Abstract

This cross-sectional study evaluated the association between salivary immunoglobulins, plaque index, and gingival index in Brazilian children with and without type 1 diabetes mellitus (DM1). The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) checklist for the reporting of observational studies was followed. The DM1 group had 38 children, and an equal number of volunteers matched by sex and age were recruited as controls. Clinical examination was performed for plaque index and gingival index determination. Non-stimulated whole saliva was collected. Concentrations of IgA, IgG, and IgM were determined by ELISA test. Data were tested by the Kolmogorov-Smirnov, Mann-Whitney, and Spearman tests and a multiple linear regression model (p<0.05) was performed. Gingival index was higher in the Control (DM1: 0.16±0.17; Control: 0.24±0.23, p=0.040). In DM1, there was a correlation between IgA and age (rho=0.371, p=0.024), IgM and IgG (rho=0.459, p=0.007), and IgM and gingival index (rho=0.394, p=0.014). In DM1, multiple linear regression showed that age (p=0.041; β=0.363), gingival index (p=0.041; β=0.398), and plaque index (p=0.008; β=-0.506) were good predictors of IgA levels in saliva. Thus, IgA was the only researched immunoglobulin that was directly associated with plaque and gingival indices in Brazilian children with DM1, but not in control subjects.

Immunoglobulins; Dental Plaque Index; Periodontal Index; Diabetes Mellitus, Type 1

Introduction

Type 1 diabetes mellitus (DM1) is a growing public health problem that accounts for 5–10% of all diabetes cases worldwide.¹1. American Diabetes Association. Classification and diagnosis of diabetes: standards of medical care in diabetes - 2021. Diabetes Care. 2021 Jan;44(1 Suppl 1):S15-33. https://doi.org/10.2337/dc21-S002
https://doi.org/10.2337/dc21-S002... Brazil has the third highest prevalence of DM1 in children and adolescents (92.3 million), after India (229.4 million) and the United States of America (157.9 million), and an annual incidence of 8.9 million in this age group.²2. International Diabetes Federation. IDF Diabetes Atlas. 10th ed. Brussels: Belgium; 2021 [cited 2022 Dec 9]. Available from: https://www.diabetesatlas.org
https://www.diabetesatlas.org...

Oral complications are common in DM1, especially in those with decompensated metabolic disease.³3. Díaz Rosas CY, Cárdenas Vargas E, Castañeda-Delgado JE, Aguilera-Galaviz LA, Aceves Medina MC. Dental, periodontal and salivary conditions in diabetic children associated with metabolic control variables and nutritional plan adherence. Eur J Paediatr Dent. 2018 Jun;19(2):119-26. https://doi.org/10.23804/ejpd.2018.19.02.05
https://doi.org/10.23804/ejpd.2018.19.02... Diabetes is the most important systemic disease that adversely affects periodontal tissues,⁴4. Borgnakke WS. IDF Diabetes Atlas: diabetes and oral health: a two-way relationship of clinical importance. Diabetes Res Clin Pract. 2019 Nov;157:107839. https://doi.org/10.1016/j.diabres.2019.107839
https://doi.org/10.1016/j.diabres.2019.1... with gingivitis and periodontitis considered well-established sequelae of this condition.⁵5. Elheeny AA. Oral health status and impact on the oral health-related quality of life of Egyptian children and early adolescents with type-1 diabetes: a case-control study. Clin Oral Investig. 2020 Nov;24(11):4033-42. https://doi.org/10.1007/s00784-020-03273-w
https://doi.org/10.1007/s00784-020-03273... Parameters such as the gingival index, which assesses the marginal and interproximal gingival condition,⁶6. Löe H, Silness J. Periodontal disease in pregnancy. I. Prevalence and severity. Acta Odontol Scand. 1963 Dec;21(6):533-51. https://doi.org/10.3109/00016356309011240
https://doi.org/10.3109/0001635630901124... and the plaque index, which measures the presence of soft debris and deposits on tooth surfaces,⁷7. Silness J, Löe H. Periodontal disease in pregnancy. II. Correlation between oral hygiene and periodontal condition. Acta Odontol Scand. 1964 Feb;22(1):121-35. https://doi.org/10.3109/00016356408993968
https://doi.org/10.3109/0001635640899396... are frequently used to assess periodontal health in these patients.⁸8. Gujjar KR, Khadija H, Suleiman MO, Amith HV. Gingival health status of 2- to 15-year-old Benghazi children with type-I diabetes mellitus. J Dent Child (Chic). 2011 Jul;78(2):96-101.,⁹9. Al-Khabbaz AK, Al-Shammari KF, Hasan A, Abdul-Rasoul M. Periodontal health of children with type 1 diabetes mellitus in Kuwait: a case-control study. Med Princ Pract. 2013;22(2):144-9. https://doi.org/10.1159/000342624
https://doi.org/10.1159/000342624...

