Monitoring Salivary Sialic Acid and Sialidase Activity to Assess Oral Health Status: Results of a Single Site Double-Blind Study

Featured Application

Sialic acid is a critical membrane constituent required to maintain mucosal integrity. This investigation describes the biochemical quantitation of salivary sialic acid as a rapid measure of oral health that corresponds with the clinical status of the patient. The test can be utilized widely including in remote facilities with limited resources. A rapid, low-cost, visual test for sialic acid using commonly available reagents complements the biochemical assessment. The test can be applied for screening large populations, monitoring oral health status, amenable to digital health and can advance patient engagement to manage their oral health.

Abstract

Objective: Sialic acid [SA] represents a critical mucosal membrane component maintaining mucosal integrity. This investigation stratified adult subjects based on clinical parameters of periodontal health to examine salivary sialic acid [SA] as a health measure and develop a corresponding rapid visual chair-side assay. Methods: Adults [n = 90] were enrolled and clinically stratified into healthy [n = 30], gingivitis [n = 29] or periodontitis [n = 31] groups. Saliva from subjects was evaluated for SA using the Ninhydrin method. A novel rapid SA spot test was developed utilizing filter paper discs soaked in a sialidase substrate. Substrate-laden disks were incubated at room temperature with saliva produced a blue color with increasing color intensities due to higher sialidase activity. Subjects were recalled weekly for clinical and salivary assessments. Results: Average baseline salivary SA in healthy, gingivitis and periodontal disease groups were 64, 95 and 102 µg/mL, respectively with significant differences (<0.05). Differences in SA concentrations among control and test groups were maintained throughout the study. Similarly, the differences in the color intensities in the rapid visual chair side spot test were also observed during the entire study period. Conclusions: Increasing levels of salivary SA were observed from healthy to periodontal disease with these differences remaining consistent over the study. These results corresponded with the chair-side visual assay, which is suitable for patient education or monitoring.

1. Introduction

Soft tissue (mucosal) surfaces such as those in the oral cavity, gastrointestinal surfaces and other regions play critical roles in routinely protecting the underlying regions and tissue i.e., blood vessels and structural components from the environment [1]. A range of factors influence the mucosal surfaces of the human mouth with its unique anatomical features that include the teeth and tongue. Environmental factors such as those found in the diet along with localized influences due to the distinctive niches and regions that include the unique structural features within the mouth comprise those routinely impacting the oral mucosa [1].

In addition to the above influences, an important constituent of the human mouth is its indigenous microbial populations that impact the oral mucosa. The mucosal surface of the oral cavity is colonized by large densities of both gram-positive and gram-negative bacteria with fungi and other constituents representing additional residents [2]. These microflora are found as biofilms in the supragingival plaque on the exposed surfaces of teeth, as subgingival plaque below the gumline and readily found within the other distinct niches of the oral cavity such as the tongue and cheek surfaces [1,2]. The salivary microbial populations can be considered planktonic constituents that are able to transport organisms between the oral surfaces.

Further to the above, the routine intake of diet and their nutritional features facilitate microbial proliferation leading to a range of by-products such as acids, toxins, microbial cell wall constituents and including those with immunogenic and other pathogenic characteristics [2]. Taken together microbial factors represent an important component of the stress and inflammatory burden of the mouth. Identified widely in the literature with information drawn from surveys and clinical studies are the relationships between the microbial load within the human mouth and disease. Contemporary practices in clinical dentistry are based on maintaining routine optimal oral hygiene to preserve oral health [2]. Self-care measures based on toothbrushing with toothpaste are widely accepted to cleanse the mouth and improve oral aesthetics [1,2]. Despite their availability and educational measures to reduce the burden of oral diseases, most populations report the significant impact of these diseases [2]. Some of the most common oral diseases reported are caries and periodontal disease. In the absence of adequate treatments these conditions can lead to tooth loss and changes in aesthetics with long-lasting impacts on the quality of life. Surveys show that despite widespread access to excellent dental care, only about 10% of UK adults register good oral health [2]. Reversible conditions such as gingivitis representing inflammation of the gums and structures that support the tooth are other commonly reported oral conditions that are reported in 90% of certain populations [2]. The role of microbial influences on the initiation and progression of these conditions represents an area of extensive laboratory and clinical investigations.

