Elevated Salivary Inflammatory Biomarkers are Associated with SARS-CoV-2 Infection Severity

High levels of inflammatory cytokines in serum have been reported in patients with severe SARS-CoV-2 infection. There is growing interest in recognizing the role of inflammatory biomarkers in saliva in diagnosing systemic diseases. This study assumed that estimating biomarkers in saliva samples from patients infected with SARS-CoV-2 would distinguish between mild and severe cases. Saliva was collected from 142 controls and 158 SARS-CoV-2 patients (mild 72 and severe 86) to measure interleukin-6 (IL-6), C-reactive protein (CRP), and C-X-C motif chemokine ligand-10 (CXCL-10). IL-6 and CXCL-10 were significantly increased in patients with mild and severe SARS-CoV-2 infections. CRP was significantly increased only in severe SARS-CoV-2 cases. All biomarkers were significantly higher in severe cases than in mild cases (p < 0.001). Among patients with SARS-CoV-2 infection, men showed significantly higher CRP and CXCL-10 levels than females (p < 0.01 and 0.05, respectively). In addition, elderly patients (40–80 years) had significantly higher IL-6, CRP, and CXCL-10 (p < 0.001). Patients with diabetes and hypertension showed elevated IL-6, CRP, and CXCL-10 (p < 0.001). There was a significant positive correlation between IL-6, CRP, CXCL-10, and between age, IL-6, CRP, and CXCL-10. Saliva may have a future value in measuring the inflammatory biomarkers associated with the severity of SARS-CoV2 infection and therapeutic monitoring.


Introduction
Coronavirus disease 2019 (COVID-19) is a fatal disease caused by the unique SARS-CoV-2 virus spreading throughout the world [1]. e disease manifests itself in a variety of symptoms and degrees of severity. While most SARS-CoV-2 virus infections are asymptomatic and mild, the full spectrum of illnesses encompasses more severe illnesses, ranging from moderate to severe, necessitating increasing medical intervention, and is potentially fatal [2][3][4][5]. In severe cases, fast clinical worsening is common, and the serious stage of the disease is characterized primarily by a systemic hyperinflammation known as a "cytokine storm" [6,7]. Inflammatory cytokines and chemokines (interleukin (IL)-6, C-X-C motif chemokine ligand 10 (CXCL-10), and IL-10), inflammasome (IL-18 andIL-1), and interferons (interferon (IFN)-α, IFN-c, and IFN-λ) are overexpressed in patients with severe SARS-CoV-2 infection [8][9][10]. Recent research has shown that C-reactive protein (CRP) concentration is higher in SARS-CoV-2-infected patients and may be related to the severity and progression of the disease [11,12]. e inflammatory biomarkers, including IL-6, CRP, and CXCL-10, could help clinicians better understand disease progression and therapeutic responses to specific therapies. Until now, these inflammatory biomarkers were only examined in serum samples of SARS-CoV-2-infected patients [13].
ere is increasing interest in recognizing the role of inflammatory biomarkers in saliva in diagnosing systemic diseases [14]. Saliva provides several advantages over blood as a noninvasive alternative biological fluid, including ease of collection from complex patients, repeatability, cost-effectiveness, the lack of a need for rapid sample processing, and the flexibility to collect samples anywhere [15]. Furthermore, collecting saliva on one's own minimizes the risk of SARS-CoV-2 infection spreading to healthcare workers.
According to Adeoye and omson's theory, biomarkers for SARS-CoV-2 infection outcome detection could be secreted in saliva via systemic-and local-based outflow mechanisms [16]. is study hypothesized that estimating biomarkers in saliva samples from SARS-CoV-2-infected patients would distinguish between mild and severe cases. erefore, the study aimed to identify and compare salivary IL-6, CRP, and CXCL-10 concentrations in mild and severe SARS-CoV-2-infected patients and compare them with the results of a group of healthy participants.

Participants.
is study was approved by the Saudi Ministry of Health (MOH) (Institutional Review Board of Research and Studies Department, Jeddah Health Affairs, Saudi Arabia). e IRB approval number is A00981. is study included 142 healthy participants who constituted the noninfected participants and 158 SARS-CoV-2-infected patients.
e SARS-CoV-2-infected patients were further divided into the mildly infected group (86 patients) and the severe infected group (72 patients). Patients were classified according to the MOH classification during the SARS-CoV-2 virus pandemic guidelines based on whether they only needed supportive measures (mild) or monitoring and/or intervention in the intensive care unit (ICU) (severe). Using the mean and standard deviation of IL-6 in healthy and SARS-CoV-2-infected patients and a power of 80% at a 5% level of significance, the number of individuals was calculated. e data analysis was carried out using ClinCalc statistic analyzer software (https://clincalc.com/stats/samplesize.aspx) [17].
Mild SARS-CoV-2-infected patients were recruited from COVID-19 isolation centers in Jeddah, Saudi Arabia. On the other hand, severe SARS-CoV-2-infected patients were recruited from many local government medical hospitals in Jeddah, Saudi Arabia ( e Field Hospital, King Fahad General Hospital, King Abdullah Medical Complex Hospital, and East Jeddah General Hospital). Before the study began, all participants signed an informed consent form. e research was carried out between September 1, 2020, and September 1, 2021.

