Analysis of IgA Anti- S mutans Serotype c and e in ECC Patients and Its Correlation towards Saliva Viscosity and DMFT Score

Aims: To analyze Immunoglobulin A anti- S mutans serotype c and e level and the correlation towards saliva viscosity and decayed, missing, and filled teeth (DMFT) score of Early Childhood caries (ECC) patients. patients titer Results: The titer of IgA anti- S mutans serotype c and e were 6 and 15.1, the Mann-Whitney test showed P<0.05. Levels of IgA anti- S mutans serotype c and e were in line with the saliva’s viscosity in which increasing viscosity resulted in higher level of IgA titer. Further regression analysis reveals that the titer of IgA with high viscosity of saliva contain higher titer of IgA anti- S mutans compare those lower viscosity. In addition, the data shows that DMFT score have a negative correlation with IgA anti- S mutans serotype c and e levels. Conclusion: In ECC patients, S mutans serotype e may be more immunogenic than serotype c and higher salivary viscosity may be due to its high IgA content.


INTRODUCTION
Early Childhood Caries (ECC) is the presence of one or more decayed, missing or filled tooth in a child 71 months of age or younger [1]. ECC started with white patches, that becoming caries on tooth surfaces [2]. ECC is concerned as oral health problem in Indonesia In 2015, ECC prevalence in Pineleng, North Sulawesi was 66.21% and ECC prevalence in Gunung Anyar, Surabaya was 60% [3]. S mutans is the main cariogenic microorganism that play important role in ECC pathogenesis [4]. This microorganism can colonize the tooth surfaces, fermenting carbohydrate, then produces acid that could undermine tooth surfaces [4]. Based on their chemical structure, S mutans classified into four serotype, c, e, f, and k [5]. The most dominant S mutans serotype that found in oral cavity is serotype c (70-80%), followed by serotype e (20%), f (<5%) and k (2-5%) [5]. S mutans population in oral cavity were affected by saliva condition [6,7]. Körber et al. [8] reported that in the distribution of serotypes of S mutans in the caries population was predominated by serotype c (53.2%), followed by e (31.9%), f (8.5%) and k (6.4%).
Saliva consist of 99% water and various electrolytes (Na, K, Ca, Mg, bicarbonate, phosphate), immunoglobulin, proteins, enzymes, mucins, and nitrogen products [9]. Saliva has a multi-function such as a taste enhancement and digestion, lubrication and protection, buffering and clearance, maintenance of tooth integrity, and as the first line of defense against bacterial and viral attack [6]. Specific humoral immunity components are IgA, IgM, and IgG, while nonspecific immunity factor consist of mucins, lactoferrin, lysozyme, peroxidase, cystatins, and hystatins [9]. The physicochemical properties of saliva, such as viscosity, pH, salivary flow rate, and buffering capacity has a relation with caries activity in children and act as markers of caries activity [8]. The protein and ion components in saliva make a solution into a viscoelastic solution [10] IgA, the predominant immunologic component of saliva act to maintain the integrity of oral surfaces, by limiting the formation of S mutans biofilm through binding the pellicle surfaces or by agglutinating the bacteria [9]. Immunoglobulin A has a role in preventing the formation S mutans biofilm [11], which is known as the main causes of ECC. Therefore, we were interested to analyse IgA anti-S mutans serotype c and e level in saliva of ECC patients, and its correlation with DMFT score and saliva viscosity in ECC patients. Here we evaluated the relationship between level IgA anti-S mutans serotype c and e and the salivary flow rate, viscosity as well as the DMFT scores in saliva of patients ECC. Clinical examination of subjects was carried out by single examiner. Decay, missing and fillings (DMFT) score determined according to World Health Organization (1986) diagnostic criteria. Resting whole saliva was collected in the morning time according to Fédération dentaire internationale (FDI). The subjects were 20 children in Early Childhood Education (ECD) Al-Ikhlas Paseban Village, Senen Sub-district, Central Jakarta, aged 3-5 years who have diagnosed as ECC. Subjects were instructed to accumulate saliva in the mouth for 2 min and then spit in the sterile container. This procedure was repeated three times and the salivary flow rates were calculated. In addition, the viscosity of saliva also recorded as high or low.