The oral mucosa provides the first barrier protecting the underlying structures and blood vessels from environmental insults including those by microbial constituents. In addition to protection, the oral mucosa is central to maintaining epithelial integrity and mucosal function [1,3]. The cell membranes of the exposed mucosal layers are studded with a membrane attached to sialic acid [SA] representing glycoproteins that play critical roles in epithelial integrity and mucosal function. SA reportedly serves as the inter-epithelial glue holding together the cells, aiding mucosal balance, response to stressors, etc. [3]. Studies indicate that SA is ubiquitous and found on the surfaces of cell membranes with densities that may approximate 100 mM [3]. Due to their critical role in membrane function SA are referred to as cytoprotectants with multiple functions [1,3,4,5] and serve as masks and ligands [5,6]. Sialidases are enzymes produced by organisms to liberate the sialic acid with the influenza virus representing the earliest and best-categorized system [5,6,7,8]. The free sialic acid is identified as a nutritional component for organisms and mucosal colonization [6,7,8,9,10].

Available in the literature are several previous reports examining SA and sialidase in the saliva and blood samples amongst patients of different ages [11,12,13] including those stratified by their health status [14,15,16,17,18,19,20,21]. These studies have utilized several analytical approaches for SA analysis [12,14,15,16,17,18,19,20,21,22]. Whereas these previous investigations report salivary SA levels, the results are from one sampling event and do not report evaluations over time. This clinical investigation examined saliva SA amongst adult subjects stratified by their clinical status with evaluations conducted weekly over a three-week period. The longitudinal assessment of clinically stratified subjects with clinical examinations during each sampling visit represents a distinctive feature of this study. An additional objective of this investigation was to develop a low-cost and rapid visual test to detect salivary sialidase applicable for chair-side use for patient education or monitoring.

2. Materials and Methods

2.1. Study Design

The protocol for this single-site double-blind study was approved by the ethics board of the JSS Dental College and Hospital, Mysuru, India, prior to the commencement of the study. The study was approved by the ethical board of the JSS Dental College and Hospital, JSS Academy of Higher Education & Research [JSS DCH/Ethical/15/2016-17].

All study-related procedures were conducted at the dental clinic of the JSS Dental College and Hospital, JSS AHER, Mysuru, Karnataka, India.

2.2. Study Subjects

Adult subjects of either gender from the local area and between the age of 18–70 years who voluntarily completed an informed consent were invited to a screening visit. The study was designed as a pilot to identify initial parameters relevant to evaluating SA in the context of study goals. During the screening visit, subjects were provided a unique identification number and interviewed for their medical history and health outcomes prior to a dental exam by a dentist. The study dentist completed an oral examination that included the entire dentition, the tongue, palate and all soft tissue regions. All subjects were scheduled for their examinations in the morning with these dental assessments conducted in a dental operatory under constant lighting conditions. Subjects who reported pregnancy or an impending pregnancy, breastfeeding, smoking, diabetes, systemic diseases, ongoing or scheduled medical or dental treatments including prescription medications were all excluded. Exclusion criteria also included those reporting allergies to oral hygiene formulations, participation in a clinical study in the 30 days preceding the screening visit, or a history of drug or alcohol abuse. Also excluded were subjects who reported ongoing treatments or were under the active care of a healthcare provider or scheduled for treatments including any procedures over the study period. All subjects who presented with significant dental conditions were excluded and referred to the dental clinics for immediate care. Correspondingly, all subjects were presented with dental education and provided information to augment their routine oral health and information regarding the dental clinic. This study enrolled 90 adults and were clinically stratified into healthy [n = 30], gingivitis [n = 29] or periodontitis [n = 31] groups.

2.3. Laboratory Evaluations

Whole unstimulated saliva samples were collected from subjects in a wide-mouth sterile screw-cap disposable tube marked with unique subject identifiers. Approximately 5 mL of saliva was collected from each subject. Each tube with collected saliva sample was registered in a log and the tubes were transported immediately to the laboratory for analysis. Samples were evaluated for SA using ninhydrin reagent with appropriate standards. Experimentally, an aliquot of suitably diluted saliva containing sialoglycoprotein was incubated with the Ninhydrin reagent (0.2% in ethanol) at 100 °C for 10 min. The stable color, which was produced upon reaction was measured at 470 nm [18].