Elevated immunoglobulin levels have also been strongly associated with the development and progression of inflammatory diseases.¹⁰10. Parizi MK, Akbari H, Malek-Mohamadi M, Kheirmand Parizi M, Kakoei S. Association of salivary levels of immunoglobulin-a and amylase with oral-dental manifestations in patients with controlled and non-controlled type 2 diabetes. BMC Oral Health. 2019 Jul;19(1):1-9. https://doi.org/10.1186/s12903-019-0868-4
https://doi.org/10.1186/s12903-019-0868-... IgA deficiency in the blood has been observed in individuals with DM1 and is usually accompanied by normal or increased serum levels of IgG and IgM.¹¹11. Landgraf LF, Rosário NA, Moura JF, Wells KA, Figueiredo BC. High prevalence of immunoglobulin A deficiency in patients with type 1 diabetes mellitus detected by ELISA. Einstein (Sao Paulo). 2008;2013:26-30. Furthermore, Ardawi et al.¹²12. Ardawi MS, Nasrat HA, Bahnassy AA. Serum immunoglobulin concentrations in diabetic patients. Diabet Med. 1994 May;11(4):384-7. https://doi.org/10.1111/j.1464-5491.1994.tb00290.x
https://doi.org/10.1111/j.1464-5491.1994... found a positive correlation between serum levels of IgA and IgG and glycated hemoglobin (A1C) in patients with DM1, suggesting that metabolic control can influence the humoral response and the synthesis of immunoglobulins. The quantification of immunoglobulin levels in diabetics can therefore improve our understanding of immune dysfunction.¹³13. Mazer NA, Welbourn D, Bernstein RK, Rubinstein A. Immunoglobulin deficiency in patients with diabetes mellitus. J Allergy Clin Immunol. 2011 Feb;127(2):AB12. https://doi.org/10.1016/j.jaci.2010.12.061
https://doi.org/10.1016/j.jaci.2010.12.0...

Determination of salivary components in diabetes is also important for describing and understanding the oral findings in this condition, ¹⁴14. Caseiro A, Vitorino R, Barros AS, Ferreira R, Calheiros-Lobo MJ, Carvalho D, et al. Salivary peptidome in type 1 diabetes mellitus. Biomed Chromatogr. 2012 May;26(5):571-82. https://doi.org/10.1002/bmc.1677
https://doi.org/10.1002/bmc.1677... ,¹⁵15. Lasisi TJ, Fasanmade AA. Comparative analysis of salivary glucose and electrolytes in diabetic individuals with periodontitis. Ann Ib Postgrad Med. 2012 Jun;10(1):25-30.since systemic diseases such as DM1 can also compromise the function of the salivary glands and influence the quantity and quality of saliva produced.¹⁶16. Tenovuo J, Lehtonen OP, Viikari J, Larjava H, Vilja P, Tuohimaa P. Immunoglobulins and innate antimicrobial factors in whole saliva of patients with insulin-dependent diabetes mellitus. J Dent Res. 1986 Jan;65(1):62-6. https://doi.org/10.1177/00220345860650011101
https://doi.org/10.1177/0022034586065001... ,¹⁷17. Mata AD, Marques D, Rocha S, Francisco H, Santos C, Mesquita MF, et al. Effects of diabetes mellitus on salivary secretion and its composition in the human. Mol Cell Biochem. 2004 Jun;261(1-2):137-42. https://doi.org/10.1023/B:MCBI.0000028748.40917.6f
https://doi.org/10.1023/B:MCBI.000002874... Compared to healthy controls, salivary concentration of IgA, IgM, and IgG did not appear to be altered in individuals with DM1.¹⁸18. Martins RA, Costa FW, Silva SM, Silva PG, Carvalho FS, Fonteles CS, et al. Salivary immunoglobulins (A, G, and M) in type 1 diabetes mellitus patients: A PROSPERO-registered systematic review and meta-analysis. Arch Oral Biol. 2021 Feb;122:105025. https://doi.org/10.1016/j.archoralbio.2020.105025
https://doi.org/10.1016/j.archoralbio.20... However, it is worth noting that, of the studies included in this systematic review with meta-analysis, only two, Belazi et al.¹⁹19. Belazi MA, Galli-Tsinopoulou A, Drakoulakos D, Fleva A, Papanayiotou PH. Salivary alterations in insulin-dependent diabetes mellitus. Int J Paediatr Dent. 1998 Mar;8(1):29-33. https://doi.org/10.1046/j.1365-263X.1998.00057.x
https://doi.org/10.1046/j.1365-263X.1998... and Javed et al.,²⁰20. Javed F, Sundin U, Altamash M, Klinge B, Engström PE. Self-perceived oral health and salivary proteins in children with type 1 diabetes. J Oral Rehabil. 2009 Jan;36(1):39-44. https://doi.org/10.1111/j.1365-2842.2008.01895.x
https://doi.org/10.1111/j.1365-2842.2008... evaluated patients younger than 20 years of age (4–17 and 10–19, respectively).