Bacterial Culture and Antigen Preparation
Glycerol stock of S mutans serotype c and e bacteria from clinical isolates was obtained from Oral Biology Laboratory Faculty of Dentistry, Universitas Indonesia. Bacteria were grown in TYS20B medium with anaerobic condition in 37°C for 72 hours. Cultures of S mutans serotype c and e bacteria then sonicated and centrifuged to get the antigen. S mutans serotype c and e antigen concentration than measured using Bradford methods. After antigen concentration obtained, then dilute the antigen with coating buffer until the concentration become 10 µg/mL [12].
100 µL of antigen of S mutans serotype c and e were coated at concentration of 10 µg/mL in each well of ELISA plate, then incubated in 4°C overnight. Wash all the well using PBST, and then 100 µL blotto as blocking solution and incubated in 37°C for one hour. After that, wash the well using PBST. Each saliva sample then diluted serially from 1:2 to 1:16. Followed by incubated in 37°C for one hour. After each well was washed using PBST, then add 100 µL of 1:2000 secondary antibody into each well and incubate the plate in 37°C for 1 hour. The plates were washed with PBS prior added by 50 µL TMB substrate. ELISA plates than were shaked in orbital shaker for 15 minutes. The enzyme reaction was stopped by adding 50 µL HCl 1 N each well, and the color that developed read at 405 nm with ELISA reader [12].

Statistical Analysis
The differences of titer IgA anti-S mutans serotype c and e were statistically tested with Mann-Whitney U Test. In addition the correlation of DMFT score and titers of IgA anti-S mutans serotype c and e were tested with simple linear regression analysis.

The Relationship between DMFT
Score with Salivary Flow Rate with Salivary Viscosity Fig. 1 shows that the salivary flow rate in ECC subject with DMFT score 4 was higher (4.7 mL/min) than those have DMFT score 5 and score 6 (4,6 mL/min, 3.6 mL/min and 4 mL/min) respectively (p>0.05).
Observation in viscosity of saliva reveals that 8 ECC subjects with DMFT score 5 have high viscosity and only 2 of them have low viscosity. Subjects with DMFT score 4 seems to be equal number in the viscosity of saliva (4 vs 4). Interestingly all subject with DMFT score 6 have high viscosity in their saliva (Fig. 2). It seems that in ECC high viscosity saliva related to the severity of caries.

Titer of IgA Anti-S mutans Serotype c and e in ECC Patients
IgA titer was determined as the higher dilution saliva sample that reach optical density (OD) 0.2 at 450 nm absorbance in ELISA reader. As shown in Fig. 3. The data reveals that in saliva of ECC subject, the average titer of IgA anti-S mutans serotype c and were 1/6 and 1/9.8 (P<0.05).
We analyse data of IgA anti S mutans of ECC's saliva subject in their relationship to DMFT score. The results show that IgA anti-S mutans serotype has stronger negative correlation with DMFT (r= -0.978) than serotype c (r= -0.577) with P <0.05. (Fig. 4).
Further analysis of IgA anti-S mutans in saliva of ECC subject and the correlation with viscosity of their saliva reveals that highers titers of IgA anti-S mutans in high viscosity of saliva both in serotype c and e. However, IgA anti-S mutans serotype e has the highest one (Fig. 5).