A spot test was developed for evaluating saliva for sialidase activity. SA spot test was developed utilizing filter paper discs (2 × 2 cm) soaked in a colorless sialidase substrate (0.63 mM 5-Bromo-4-chloro-3-indoly-alpha-D-N-acetyl neuraminic acid-prepared by dissolving 3.52 mg in 10 mL of 150 mM sodium acetate buffer pH-5.5 containing 25 mM calcium chloride and 1 mM sodium chloride) and air-dried for use. Substrate (50 µL) laden disks were incubated at room temperature with saliva (10 µL) from subjects for about 40 min and the development of color was monitored. Sialidase-positive saliva produced a greenish-blue color with increasing color intensities due to higher salivary sialidase activity [23].

2.4. Clinical Measurements

2.4.1. Whole Mouth Gingival Index Assessment

A calibrated dentist conducted clinical measurements for dental plaque and gingivitis on all scorable teeth during all visits [24]. A primary outcome measure was the gingival index [Loe-Silness Index] using a 3-point scale on 6 surfaces per tooth: (1) mesio-facila; (2) mid-facial; (3) disto-facial; (4) mesio-lingual; (5) mid-lingual; and (6) disto-lingual. The maximum score per tooth is 18. All teeth were evaluated but assessments excluded those with cervical restorations or prosthetic crowns and third molars.

2.4.2. Whole Mouth Dental Plaque Evaluation

Another primary outcome measure was the whole mouth plaque examination based on the Turesky Modified Quigley-Hein plaque index [25]. A red dye was used to disclose the dental plaque and all scorable surfaces of the maxillary and mandibular teeth were evaluated. Dental plaque was assigned a score of 0 to 5 by a dental examiner using a dental light and dental mirror. All teeth were scored on six surfaces: (1) mesio-facial; (2) mid-facial; (3) disto-facial; (4) mesio-facial; (5) mid-lingual; and (6) disto-lingual. Scoring procedures evaluated all teeth for plaque but excluded third molars and those with cervical restorations or crowns. Mean dental plaque scores for each subject were determined by adding the values given by the dental examiner to scorable surfaces and dividing them by the total number of scored surfaces.

2.4.3. Periodontal Evaluations

Subjects underwent a whole mouth periodontal examination with assessments of clinical attachment loss and community periodontal index in accordance with previously reported procedures by Armitage GC [26]. Periodontal pocket depths were recorded as part of periodontal charting. Additionally, evaluations included bite-wing radiographs.

Subjects were characterized as healthy, gingivitis or periodontal disease based on observations during their screening examination.

• Healthy: Gingival index scores less than 1.0; pocket depths less than 3 mm; and no CAL.
• Gingivitis: Gingival index scores more than 1.0; pocket depths less than 3 mm; and no CAL.
• Periodontal disease: Gingival index scores more than 1.0; pocket depths of 4 mm or more and CAL of 3 mm or more.

2.4.4. Statistical Analysis

Summary statistics were derived for the entire population enrolled in the study with results summarizing outcomes from each of the evaluation visits. Correspondingly, summaries were calculated separately for each of the three subject groups and included all clinical and laboratory assessments collected over the entire study duration. Qualitative variables included the demographic characteristics of subjects. All clinical parameters evaluated i.e., plaque scores, gingivitis scores, and periodontal assessments comprised quantitative variables. The biochemical measures of SA comprised quantitative variables for statistical analyses. At each recall visit, statistical comparisons were conducted by one-way ANOVA to compare the quantitative results between each subject group. Comparisons were separately conducted for each clinical assessment and SA parameters. Statistically significant differences were further analyzed for inter-group comparisons by Tukey-Honestly Significant Difference (HSD). All statistically significant outcomes are reported at p < 0.05.

3. Results

The demographics of subjects who completed this study are shown in

Table 1. Demographic features of subjects enrolled in the study.

	Healthy	Gingivitis	Periodontal Disease
Entire Population	n = 30	n = 29	n = 31
Mean Age (SD *)	24.00 (2.477)	30.758 (8.360)	39.612 (9.094)
Age Range	22–34	18–45	23–56
Female Subjects	n = 21	11	10
Mean Age (SD *)	23.523 (1.470)	33.00 (8.854)	38.300 (9.238)
Age Range	22–26	18–45	29–55
Male Subjects	n = 9	18	21
Mean Age (SD *)	25.111 (3.855)	29.388 (7.986)	40.238 (9.186)
Age Range	22–34	22–45	23–56

* SD = Standard Deviation.

