Salivary Immunoglobulin A and Streptococcus mutans Levels among Lebanese Preschool Children with Early Childhood Caries

INTRODUCTION

Saliva plays an essential role in maintaining a good oral health. It controls metabolism, adhesion, and proliferation of local microorganisms, improves the elimination of residues of carbohydrates, ensures the remineralization of teeth, and neutralizes the organic acids produced by cariogenic microorganisms causing the demineralization of tooth enamel.¹ Recently, and despite the worldwide effort to control its prevalence, tooth decay remains a major problem in many developing countries,^2–4 especially among young children where it is known as early childhood caries (ECC).^5,6 In Lebanon, and according to a study conducted by Chedid et al., 2011,⁷ more than half of the children aged between 1 years and 4 years present a high risk of caries with 74.7% having at least one decay.

Early childhood caries is a special form of carious lesions.⁸ It is encountered in patients where the food mode is characterized by bottle and breastfeeding in an inappropriate way.^3,5,9 In 2008, Caplan et al.¹⁰ defined “inappropriate” as the frequent use of bottle or glass containing milk or other sweet drink before and after meals or sleep. The infectious nature of caries in general and ECC, in particular, implies a potential involvement of salivary antimicrobial components in the phenomena of pathogenesis.^11–13 However, studies about the correlation between these immunological factors (immunoglobulin G, M, A) and tooth decay in children are inconclusive, with a variety of results based on different methods of sampling and assays used.^14–16 The American Academy of Pediatric Dentistry considered, in a report published in 2008, that the presence of any decayed deciduous tooth, missing or filled teeth in children aged <71 months, is a sign of ECC.^17,18 The answer to the question “what causes this condition?” is important and complex. The risk factors involved in ECC have been widely described in the literature.^{6,8,9,19–23}

The objective of our study was to clarify the role of the salivary immune system in the declaration of ECC, and its possible correlation with the level of salivary Streptococcus mutans (SM) and the number of cavities in a sample of young Lebanese preschoolers. In other words, we tried to provide an answer to the question: “why for a group of children from the same environment with the same eating habits, some present early childhood caries, while others do not?”

MATERIALS AND METHODS

The studied sample included 60 children aged between 18 months and 49 months (31 boys and 29 girls) (Table 1). Regarding the gender of the children, the choice was made arbitrarily. We obtained 13 girls and 17 boys in the “control” group. On the other hand, 16 girls and 14 boys were included in the “cavities” group (Table 1).

The inclusion criteria (Appendix 1) were absence of antibiotic intake for at least 2 weeks, good health, no previous dental treatment, and inappropriate diet consisting of a frequent use of bottle or glass containing milk or other sweetened drink before and after meals or bedtime. The exclusion criteria were (1) age different from 18 to 48 months; (2) antibiotic usage for at least 2 weeks before sampling; (3) presence of systemic disorders; and (4) exposure to previous dental treatment. The hygiene habits of the participants are summarized in Appendix 2. Before any dental and immunological examinations, an informed consent was signed by the parents.

A six-part questionnaire (Appendix 3) was completed for each child by one of the parents concerning his/her identity, siblings, socioeconomic background, medical, and dental history.

The dental clinical examination was performed by a single operator and was limited to a visual examination of the teeth using a mirror and a probe at daylight. Decayed teeth were recorded. No radiological examination was performed.

After dental examination, children were divided into two groups depending on whether they have ECC (caries group) or not (no caries group).

The SM Dentocult Strip Mutans test^24,25 was used to detect SM in the saliva and plaque. This method is based on the ability of the microorganisms to adhere and grow on strips delivered with the kit after incubation. Using a sterile probe, the plaque was removed from the interdental surfaces and gently spread over the ribbed strip. Plaque collections were denoted as P1, P2, P3, and P4 corresponding to the teeth number 51, 61, 64, and 84, respectively. Saliva was collected by impregnation. After an incubation period of 48 hours at a temperature of 37°C, the scores of SM were recorded by comparing the culture media to a charter issued with the microbiological test kit.