DISCUSSION
The main bacteria that causes caries is S mutans classified into 4 serotype, one of them is serotype c [12]. Previous study reported that S mutans serotype c is the most commonly found bacteria in oral cavity [5]. In Indonesia S mutans serotype f is the most commonly found bacteria in child's oral cavity [14].
This study found that that in saliva of ECC subject, the average titer of IgA anti-S mutans serotype c is lower than IgA anti-S mutans serotype e. This may indicated that due to S mutans serotype e play predominant role in ECC patient. Level IgA against antigens of S mutans have been studied previously, and low levels of IgA are associated with a high risk of caries in children. IgA antibody specificities may be critical in modulating initial S mutans infection [15]. This study showed that low levels of IgA anti mutans serotype e of in patients with high scores. With the decrease of the level of IgA anti S mutans serotype e at high DMFT be seen that the risk of caries owned is certainly higher. Meanwhile, According to Ranadheer et al. [15] provide mechanical protection against bacteria S mutans on the teeth causing caries is the purpose of the IgA so it would be good if in the salivary IgA level is higher than the score DMFT. Besides the observations that have been obtained is also consistent with studies that have been done before by Doifode D. states that the IgA antibody contained in saliva naturally have an important role in the immunologic control of dental caries that in children such as ECC. Koga-Ito Cristiane et al [17]. reported that anti-S mutans IgA levels are higher in caries-free individuals than in individuals with karies and low level of IgA hav a correlation towards the high risk of caries in children.

Number of caries subjecs dmft
In this study found there is an association between high viscosity and the high levels of IgA. While, a negative correlation between IgA anti mutans serotype c levels with saliva viscosity and DMFT score founded in this study. The caries risk assessment using saliva in terms of pH (stimulated and unstimulated), consistency and buffering capacity assists dental professionals in determining low, moderate or extreme high caries risk caries [18]. Some study supported about this study results, that the Bachtiar et al.;ARRB,12(6): [1][2][3][4][5][6][7]2017; Article no.

-S mutans serotype c and e titers of ECC subject with high and low saliva viscosity. The highest S mutans serotype e in subject with high viscosity of saliva (P <
This study showed that low levels of IgA anti-S serotype e of in patients with high DMFT With the decrease of the level of IgA anti-DMFT scores can be seen that the risk of caries owned is certainly Ranadheer et provide mechanical protection against on the teeth causing caries is the purpose of the IgA so it would be good if in salivary IgA level is higher than the score . Besides the observations that have been obtained is also consistent with studies that have Doifode D. [16] which states that the IgA antibody contained in saliva tant role in the immunologic control of dental caries that occurs Ito Cristiane et al. IgA levels are free individuals than in individuals with karies and low level of IgA have a correlation towards the high risk of caries in In this study found there is an association between high viscosity and the high levels of IgA. While, a negative correlation between IgA anti-S serotype c levels with saliva viscosity score founded in this study. The caries risk assessment using saliva in terms of pH (stimulated and unstimulated), consistency and buffering capacity assists dental professionals in determining low, moderate, high or extreme high caries risk caries [18]. Some study supported about this study results, that the higher DMFT score, the lower IgA anti serotype c levels [19]. Decreased IgA levels, reflect the high caries risk factor in patients [20,21]. More appropriate protein intake might be need for patient with ECC. As reported by previous study that multivitamin usage and tooth decay relation is not a widely questioned phenomena in ECC [22]. Our finding might be valuable for the strategy to prevent ECC focusing on S mutans serotype c and the role of specific IgA against this S mutans saliva.

CONCLUSION
In ECC patients, S mutans serotype e may be more immunogenic than serotype c and higher salivary viscosity may be due to its high protein contain as well as IgA content. score, the lower IgA anti-S mutans Decreased IgA levels, sk factor in patients re appropriate protein intake might be As reported by previous study that multivitamin usage and tooth decay relation is not a widely questioned phenomena in ECC [22]. Our finding might be valuable for the strategy to prevent ECC by serotype c and the role of S mutans serotype in serotype e may be more immunogenic than serotype c and higher salivary viscosity may be due to its high protein Early childhood caries: , clinical considerations, consequences and management. Int Dent.