. Ninety adults between the age of 18–56 years participated comprising forty-two females and forty-eight males. The healthy group included 30 subjects registering the lowest average age of 24 years. The healthy group included 9 males [average age 25] and 21 females [average age 23] and an age range of 22–34 years for these subjects. The gingivitis group comprised 29 adults with an average age of 30.75 years and an age range of 18–45 years. There were 18 male subjects enrolled in the gingivitis group [average age 29] and an age range of 22–45 Eleven female subjects were enrolled in the gingivitis group with an average age of 33 and an age range of 18–45 years. The average age of subjects with periodontal disease [n = 31] was 39.61 years with the highest age range of 23–56 years. There were 21 male subjects in the periodontitis group [average age 40.23 years] and an age range of 23–56 years. The periodontitis group also included 10 female subjects [average age 38.3 years] and an age range of 29–55 years. All subjects completed the entire study providing evaluable results. No adverse events were noted by the clinical examiner or reported by the subjects during the recall visits or over the course of their study participation.

Shown in

Table 2. Summary of clinical evaluations over study period.

Clinical Group	Parameter	GI, D0	PI, D0	CAL, D0	CPI, D0	GI, D8	PI, D8	CAL, D8	CPI, D8	GI, D15	PI, D15	CAL, D15	CPI, D15
Healthy	Mean	0.27	2.66	0.04	1.51	0.15	2.87	0.04	1.44	0.21	2.60	0.04	1.36
	SD	0.18	0.91	0.11	0.31	0.16	0.59	0.11	0.35	0.19	0.72	0.11	0.30
Gingivitis	Mean	1.60	2.98	0.07	1.83	1.46	2.81	0.06	1.90	1.35	2.54	0.05	1.70
	SD	0.49	1.03	0.15	0.48	0.35	0.79	0.14	0.52	0.32	0.74	0.10	0.51
Periodontal Disease [mm]	Mean	1.95	3.96	1.90	4.03	1.92	3.68	1.84	4.03	1.89	3.75	1.90	4.11
	SD	0.40	0.57	1.26	1.07	0.37	0.77	1.22	1.08	0.33	0.59	1.26	1.04

Table represents results from GI [Gingival Index], PI [Dental Plaque Index], CAL [Attachment Loss], CPI [Community periodontal index] with evaluations conducted on baseline [D0], with recall visits D8 and D15 conducted after 8 and 15 days, respectively, after the baseline. Clinical evaluations and indices based on previously published scales [18,19,20].

are the results from the clinical examinations. The healthy group registered average whole mouth gingival indices of 0.27 at baseline Gingival index scores at recall evaluations conducted after 8 and 15 days of standardized oral hygiene with a fluoride toothpaste were 0.15 and 0.21, respectively. In comparison to the healthy group, higher gingival index scores were registered in the gingivitis subjects with average scores of 1.6, 1.46 and 1.35 at the baseline day 8, and day 15 recall visits, respectively. Higher average gingival index scores were registered amongst subjects stratified to the periodontal disease group. This group presented scores of 1.95, 1.92 and 1.89 at the baseline, day 8 and day 15 evaluations, respectively. Plaque index scores of the healthy group ranged between 2.66–2.87 with the gingivitis group ranging from 2.54–2.98 over the evaluations. The periodontal disease group registered the highest plaque index scores between 3.68–3.96 during the study period.

There were no differences in average clinical attachment level (CAL) score registrations in the health group over the study with an average of 0.04 recorded at all evaluations. The CAL scores amongst gingivitis subjects were slightly higher and ranged between 0.05–0.07 with the highest scores noted in the periodontal disease group with a range between 1.84–1.90. Correspondingly, the average community periodontal index (CPI) for the healthy subjects was the lowest with baseline values of 1.51 that decreased slightly to 1.44 and 1.36 at the day eight and fifteen recall visits, respectively. Higher CPI scores were registered amongst the gingivitis subjects with 1.83 recorded at baseline and scores of 1.90 and 1.70 at the day eight and fifteen recall visits, respectively. The highest CPI scores were amongst the periodontal disease group with a score of 4.03 at both the baseline and day eight recall visits and an average of 4.11 recorded at the final visit.

Shown in

Table 3. Summary of Sialic acid (µg/mL) analyses from each clinical group over the study period.

