highly accurate, they may not always be feasible in certain settings or for regular monitoring. In contrast, salivary biomarker analysis offers a less invasive and more accessible approach. This study's results suggest that salivary diagnostics can complement or, in some cases, substitute for traditional diagnostic techniques, particularly for the initial screening or for monitoring disease progression in real-time.

For example, CRP and IL-6 levels in saliva can be measured easily using ELISA, which is both sensitive and cost-effective, and may offer an alternative to the more expensive and invasive blood tests. Additionally, the non-invasive nature of saliva collection makes it particularly appealing for vulnerable populations, such as the elderly, children, and individuals with needle phobias. Moreover, saliva can be easily collected multiple times during the day, providing a dynamic measure of disease markers, whereas blood samples often require clinical visits.

Limitations and Future Directions

Despite promising results, there are several limitations to this study. The relatively small sample size (200 patients and 50 controls) may limit the generalizability of the findings. Moreover, while the biomarkers studied here are promising, further studies are needed to explore the broader range of salivary biomarkers and their association with other systemic diseases. Standardization of sample collection techniques, processing protocols, and assay methods is crucial to ensure reproducibility and reliability across different settings. Longitudinal studies are also necessary to determine the utility of salivary biomarkers in monitoring disease progression and assessing treatment responses over time.

In addition, the potential for confounding factors—such as diet, medication, or circadian rhythms—should be carefully considered in future studies. These factors can influence the concentration of salivary biomarkers and may affect diagnostic accuracy. Advances in salivary diagnostic technologies, such as the integration of nanotechnologies and high-throughput omics-based approaches, may further enhance the sensitivity and specificity of these tests.

Salivary biomarkers hold great promise as a diagnostic tool for systemic diseases, offering a non-invasive, cost-effective, and easily accessible alternative to traditional diagnostic methods. Our study demonstrates that CRP, IL-6, cortisol, and glucose levels in saliva are significantly elevated in patients with systemic diseases and exhibit strong diagnostic accuracy. These findings suggest that salivary biomarkers can be effectively integrated into clinical practice, particularly for early disease detection and monitoring disease progression. However, further research is needed to validate these findings in larger, more diverse populations and to explore the clinical utility of salivary diagnostics in other disease contexts.

Acknowledgments: The authors thank the staff and patients of GCRG Institute of Medical Sciences for their cooperation during the study.

Conflict of Interest Statement: The authors declare no conflict of interest.

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