The N-latex immunoglobulin A (IgA) assay was carried out in the laboratory of chemistry at the American University of Beirut Medical Center. After the collection of all the samples, the tubes were analyzed in a machine called “nephelometry” (the nephelometry technique is a test quickly and accurately measures the serum concentration of antibodies).

A major difficulty that we encountered when carrying out this work was being able to take saliva and plaque samples at the same time for all children. We opted for the morning between 9 a.m. and 11 a.m. to be sure that we could guarantee the 2 hours of abstention from any ingestion of food or drink (other than water), because according to previous studies,^26,27 the level of SM varies depending on the nature and duration of ingestion of the last meal. Thus, the samples of unstimulated saliva were taken using a sterile spoon in which we ask the child to spit or using a pipette if the child’s cooperation is negative, to remove saliva under the tongue (this technique was previously described).²⁸ Finally, if during salivary samples, the child becomes non-cooperating and begins to cry, the test was postponed because stress modifies the salivary components, which risks distorting the microbiological and immunological results.²⁹

For each variable (P1 to P4 and S), the children were classified into two categories: “low score” category if the value of the variable is between 0 and 1; “high score” category if the variable is equal to 2 or 3.³⁰

RESULTS

This study included 60 participants who were divided into two groups. Group I (control group) included 30 participants [13 (43.3%) girls and 17 (56.7%) boys] who have no caries. Group II (caries group) contained 30 participants [16 (53.3%) girls and 14 (46.7%) boys] who have caries (Table 1). Gender difference was not statistically significant (p = 0.438) between the two groups. The average age (29.3 months) in the control group was significantly lower than that (39.7) in the “caries” group (p < 0.001) (Table 1).

Table 1 contains summary statistics about the distribution of SM levels among members for each group along with the comparisons between the groups.

Although the “caries” group had a higher average IgA than that of the control group (23.6 vs 30.4), the difference was not statistically significant (p = 0.170). As for S and P1–P4, we found that most people scored in the lower range (0 or 1) on those variables in the control group and in the higher range (2 and 3) in the “caries” group. The differences between the two groups were significant for all those variables. Moreover, the average P was significantly higher (p < 0.001) in the “caries” group (average = 2.6) as compared to the control group (average = 1.1).

The results of comparing IgA among children with low vs high P1–P4 in the “caries” group are summarized in Table 2. It is clear that for all the variables, the mean IgA was always higher in the high category as compared to the low category. However, marginal significance was only seen for P1 (p = 0.085) and P4 (p = 0.095). A similar comparison was performed for the control group and none of the variables reached statistical or marginal significance (Table 3). As for S, there was no significant difference in IgA levels between the “low” and the “high” S categories in both of the groups (p = 0.350 for the “caries” group and p = 0.933 for the “control” group).

We further studied the correlation between IgA and each of age, average P, and number of caries for each of the two groups. In both groups, there was no significant correlation between IgA and age (p = 0.619 for the control group and p = 0.849 for the “caries” groups). In the control group, there was no significant correlation (p = 0.940) between IgA and average P; however, in the “caries” groups, the correlation was positive (rho = 0.33) and marginally significant (p = 0.077). The number of caries was not significantly correlated (p = 0.633) with total IgA levels. It is worthy to note that there was also a marginally significant (p = 0.064) positive correlation (rho = 0.343) between the number of caries and age.

DISCUSSION

Till date, comparison between two populations with similar inappropriate diet habits but with different dental status (one population is free from decay while the second is affected by the ECC) has never been described in the literature. Performing such comparison was therefore the aim of the present study. The originality of our work lies in the choice of the sample and particularly the control group. This study compares for the first time, two populations with a similar diet but one of which is free from caries while the second has ECC.