Inclusion Criteria.
All included participants were unvaccinated. Non-SARS-CoV-2-infected participants were not previously infected with the coronavirus. Negative PCR results confirmed non-SARS-CoV-2-infected participants. Positive PCR results confirmed patients infected with SARS-CoV-2. e PCR was done using either nasopharyngeal or oropharyngeal swabs.

Exclusion Criteria.
Participants were vaccinated against the SARS-CoV-2 virus, under 18 years of age, or had autoimmune disease, periodontitis, oral mucosal diseases, and oral cancer. Patients treated with any drug that affects IL-6, CXCL-10, and CRP.

Collection of Demographic and Clinical Data.
Demographic and baseline clinical data of all participants, including nationality, age, gender, weight, height, and history of any chronic disease, were collected through a form created using the SurveyMonkey website and delivered through each patient's WhatsApp number.

Collection of Saliva Samples.
All participants were asked to abstain from smoking, eating, drinking, and oral hygiene for at least one hour before sample collection. According to preestablished protocols of [15,18], saliva samples were taken between 7 and 9 a.m. using the passive drooling procedure. Approximately 2 ml of total unstimulated saliva was collected. e samples were immediately frozen at −20°C and then stored at −80°C. To remove insoluble material and cell debris, the saliva samples were centrifuged at 3000 rpm and 4°C for 10 min. All experiments were carried out on the supernatants. e test was repeated twice to ensure accuracy. All tests were performed according to the manufacturer's recommendations.

Statistical Analysis of Data.
Concerning demographic and baseline clinical data, descriptive statistics were performed, and data were expressed as frequency (number and percentage). Chi-squared and Fisher's tests were used to compare the demographic and clinical data of mild and severe SARS-CoV-2-infected groups.
Concerning the IL-6, CRP, and CXCL-10 measurements, the normality of the distributions was evaluated using the Kolmogorov-Smirnov normality test. e ANOVA test was used to compare the results of the non-SARS-CoV-2-infected participants, mild and severe SARS-CoV-2-infected patients, followed by Tukey's multiple comparisons test. e unpaired t-test was used to compare the data of non-SARS-CoV-2-infected participants and all SARS-CoV-2-infected patients. Regression and Pearson correlation were also performed.
Statistical data evaluation was performed using Prism ® (version 8.4.0, GraphPad Software Inc., La Jolla, CA, USA). e level of significance was established at p < 0.05.
Statistical analysis of data showed no significant differences between gender and salivary concentrations of IL-6, CRP, and CXCL-10 within the non-SARS-CoV-2, mild SARS-CoV-2, and severe SARS-CoV-2 groups (data not shown).
Statistical analysis of data showed no significant differences between age and salivary concentrations of IL-6, CRP, and CXCL-10 within the non-SARS-CoV-2, mild  SARS-CoV-2, and severe SARS-CoV-2 groups (data not shown).
Statistical analysis of data showed no significant differences between BMI and salivary concentrations of IL-6, CRP, and CXCL-10 within the non-SARS-CoV-2, mild SARS-CoV-2, and severe SARS-CoV-2 groups (data not shown).

Effect of DM on Concentrations of Salivary IL-6, CRP, and CXCL-10.
ere were no significant differences in the salivary concentrations of IL-6, CRP, and CXCL-10 between   diabetic and nondiabetic participants in the non-SARS-CoV-2 participants. On the other hand, salivary concentrations of IL-6, CRP, and CXCL-10 were significantly increased (p < 0.001) in SARS-CoV-2 patients with DM compared to SARS-CoV-2 patients without DM (Figures 5(a)-5(c)).
Statistical analysis of data showed no significant differences between DM and salivary concentrations of IL-6, CRP, and CXCL-10 within the non-SARS-CoV-2, mild SARS-CoV-2, and severe SARS-CoV-2 groups (data not shown).

Effect of HT on Concentrations of Salivary IL-6, CRP, and CXCL-10.
ere were no significant differences in the salivary concentrations of IL-6, CRP, and CXCL-10 between participants with and without HT in the non-SARS-CoV-2 participants. On the other hand, salivary concentrations of IL-6, CRP, and CXCL-10 were significantly increased (p < 0.001) in SARS-CoV-2 patients with HT compared to SARS-CoV-2 patients without HT (Figures 6(a)-6(c)).
Statistical analysis of data showed no significant differences between HT and salivary concentrations of IL-6, CRP, and CXCL-10 within the non-SARS-CoV-2, mild SARS-CoV-2, and severe SARS-CoV-2 groups (data not shown).