Evaluation	Clinical Group	Mean	Std. Deviation	Std. Error	95% Confidence Interval for Mean		Minimum	Maximum
					Lower Bound	Upper Bound
	Healthy	64.887	20.626	3.766	57.185	72.589	21.102	98.220
Baseline	Gingivitis	95.395	46.557	8.960	76.978	113.813	38.051	205.000
	Periodontitis	102.034	35.143	6.416	88.911	115.156	41.441	187.203
	Healthy	53.701	25.733	4.698	44.092	63.310	15.169	97.373
Day 8	Gingivitis	99.947	41.322	7.952	83.600	116.293	28.729	202.458
	Periodontitis	109.407	51.491	9.401	90.180	128.634	25.339	280.424
	Healthy	57.768	21.710	3.964	49.662	65.875	19.407	97.373
Day 15	Gingivitis	103.023	46.289	8.908	84.711	121.334	20.254	230.424
	Periodontitis	113.955	50.813	9.277	94.981	132.929	43.136	262.627

The healthy group demonstrated significantly lower levels of SA than the remaining clinical groups at each evaluation (p < 0.05). No significant differences were observed between the gingivitis or periodontal disease groups at any evaluation (p > 0.05).

are the results of SA assessments reported as µg/mL. Average salivary SA levels were lowest amongst the healthy group at all evaluations with baseline values of 64 µg/mL that were significantly lower than the gingivitis and periodontal disease subjects with 95 µg/mL and 102 µg/mL, respectively (p < 0.05). The range for SA amongst healthy subjects was 21–98 at baseline. The SA ranged from 38–205 amongst gingivitis and 41–187 amongst periodontal disease subjects, respectively, in the baseline. No significant differences were noted between SA levels of the gingivitis and periodontal disease groups at baseline (p > 0.05). SA levels in the healthy group were significantly lower than in the other groups at all recall visits (p < 0.05). On days eight and fifteen, average SA levels in the healthy group were 53 µg/mL and 57 µg/mL, respectively, with a range of 15–97 µg/mL. In contrast, the average SA values amongst gingivitis subjects were 99 µg/mL and 103 µg/mL, respectively, on day eight and fifteen evaluations, respectively with a range of 20–230 µg/mL. The periodontal disease group registered the highest average values for SA over the study with scores of 109 µg/mL and 113 µg/mL at the day eight and fifteen evaluations, respectively. Correspondingly, this group registered the higher ranges for SA between 25–280 µg/mL. No significant differences in salivary SA were noted between the gingivitis and periodontal disease groups at all evaluations (p > 0.05).

Results from the spot test for sialidase are shown in Figure 1 and include a representation from subjects clinically stratified into the three clinical groups. Incubation of saliva samples with substrate-laden filter paper strips resulted in a blue color, which is indicative of the presence of sialidase. Notably, samples from healthy subjects [marked H] had little sialidase activity represented by a slight blue color. In contrast, samples from gingivitis [marked G] and periodontal diseases [marked P] resulted in a more intense blue color with limited discrimination in color intensities between these populations.

4. Discussion

Dynamic factors of the human mouth influencing periodontal status include the influences of dietary factors, microbial activity, oral hygiene behaviors and environmental components [1,2]. The oral mucosa serves as a critical barrier maintaining the integrity and associated barrier functions to reduce deleterious effects [1]. Components of the mucosa facilitating these functions include SA, a key molecule reported in the maintenance of oral mucosal integrity [1,3].

In addition to its role in maintaining host physiology, oral bacteria are known to bind SA. For example, Streptococcus gordonii, a gram-positive bacterium that is identified as an early colonizer of oral biofilms has SA binding regions. A recent study evaluated 186 clinical strains of S. gordonii, collected from human subjects and reported variations in SA binding regions [6,7]. Whereas the specific clinical outcomes of these variations remain unclear, it is possible that these variations likely contribute to the factors that influence the progression of disease. These proposals have gained recognition due to the immune disruption noted in periodontal disease due to microbial dysbiosis as an identified virulence mechanism [1,2]. Furthermore, the sialic acid found in the glycans of the exposed epithelial surfaces of oral, gut and other mucosal barriers serves as a source of energy for microbial growth and the initial steps of microbial pathogenesis. For instance, it is estimated that more than 70 different oral organisms utilize exogenous sialic acid [7] and is known to stimulate the growth of organisms such as Tannerella forsythia [8,10].