According to the primary statistical analysis, the difference between genders was not statistically significant, similar to what was previously reported by Gonçalves de Farias and Barreto Bezerra in 2003.¹³ On the other hand, there was a statistically significant difference in age between the control group (29.3 months) and the ECC group (39.7 months) with p < 0.001. We can explain this by the fact that for most parents, caries lesions at the initial stage go unnoticed, and therefore children with leukoma are almost never treated at this stage. In the literature, the average age of recruited children affected by ECC varies according to the authors: for Gonçalves de Farias and Barreto Bezerra (2003) the average age is 28 months for both groups (free or affected by caries) while it is 36.8 months for van Everdingen et al.,³¹ 23.6 months for Febres et al.,³² and 53 months for Elarabi et al.³³

Regarding our first objective to evaluate and compare SM levels taken from the saliva and plaque, we obtained a significant difference between the two groups for all variables (p < 0.001). This allows us to confirm the direct relationship between the level of SM and dental caries. These results are similar to those of Seki et al.²⁴

In this study, we compared the total IgA levels between the two groups of children. We found out a higher level of total IgA in the caries group compared to the control group (30.6 mg/L against 23.6 mg/L). Nevertheless, the individual values that we obtained were very variable: one of the children had a level of 251 mg/L whereas the average was 89.6 in the caries group. His score of SM was 3 as he was a premature infant (this factor is considered promoting decay disease).³⁴ However, the difference was not significant (p = 0.17). Some studies^13,35 confirm the positive correlation between these two variables (IgA and caries), while others have found conflicting results.³⁶

The lack of correlation between the level of IgA and the saliva should not exclude the role of the immune system as our results could be explained by the following factors:

• The assessment was limited to the rate of total IgA and not that of the SM-specific IgA.^37,38
• The lack of standardization and reproducibility problems.
• The effect of salivary flow on the composition of saliva that was not considered in our study.³⁸
• The immaturity of the immune system in children.³⁹
• High levels of salivary IgA may be the reflection of past exposure to SM.^40,41

However, the marginal correlation obtained by comparing the level of total IgA and scores of SM taken from the plaque, opens up new research perspectives.

CONCLUSION

The particularity of our work lies in the choice of the population. The direct connection between the decay in general and ECC in particular and the high levels of SM was evident. Thus, the lack of correlation between IgA and SM collected from the saliva does not eliminate the importance of the preventive role of salivary immune system. The marginal correlation between IgA and SM collected in the plaque shows a boundary link and needs further research. Prevention, by means commonly recognized as dental hygiene tools and control of diet, stays after all, the basic of all therapeutic measures.

CLINICAL SIGNIFICANCE

Early childhood caries is a public dental health problem that has been affecting preschool children all over the world. Its prevention must be a priority for all professionals in the medical and dental community. This study highlights the absence of correlation between total IgA and SM collected from the saliva. This does not completely exclude the preventive role of salivary immune components, but further studies are required to better understand this relationship.

APPENDIX 1: INCLUSION CRITERIA

APPENDIX 2: HYGIENE HABITS AND NUTRITION MODE

APPENDIX 3: QUESTIONNAIRE

REFERENCES

1. Shore RC, Kirkham J, Brookes SJ, et al. Distribution of exogenous proteins in caries lesions in relation to the pattern of demineralisation. Caries Res [Internet] 2000;34(2):188–193. DOI: 10.1159/000016588 .Available from http://www.ncbi.nlm.nih.gov/pubmed/10773638.

2. El Tantawi M, Folayan MO, Mehaina M, et al. Prevalence and data availability of early childhood caries in 193 United Nations countries, 2007-2017. Am J Public Health [Internet] American Public Health Association; 2018;108(8):1066–1072.Available from https://ajph.aphapublications.org/doi/10.2105/AJPH.2018.304466.

3. Finlayson TL, Gupta A, Ramos-Gomez FJ. Prenatal maternal factors, Intergenerational transmission of disease, and child oral health outcomes. Dent Clin North Am [Internet] 2017;61(3):483–518. DOI: 10.1016/j.cden.2017.02.001 .Available from: http://www.ncbi.nlm.nih.gov/pubmed/28577633.