Discussion
COVID-19 is a fatal respiratory illness caused by the SARS-CoV-2 virus. e severity of the COVID-19 disease is related to the triggering of a cytokine storm. e cytokine storm associated with SARS-CoV-2 virus infection is currently believed to be dominated by IL-6, CXCL-10, and infiltrating macrophages. Serum CRP has been identified as a critical inflammatory measure that fluctuates dramatically in severe SARS-CoV-2 virus infection [19][20][21]. Inflammation, reflected by cytokine storms and CRP in COVID-19 patients, could worsen the disease [22]. Saliva has been studied and described as a possible source of diagnosis for a wide variety of conditions [23]. e current study suggests saliva as a reliable method to detect the severity of SARS-CoV-2 virus infection due to the convenience of taking samples, the potential for self-collection, the low cost, the elimination of special equipment, and the reduced risk of transmission for healthcare professionals [15].
is study included 158 SARS-CoV-2 infection cases with symptoms ranging from mild (72) to severe (86) and 142 healthy participants. IL-6, CRP, and CXCL-10 were measured in the saliva samples of all participants. Saliva IL-6 and CXCL-10 were significantly increased in both mild and severe SARS-CoV-2 patients compared to the control non-SARS-CoV-2 participants. Saliva CRP was significantly increased only in severe SARS-CoV-2 patients. In severe cases, all of the biomarkers were significantly higher than the mild cases. A significant positive correlation was found between salivary IL-6 and both CRP and CXCL-10 in all SARS-CoV-2 patients.
Our results are similar to recently published results, which proposed that patients with COVID-19 could be distinguished from the noninfected people using a panel of 27 cytokines, including chemokines and growth factors. IL-6, IL-5, GCSF, IL-2, TNF-α, GMCSF, and IFN-c were identified as COVID-19-related distinguishable cytokines, but only one (IL-12p70) was detected in the noninfected people. e most predictable cytokines for COVID-19 and control were IL-6 and IL-12p70. Results of serum cytokine profiling in patients with severe symptoms and complications showed a cytokine profile that closely paralleled the saliva cytokine profile at the time of infection [24]. e present study results also agree with those of Santa Cruz et al. [25], who reported a significant increase in serum IL-6 concentration along with the COVID-19 disease stage (I, IIa, IIb, and III). However, they only declared a significant increase in serum CRP concentration in stage III of the disease, the most severe stage. eir results also confirmed a positive correlation between serum IL-6 and serum CRP. Our results also agree with Han et al. [22], who found that SARS-CoV-2infected patients had higher serum concentrations of IL-6 and CRP than healthy controls.
Furthermore, serum IL-6 concentration in SARS-CoV-2-infected patients was significantly increased in the critical cases than in the moderate and severe cases. Compared to patients treated outside the ICU, SARS-CoV-2-infected patients who were eventually admitted to the ICU had significantly higher serum concentrations of IL-6 and CRP [26]. Like our results, Tripathy et al. [27] observed that asymptomatic and mildly symptomatic SARS-CoV-2-infected patients had significantly higher plasma concentrations of IL-6 and CXCL-10 than controls; furthermore, CXCL-10 was significantly higher in mildly symptomatic patients. In contrast to the result of the present study, there were no changes in plasma IL-6 concentration between asymptomatic and mildly symptomatic patients [27].
is study found less saliva concentrations of CRP and CXCL-10 in SARS-CoV-2-infected females than males. Several investigations around the world have suggested a gender discrepancy in the severity and prognosis of patients with SARS-CoV-2 infection due to the mechanisms of viral infection, the immune response to the virus, the establishment of the inflammatory process, and subsequent systemic consequences, including the thromboembolism. COVID-19 has a gender difference in severity, with women having a better prognosis than men, according to epidemiological statistics [28]. Male mice inoculated with SARS-CoV-MA15, which mimics the clinical symptoms of SARS, are more vulnerable to infection than females. e higher susceptibility of male mice was associated with higher virus titers, neutrophil infiltration, and concentrations of proinflammatory cytokines (IL-1, IL-6, and TNF) in the lungs, suggesting a poorer natural and adaptive immune response in males than in females, most likely due to differences in expression and activity of immune-related genes on the X chromosome [29].
ere were no significant differences in salivary IL-6, CRP, and CXCL-10 concentrations among overweight, obese, and normal weight SARS-CoV-2-infected patients in the mild and severe cases. Similar to our results, McNeill et al. [30] observed no significant association between obesity and serum IL-6 concentration. On the other hand, the same authors indicated that obese people had higher baseline and peak serum CRP concentrations than nonobese people [30]. Frasca et al. [31] also found a significant positive association between BMI and serum CRP concentration in PCR-positive COVID-19 patients.