In addition to the role of SA in host-microbial interactions, the research identifies “siglecs” representing sialic acid-binding immunoglobulin-type lectins expressed on the majority of white blood cells [9]. Siglecs mediate immune responses with critical roles in cell signaling with important functions in distinguishing between self and non-self which are mediated by the recognition of sialic acid-containing ligands of glycoproteins and glycolipids. Due to their expression on limited cell types, they are actively investigated for their roles in many human diseases including chronic conditions such as Alzheimer’s disease, autoimmunity, neurodegeneration, etc. [3,9,10]. These are relevant research avenues based on recent reports exploring the potential associations between oral microbial dysbiosis and chronic diseases [1,2,10].

Microbial dysbiosis is a factor implicated in commonly reported oral health disease conditions such as gingivitis and periodontal disease [1,2]. To facilitate new avenues for diagnosis and treatments, recent research directions in clinical dentistry include an emphasis on biomarkers and emerging technologies to identify the transition from health to disease [14,16,26,27]. The present investigation determined salivary SA amongst subjects clinically stratified based on periodontal status. Included in this investigation was the development of a rapid chair-side visual test for patient engagement and education. This investigation enrolled adults over the age of 18 years who did not require dental or medical care and excluded those reporting systemic diseases, pregnancy and other chronic conditions. At the dental clinic, subjects underwent clinical examinations for dental plaque, gingival inflammation and bleeding utilizing established indices. Following study enrollment, subjects were provided a fluoride toothpaste and a toothbrush to standardize their oral hygiene routines. Other study standardization features included scheduling subjects for their clinical and biochemical assessments in the morning at the dental clinic. All subjects were recalled to the clinic in the morning for weekly evaluations of SA and clinical parameters to identify changes over time. Notably, in interviews subjects revealed that they had never participated in any clinical study. In this regard, the enrolled population represented a naive population and included those who reported both urban and rural domicile reporting oral hygiene with toothpaste and a toothbrush. They demonstrated a limited understanding of dental hygiene but were receptive to learning.

Biochemical tests for SA described in this investigation were conducted with small volumes of saliva, a sample that can be collected easily in remote settings. Additionally, the biochemical tests for SA identified in the literature are rapid and facilitate the inclusion of multiple replicates to improve accuracy. Long-term storage of collected saliva samples offers other flexibility factors in the analysis of SA and can facilitate longitudinal analyses monitoring patient health over time. Furthermore, the analytical features described in this investigation allow procedural adaptations for screening larger populations.

Total sialic acid (SA) is a marker of the acute-phase response and is a predictor of several systemic disorders that include cardiovascular events, rheumatoid arthritis and diabetes [28,29]. Studies have demonstrated increased SA in the serum of head & neck cancer patients [30]. In our present study, we estimated salivary SA. The mean salivary SA level of the periodontal group was significantly higher than gingivitis and normal group. Even though SA levels were detected in the saliva of healthy controls these levels were significantly lower than those in the gingivitis and periodontitis groups, indicating that salivary SA could be a potential marker to categorize patients into gingivitis and periodontitis groups. Results from this investigation align with prior research reported from different populations. Subjects with periodontitis consistently registered the highest levels of SA as reported by Rathod et al., 2014 [20] and Naresh et al., 2019 [15]. In all these studies, the least amount of SA was noted amongst healthy subjects with gingivitis subjects reporting SA levels between these two groups. Correspondingly, in a study with a larger number of subjects, Ancy et al. (2021) reported the lowest levels of SA amongst healthy subjects with no significant effects of age or gender on these outcomes [14]. Other reports indicate the lack of an association between salivary SA among other groups of subjects evaluated after swimming representing physical exertion [21] or children with Down’s syndrome [22].

The levels of sialic acid in healthy individuals were compared to those individuals suffering from periodontal health disease in a study reported by Varma et al., 2019 [16]. Results from that investigation described increased SA in periodontal disease in comparison to those who were clinically healthy. However, only a marginal difference between early and moderate periodontitis was reported. In the present investigation, our observations corroborate these results and demonstrate an increase in SA concentration from health to disease groups with the highest level observed in periodontitis. These observations were maintained over the entire study period representing stability in oral SA as a viable assessment of health status and were related to the clinical indices of oral health.