4. Ganesh A, Muthu MS, Mohan A, et al. Prevalence of early childhood caries in india – a systematic review. Indian J Pediatr [Internet] 2019;86(3):276–286.Available from http://link.springer.com/10.1007/s12098-018-2793-y.

5. Seow WK. Early childhood caries. Pediatr Clin North Am [Internet] 2018;65(5):941–954. DOI: 10.1016/j.pcl.2018.05.004 .Available from http://www.ncbi.nlm.nih.gov/pubmed/30213355.

6. Warren JJ, Weber-Gasparoni K, Marshall TA, et al. Factors associated with dental caries experience in 1-year-old children. J Public Health Dent [Internet] 2008;68(2):70–75.Available from: http://doi.wiley.com/10.1111/j.1752-7325.2007.00068.x.

7. Chedid NR, Bourgeois D, Kaloustian H, et al. Caries Prevalence and caries risk in a sample of Lebanese preschool children. Odontostomatol Trop [Internet] 2011;34(134):31–45.Available from http://www.ncbi.nlm.nih.gov/pubmed/22003807.

8. Anil S, Anand PS. Early childhood caries: Prevalence, risk factors, and prevention. Front Pediatr [internet] Frontiers Media SA; 2017;5:157. DOI: 10.3389/fped.2017.00157 .Available from http://www.ncbi.nlm.nih.gov/pubmed/28770188.

9. Tinanoff N. Introduction to the early childhood caries conference: initial description and current understanding. Commun Dent Oral Epidemiol [Internet] 1998;26 (S1):5–7.Available from: http://doi.wiley.com/10.1111/j.1600-0528.1998.tb02089.x.

10. Caplan LS, Erwin K, Lense E, et al. The potential role of breast-feeding and other factors in helping to reduce early childhood caries. J Public Health Dent [Internet] 2008;68(4):238–241. DOI: 10.1111/j.1752-7325.2007.00080.x .Available from http://www.ncbi.nlm.nih.gov/pubmed/18384535.

11. Russell MW, Hajishengallis G, Childers NK, et al. Secretory immunity in defense against cariogenic Mutans Streptococci. Caries Res [Internet] 1999;33(1):4–15. DOI: 10.1159/000016490 .Available from http://www.ncbi.nlm.nih.gov/pubmed/9831775.

12. Michalek SM, Katz J, Childers NK. A vaccine against dental caries. BioDrugs [Internet] 2001;15(8):501–508. DOI: 10.2165/00063030-200115080-00002 .Available from http://www.ncbi.nlm.nih.gov/pubmed/11543691.

13. Gonçalves de Farias D, Barreto Bezerra AC. Salivary antibodies, amylase and protein from children with early childhood caries. Clin Oral Investig [Internet] 2003;7(3):154–157. DOI: 10.1007/s00784-003-0222-7 .Available from http://www.ncbi.nlm.nih.gov/pubmed/12915963.

14. Alaluusua S. Longitudinal study of salivary IgA in children from 1 to 4 years old with reference to dental caries. Eur J Oral Sci [Internet]. John Wiley and Sons, Ltd 1983;91(3):163–168.Available from: http://doi.wiley.com/10.1111/j.1600-0722.1983.tb00796.x.

15. Rose PT, Gregory RL, Gfell LE, et al. IgA antibodies to Streptococcus mutans in caries-resistant and -susceptible children. Pediatr Dent [Internet] 2020;16(4):272–275.Available from: http://www.ncbi.nlm.nih.gov/pubmed/7937259.

16. Vadiakas G. Case definition, aetiology and risk assessment of early childhood caries (ECC): a revisited review. Eur Arch Paediatr Dent [Internet] 2008;9(3):114–125. DOI: 10.1007/BF03262622 .Available from http://www.ncbi.nlm.nih.gov/pubmed/18793593.