e present findings showed significantly higher salivary IL-6, CRP, and CXCL-10 in diabetic and hypertensive SARS-CoV-2-infected patients. Diabetes, cerebrovascular disease, and hypertension are among the most prevalent comorbidities noted in patients with confirmed COVID-19 [32]. Metabolic diseases, including type 2 DM and HT, are associated with chronic low-grade systemic inflammation or meta-inflammation. ese diseases constitute deadly systemic scenarios for SARS-CoV-2 infection results [33,34]. Meta-inflammation develops after activating adipose tissueresident macrophages, promoting the recruitment of M1polarized macrophages, which is a more proinflammatory phenotype, and increasing the production of proinflammatory cytokines such as TNF-α, IL-6 and chemokines both locally and systemically [35]. e number of white blood cells, acute-phase proteins such as CRP, and plasma concentrations of coagulation components (fibrinogen, D-dimers) also increase during inflammation [36]. In a case series from China, hospitalized SARS-CoV-2-infected cases with DM as the only comorbidity had high CRP and IL-6 [37]. Compared to normoglycemia, diabetes was significantly associated with severe COVID-19, while prediabetes was not a risk factor. CRP mediated 32.7% of the total association between diabetes and severe COVID-19 outcome [38]. e pathogenic link between diabetes and COVID-19 severity can be described by the following mechanisms: (i) immune dysfunction; (ii) reduced viral clearance; and (iii) heightened inflammatory state [39,40]. Diabetes is associated with higher CRP levels, [39] similar to our findings. Chronic hyperglycemia is known to incite a proinflammatory, prooxidative state, which is linked to adverse outcome in the critical care setting [41,42]. Hypertension is more common in patients with severe COVID-19 infections.
e clinical presentation of severe COVID-19 presentations has been linked to cytokine storm. Given the essential role of inflammation in the development and pathogenesis of hypertension, it is conceivable that hypertension may represent an immunologically vulnerable condition that predisposes patients to more severe presentations [32]. An increased serum level of proinflammatory cytokines such as IL-1, IL-6, IL-8, IL-17, IL-23, TGF-β, and TNF-α was observed in hypertensive patients [43].  e present data showed a direct relationship between patients' age and IL-6, CRP, and CXCL-10 concentrations in SARS-CoV-2-infected patients. Aging causes a mild, chronic, systemic proinflammatory reaction known as inflammaging, associated with a progressive decline and dysregulation of immune function. Increased concentrations of systemic proinflammatory cytokines such as IL-1, IL-6, and TNF-α characterize inflammaging [44]. Inflammaging creates a favorable setting for developing a cytokine storm in the elderly [34].
Within each COVID-19 disease category, there were no significant differences in salivary levels for IL-6, CRP, and CXCL-10 due to gender, age, DM, and HT differences (data not shown). Accordingly, the effects of gender, age, DM, and HT on the salivary levels of IL-6, CRP, and CXCL-10 observed in SARS-CoV-2 patients might be consequences of their impacts on COVID-19 disease severity.
In conclusion, the present study findings revealed that salivary concentrations of IL-6, CRP, and CXCL-10 measured in SARS-CoV-2 infected patients are comparable to those previously assessed in the blood. e saliva concentrations of these inflammatory biomarkers are associated with SARS-CoV-2 infection severity. Inflammatory markers were also higher in males, patients with diabetes, individuals with high blood pressure, and older patients. In the future, saliva could be used to assess various biomarkers of inflammation related to SARS-CoV-2 infection as a noninvasive diagnostic fluid. e current study had certain drawbacks. First, the number of patients was small, but a post hoc power analysis utilizing the values obtained to verify the null hypothesis using the mean and standard deviation indicated that the sample was large enough to give 80% power at a 5% significance level. We did not measure the concentrations of IL-6, CRP, or CXCL-10 in the serum, and it is best to do so simultaneously with the saliva sample. A combination of both saliva and serum estimation of those biomarkers will provide more information regarding the correlation between systemic and local events associated with the viral infection and help to prove if this technique could be used in the future as a noninvasive tool to differentiate between different stages of SARS-CoV-2 infection for better selection of management plan. Furthermore, knowing the serum values would have been proper to evaluate which markers are the best in the two biological fluids.

Data Availability
All data used to support the findings of the study can be obtained from the corresponding author upon request.

Conflicts of Interest
e authors declare that there are no conflicts of interest.