Oktay et al., 2020 [19] evaluated SA levels at different stages of periodontal disease and showed elevated SA in periodontal disease is due to enhanced cleavage of SA molecules from the cell membranes of oral tissues by invading periodontal pathogens. Similarly, Rathod et al. [20] also showed elevated total sialic acid [TSA] in periodontal disease and mentioned that high TSA is because of elevated sialidase activity and inflammatory responses in individuals suffering from periodontitis. Many recent studies have also reported increased sialic acid in chronic periodontitis in subjects who had high oxidative stress due to smoking. An increase in the mean concentration of SA might be also due to a rapid synthesis of sialoproteins [31,32,33]. Elevated levels of TSA in saliva may play other roles in oral disease [33]. A separate study suggested that high TSA could influence oxidative stress noted in inflammatory diseases including periodontitis [16]. Increased TSA levels in the saliva may play a role in the pathogenesis of periodontal disease [16,18]. However, the present study did not discriminate between gingivitis and periodontitis based on baseline values. Whereas, a variety of clinical and other variables likely impact oral SA, a likely reason includes the enrollment of less severe forms of periodontitis (mild and moderate) with a mean probing depth of 3.9 and attachment loss of 1.9 leading to statistical insignificance [20] between the evaluated subjects.

The development of rapid visual chair-side tests for patient engagement is an identified goal in managing patients [1,2,28]. Notable advantages of these tests are their benefits to engage patients, enable personalization and facilitate their ability to manage their dental health. The literature recognizes previous initiatives in chair-side tests to aid patient education. A specific goal of this investigation was to develop a visual test that can be conducted with limited laboratory resources. The visual test procedures allow preparation of the filter paper disks laden with appropriate reagents at the central laboratory which can be readily transported to dental clinics under ambient conditions. No specialized equipment is needed to maintain and store these disks with the test readily performed chair-side for rapid visual results under resource-limited settings. Outcomes from this investigation outline the feasibility of utilizing this chair-side test to screen larger populations for their dental health status. A critical unmet need in the region where this investigation was conducted is the limited availability of dental health services with this requirement increasing in concern at more rural regions. It is likely features of the described test will allow for distance-based readouts using image capture and its transmittal using secure data transfer platforms facilitating longitudinal monitoring.

The current results will support the design of future efforts to assess larger populations and gain insights relevant to assay design. It is estimated that these efforts will help with improving the test procedures for more sensitive and rapid assessment of collected samples. Assessment of large populations will provide insights on sialic acid status as they align with disease severity with particular reference to subjects who have marginal differences in clinical findings. Advancing research in the analysis of sialic acids and developments in chemistry will be an important factor in improving the methods presented in this effort. These advances will facilitate the selection and validation of the most appropriate and economically feasible procedures. An important focus of our future research is to study the effects of interventions on salivary sialic acid. Our interests are in assessing interventions provided to patients at the dental clinic and others that are appropriate for self-care by the subjects and available for routine use. A longer-term goal would be to utilize these results to develop appropriate testing protocols that can be used by patients to monitor their oral health status in a setting that does not require extensive laboratory resources. In addition to the above, a possible avenue for future research direction is from a recent investigation that reports sialic acid in other body fluids such as sweat. Results from that investigation examined the binding of skin bacteria to these ligands with the intent of identifying future therapeutic mechanisms to mitigate the effects of skin bacteria [34].

Limitations

This study enrolled a curated group of adult subjects to identify trends in salivary sialic acid levels in health and disease. The selected study population did not include children and others with mixed dentition or implants. This study enrolled more female subjects representing the general population and the availability of subjects who were willing to participate. Future studies can seek to enroll a gender-balanced study population. Furthermore, future research is required to evaluate study parameters in other populations and the inclusion of subjects with dental implants and prostheses. Additionally, studies are required to examine the effects of interventions designed to improve oral health on salivary sialic acid.

5. Conclusions

The present investigation aligns with priorities in clinical dentistry to develop easy-to-implement approaches that distinguish health from disease [1,2]. Results indicate that salivary SA is an adjunct to differentiate subjects based on periodontal health. A notable outcome of this study is the rapid visual chair-side test that can be conducted with limited laboratory resources to facilitate patient education. Future studies will augment the present observations in establishing the effects of oral hygiene or therapeutic interventions with the rapid visual chair-side test enhancing patient education to improve periodontal health.