17. Kaste LM, Drury TF, Horowitz AM, et al. An Evaluation of NHANES III estimates of early childhood caries. J Public Health Dent [Internet] 1999;59(3):198–200.Available from: http://doi.wiley.com/10.1111/j.1752-7325.1999.tb03269.x.

18. Folayan M, Olatubosun S. Early childhood caries - a diagnostic enigma. Eur J Paediatr Dent [Internet] 2018;19(2):88. DOI: 10.23804/ejpd.2018.19.02.00 .Available from http://www.ncbi.nlm.nih.gov/pubmed/30063145.

19. Kirthiga M, Murugan M, Saikia A, et al. Risk factors for early childhood caries: a systematic review and Meta-analysis of case control and cohort studies. Pediatr Dent [Internet] 2019;41(2):95–112.Available from http://www.ncbi.nlm.nih.gov/pubmed/30992106.

20. Miyaji H, Azuma T, Ito S, et al. Helicobacter pylori infection occurs via close contact with infected individuals in early childhood. J Gastroenterol Hepatol 2000;15(3):257–262. DOI: 10.1046/j.1440-1746.2000.02070.x.

21. Sun HB, Zhang W, Zhou XB. Risk factors associated with early childhood caries. Chin J Dent Res [Internet] 2017;20(2):97–104.Available from http://www.ncbi.nlm.nih.gov/pubmed/28573263.

22. Schroth RJ, Cheba V. Determining the prevalence and risk factors for early childhood caries in a community dental health clinic. Pediatr Dent [Internet] 2007;29(5):387–396.Available from http://www.ncbi.nlm.nih.gov/pubmed/18027773.

23. Tiberia MJ, Milnes AR, Feigal RJ, et al. Risk factors for early childhood caries in Canadian preschool children seeking care. Pediatr Dent [Internet] 2007;29(3):201–208.Available from http://www.ncbi.nlm.nih.gov/pubmed/17688016.

24. Seki M, Karakama F, Terajima T, et al. Evaluation of mutans streptococci in plaque and saliva: correlation with caries development in preschool children. J Dent [Internet] 2003;31(4):283–290. DOI: 10.1016/s0300-5712(03)00033-2 .Available from http://www.ncbi.nlm.nih.gov/pubmed/12735923.

25. Tedjosasongko U, Kozai K. Initial acquisition and transmission of Mutans Streptococci in children at day nursery. ASDC J Dent Child [Internet] ASDC J Dent Child 2002;69(3).Available from: https://pubmed.ncbi.nlm.nih.gov/12613313/?from_term=Tedjosasongko+Uandfrom_pos=3.

26. SJ W, GZ W. Variables influencing Streptococcus Mutans testing. Pediatr Dent [Internet]. Pediatr Dent 1990;12(5).Available from: https://pubmed.ncbi.nlm.nih.gov/2095541/?from_term=Weinberger+SJandfrom_cauthor_id=2095541andfrom_pos=1.

27. Smith DJ, King WF, Akita H, et al. Association of salivary immunoglobulin A antibody and initial Mutans Streptococcal infection. Oral Microbiol Immunol [Internet] Oral Microbiol Immunol 1998;13(5).Available from: https://pubmed.ncbi.nlm.nih.gov/9807119/?from_term=Smith+DJandfrom_cauthor_id=9807119andfrom_pos=1.

28. Bosch JA, Ring C, de Geus EJ, et al. Stress and secretory immunity. Int Rev Neurobiol [Internet] Int Rev Neurobiol 2002;52:213–253. DOI: 10.1016/s0074-7742(02)52011-0 .Available from: https://pubmed.ncbi.nlm.nih.gov/12498106/?from_term=Bosch+JAandfrom_cauthor_id=12498106andfrom_pos=1.

29. Tanabe Y, Park JH, Tinanoff N, et al. Comparison of chairside microbiological screening systems and conventional selective media in children with and without visible dental caries. Pediatr Dent [Internet] 2006;28(4):363–368.Available from http://www.ncbi.nlm.nih.gov/pubmed/16903447.