The relationship between levels of IgA, IgG, and IgM in saliva and plaque and gingival indices has already been investigated in systemically healthy children (3 to 12 years old) with and without gingivitis.²¹21. Romero MR, Lozano ML, Posada C, Rueda PA, Roa NS, Rodríguez A. Immunoglobulin A, G and M levels in saliva in children between 3 - 12 years of age, healthy and with gingivitis. Acta Odontol Latinoam. 2011;24(2):176-82. PMID:22165316 Analysis of the relationship between immunoglobulin levels and clinical parameters of gingivitis showed that there was a direct correlation between IgM and gingival index (p < 0.05) in children with gingivitis. However, in children with DM1, this relationship has not yet been elucidated. To date, only the study by Wang et al.²²22. Wang MX, Wang X, Zhang Z, Qin M. [The salivary factors related to caries and periodontal disease in children and adolescents with diabetes mellitus]. Chung Hua Kou Chiang Hsueh Tsa Chih. 2013 Sep;48(9):545-9. Chinese. compared plaque and gingival indices and salivary factors (pH, buffering capacity, glucose, total protein, immunoglobulins, lysozyme, and lactate dehydrogenase) in children (7 to 15 years old) with and without DM1, but did not investigate possible associations between these variables. This gap in children with DM1 must thus be clarified.

The null hypothesis of this study was that there is no relationship between salivary immunoglobulins and periodontal indices in pediatric patients with DM1. Thus, the aim of the present study was to evaluate the association between salivary immunoglobulins (IgA, IgG e IgM), plaque index, and gingival index in children with and without DM1.

Methodology

The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) checklist was followed in this study.

Study design and ethical considerations

This was a comparative cross-sectional study. The present study was conducted ethically in accordance with the Declaration of Helsinki (World Medical Association) and approved by the Ethics Committee in Research Involving Human Beings of the Federal University of Ceará (protocol # 1.020.102), following the rules and regulations of the National Health Council. The parents or legal guardians of all participants signed an informed consent form before the child’s enrollment in the research.

Sample calculation and patient recruitment

A sample calculation was performed using an independent samples t-test based on the gingival IgA levels of patients with periodontal disease and diabetes (11.43 ± 1.51 mg/dL) compared to patients without diabetes (10.16 ± 1.58 mg/dL )²³23. Anil S. Immunoglobulin concentration in gingival tissue of type 2 diabetic patients with periodontitis. Indian J Dent Res. 2006;17(4):151-4. https://doi.org/10.4103/0970-9290.29872
https://doi.org/10.4103/0970-9290.29872... with 32 patients per group, a power of 90%, and a confidence interval of 95%. However, to account for losses, the sample was increased by 20% getting a final sample of 38 patients per group. Based on the IgA concentration in diabetic patients older than seven years observed in this study (59,200 ± 4,960 ng/mL) and controls (48,469 ± 4,891 ng/mL), a power of 100% was estimated for a sample of 38 patients per study group to reject the null hypothesis.

Thus, 38 children of both sexes, 2–10 years old and diagnosed with DM1 were selected to participate in the study. These children were spontaneously invited and examined in 2019 during routine consultations with the multidisciplinary team of the Integrated Center for Diabetes and Hypertension (Fortaleza, Brazil). Data collection was performed in 2019 and 2020. The eligibility criteria were: a) diagnosis of DM1; b) non-use of any medication that could alter salivary flow and/or composition; and c) absence of associated comorbidities at the time of study entry. The first 38 children who attended the service and met the eligibility criteria and whose parents consented to the participation in the study were selected. Such children made up the DM1 group. For the control group, the first 38 children without DM1 who attended the service at the pediatric dentistry clinic of the Dentistry course of the Faculty of Pharmacy, Dentistry and Nursing of the Federal University of Ceará in Fortaleza were selected, matched by gender, age and socioeconomic profile to children with diabetes, without comorbidities, meeting the previously defined eligibility criteria, and whose parents consented to participate in the study. Data obtained from these healthy participants were used for comparison.