30. Becker MR, Paster BJ, Leys EJ, et al. Molecular analysis of bacterial species associated with childhood caries. J Clin Microbiol [Internet] 2002;40(3):1001–1009.Available from http://jcm.asm.org/cgi/doi/10.1128/JCM.40.3.1001-1009.2002.

31. van Everdingen T, Eijkman MA, Hoogstraten J. Parents and nursing-bottle caries. ASDC J Dent Child [Internet] 63(4):271–274.Available from http://www.ncbi.nlm.nih.gov/pubmed/8893981.

32. Febres C, Echeverri EA, Keene HJ. Parental awareness, habits, and social factors and their relationship to baby bottle tooth decay. Pediatr Dent [Internet] 19(1):22–27.Available from http://www.ncbi.nlm.nih.gov/pubmed/9048409.

33. Elarabi S, Elhamid A, Harchaoui J, et al. La polycarie infantile: facteurs de risque. J Dent du Québec 2001;37:363–371.

34. Occhi‐Alexandre IGP, Cruz PV, Bendo CB, et al. Prevalence of dental caries in preschool children born preterm and/or with low birth weight: A systematic review with meta-analysis of prevalence data. Int J Paediatr Dent [Internet] 2020;30(3):265–275. DOI: 10.1111/ipd.12610 .Available from: http://www.ncbi.nlm.nih.gov/pubmed/31854043.

35. Letieri A, dos S, Freitas-Fernandes LB, et al. Longitudinal evaluation of salivary Iga-S in children with early childhood caries before and after restorative treatment. J Clin Pediatr Dent [Internet] 201943(4):239–243.Available from http://jocpd.org/doi/10.17796/1053-4625-43.4.3.

36. Kirstilä V, Häkkinen P, Jentsch H, et al. Longitudinal analysis of the association of human salivary antimicrobial agents with caries increment and cariogenic micro-organisms: a two-year cohort study. J Dent Res [Internet] 1998;77(1):73–80.Available from: http://journals.sagepub.com/doi/10.1177/00220345980770011101.

37. Muthu M, Amarlal D, Prabhu VR, et al. Quantitative assessment of IgA levels in the unstimulated whole saliva of caries-free and caries-active children. J Indian Soc Pedod Prev Dent [Internet] 2008;26(4):158. DOI: 10.4103/0970-4388.44031 .Available from http://www.ncbi.nlm.nih.gov/pubmed/19008624.

38. Sundaresan C, Mathai V, Khosla E, et al. A comparative study of salivary buffering capacity, flow rate, resting pH, and salivary immunoglobulin A in children with rampant caries and caries-resistant children. J Indian Soc Pedod Prev Dent [Internet] 2013;31(2):69. DOI: 10.4103/0970-4388.115697 .Available from http://www.ncbi.nlm.nih.gov/pubmed/23886715.

39. Childers NK, Greenleaf C, Li F, et al. Effect of age on immunoglobulin A subclass distribution in human parotid saliva. Oral Microbiol Immunol [Internet] 2003;18(5):298–301. DOI: 10.1034/j.1399-302x.2003.00084.x .Available from http://www.ncbi.nlm.nih.gov/pubmed/12930521.

40. Kirtaniya B, Chawla H, Tiwari A, et al. Natural prevalence of antibody titers to glucostltransferase of Streptococcus mutans in serum in high and low caries active children. J Indian Soc Pedod Prev Dent [Internet] 2010;28(2):91. DOI: 10.4103/0970-4388.66744 .Available from http://www.ncbi.nlm.nih.gov/pubmed/20660974.

41. Thaweboon S, Thaweboon B, Nakornchai S, et al. Salivary secretory IgA, pH, flow rates, mutans streptococci and Candida in children with rampant caries. Southeast Asian J Trop Med Public Health [Internet] 200839(5):893–899.Available from http://www.ncbi.nlm.nih.gov/pubmed/19058586.