Currently, diabetes may be diagnosed based on plasma glucose criteria, either fasting plasma glucose (FPG) value or the 2-h plasma glucose (2-h PG) value during a 75-g oral glucose tolerance test (OGTT) or A1C criteria.²⁴24. ElSayed NA, Aleppo G, Aroda VR, Bannuru RR, Brown FM, Bruemmer D, et al.; on behalf of the American Diabetes Association. 2. Classification and diagnosis of diabetes: standards of care in diabetes-2023. Diabetes Care. 2023 Jan;46 Suppl 1:S19-40. https://doi.org/10.2337/dc23-S002
https://doi.org/10.2337/dc23-S002... The criteria for diagnosing diabetes are: FPG ≥ 126 mg/dL (7.0 mmol/L) or 2-h PG ≥ 200 mg/dL (11.1 mmol/L) during OGTT or A1C ≥ 6.5% (48 mmol/mol) or a random plasma glucose ≥ 200 mg/dL (11.1 mmol/L) in a patient with classic symptoms of hyperglycemia or hyperglycemic crisis.²⁴24. ElSayed NA, Aleppo G, Aroda VR, Bannuru RR, Brown FM, Bruemmer D, et al.; on behalf of the American Diabetes Association. 2. Classification and diagnosis of diabetes: standards of care in diabetes-2023. Diabetes Care. 2023 Jan;46 Suppl 1:S19-40. https://doi.org/10.2337/dc23-S002
https://doi.org/10.2337/dc23-S002...

Data collection

Initially, information concerning the general health status of the participants was obtained. Subsequently, gingival health was assessed using the plaque index, as described by Silness & Löe⁷7. Silness J, Löe H. Periodontal disease in pregnancy. II. Correlation between oral hygiene and periodontal condition. Acta Odontol Scand. 1964 Feb;22(1):121-35. https://doi.org/10.3109/00016356408993968
https://doi.org/10.3109/0001635640899396... and the gingival index was assessed according to the study by Löe & Silness⁶6. Löe H, Silness J. Periodontal disease in pregnancy. I. Prevalence and severity. Acta Odontol Scand. 1963 Dec;21(6):533-51. https://doi.org/10.3109/00016356309011240
https://doi.org/10.3109/0001635630901124... . The clinical oral examination was carried out in the dental offices of the patients’ services, with adequate lighting (reflector), mouth mirror, and appropriate probe. The plaque index was recorded for each tooth in 4 areas (distobuccal, buccal, mesiobuccal, and lingual). The sum of the plaque indices of each tooth was divided by 4 to obtain the plaque index per tooth. The plaque index per individual was obtained by adding the plaque indexes per tooth, followed by its division by the number of teeth examined.⁷7. Silness J, Löe H. Periodontal disease in pregnancy. II. Correlation between oral hygiene and periodontal condition. Acta Odontol Scand. 1964 Feb;22(1):121-35. https://doi.org/10.3109/00016356408993968
https://doi.org/10.3109/0001635640899396... The gingival index, in turn, was recorded for each tooth in 4 areas (distobuccal papilla, buccal margin, mesiobuccal papilla, and complete lingual margin). For each tooth, the gingival index scores of each area were added and then divided by 4, to obtain the gingival index per tooth. The score per individual was obtained by adding the scores per tooth and then dividing by the number of teeth.⁶6. Löe H, Silness J. Periodontal disease in pregnancy. I. Prevalence and severity. Acta Odontol Scand. 1963 Dec;21(6):533-51. https://doi.org/10.3109/00016356309011240
https://doi.org/10.3109/0001635630901124... Teeth about to exfoliate, with pathological mobility, in eruption or with purulent exudate according to Romero et al.²¹21. Romero MR, Lozano ML, Posada C, Rueda PA, Roa NS, Rodríguez A. Immunoglobulin A, G and M levels in saliva in children between 3 - 12 years of age, healthy and with gingivitis. Acta Odontol Latinoam. 2011;24(2):176-82. PMID:22165316 were not included. The gingival health was assessed by a single calibrated examiner with an intra-examiner Kappa of 0.81²⁶26. Damasceno JX, Guedes MI, Leite CA, Monteiro AJ, Fonteles MC, Jimenez ME, et al. Salivary immunoglobulin levels in juvenile autoimmune hepatitis. Arch Oral Biol. 2018 Aug;92:51-6. https://doi.org/10.1016/j.archoralbio.2018.04.014
https://doi.org/10.1016/j.archoralbio.20... for both indices. The calibration was performed with 18 healthy children. The evaluators received training before the calibration. The interval between the evaluations was one week.

The collection of saliva for immunoglobulin evaluation was performed by a single researcher after gingival assessment in the dental offices of the services where the patients came from. A sample of unstimulated whole saliva was collected for each participant between 8 and 10 am to reduce possible circadian effects after a maximum of 2 hours of fasting (due to the risk of hypoglycemia in the group with DM1). Parents were asked to perform routine oral hygiene in the child one hour before collection. The volunteer remained at rest for 30 minutes and then saliva was collected with the aid of a plastic Pasteur pipette and stored in Eppendorf^® microtubes (Sigma – Aldrich Brasil LTDA, São Paulo, Brasil), followed by the addition of 5 μL enzyme inhibitor (Sigma – Aldrich Brasil LTDA, São Paulo, Brasil) to each 1 mL of collected saliva. The samples were kept and transported on ice to the laboratory for subsequent centrifugation at 12,000g for 10 minutes at 4°C, lyophilization of the supernatant, and storage at -80°C until analysis. This standardized protocol was used to control for environmental and circadian rhythm influences on the qualitative and quantitative salivary components.²⁶26. Damasceno JX, Guedes MI, Leite CA, Monteiro AJ, Fonteles MC, Jimenez ME, et al. Salivary immunoglobulin levels in juvenile autoimmune hepatitis. Arch Oral Biol. 2018 Aug;92:51-6. https://doi.org/10.1016/j.archoralbio.2018.04.014
https://doi.org/10.1016/j.archoralbio.20...

Quantitative analysis of immunoglobulins – ELISA

Each saliva sample was divided into 3 parts to be analyzed individually, being subjected to the analysis of immunoglobulin titers using the enzyme-linked immunosorbent assay (ELISA), using saliva as the primary antibody and a human antibody component as the capture antibody. The following salivary assay protocols were used: human protein IgA, Abcam^®, Cat. Num. ab137980; human IgG protein, Abcam^®, Cat. Num. ab195215; and, human IgM protein, Abcam^®, Cat. Num. ab137982 (Sigma – Aldrich Brasil LTDA, São Paulo, Brasil). Saliva and salivary standards were added to the wells followed by antibodies; wash buffer was used 5 times and stop solution was added to stop the reaction. Finally, the results were read in a spectrophotometer at 450 nm wavelength. All analyses were performed on a single day by the same researcher.

Statistical analysis

Data analysis was performed using SPSS software (Statistical Package for the Social Sciences), version 20.0 for Windows^®. Data were submitted to the Kolmogorov-Smirnov normality test, expressed as mean and standard deviation, and compared using the Mann-Whitney test (nonparametric data). Additionally, Spearman’s correlation was used to correlate clinical parameters and immunoglobulin profile. The variables that were correlated with the immunoglobulin profile were selected for the multiple linear regression model. The ROC (receiver operating characteristic) curve was also used to determine sensitivity, specificity, and accuracy. A cut-off point of 7 years old was established because 7 was the age median. The significance level was p < 0.05 for all tests.

Results

The sample consisted of 76 children, 38 with DM1 and 38 without the disease. All of them were residents of Fortaleza (Brazil). There was no statistically significant difference for age according to sex (Control group: 7.20 ± 2.17 for female and 6.78 ± 2.26 for male, p = 0.657; DM1 group: 7.15 ± 2.01 for female and 6.67 ± 1.97 for male, p = 0.544) and age of the two groups (7.00 ± 2.19 for DM1 and 6.92 ± 1.98 for Control, p = 0.785).

Both the DM1 group and Control group had higher concentrations of IgA (43,005 ± 21,236 ng/mL and 43,857 ± 24,585 ng/mL) (Figure 1).

There was no statistically significant difference between the groups for concentrations of IgA, IgM, and IgG (Figure 2).

There was no statistically significant difference between the groups for the gingival (0.17 ± 0.17 DM1 and 0.22 ± 0.20 Control; p = 0.145) and plaque indices (0.34 ± 0.32 DM1 and 0.22 ± 0.23 Control; p = 0.139) (Mann-Whitney test, p < 0.05). The mean gingival index in the Control group was 0.24 and in the DM1 group was 0.15. The median gingival index in the Control group was 0.19 and in the DM1 group was 0.10. The mean plaque index in the Control group was 0.22 and in the DM1 group was 0.34. The median plaque index in the Control group was 0.14 and in DM1 group was 0.23.

In the DM1 group, there was a correlation between IgA and age, between IgG and IgM, and between gingival index and IgM (Figure 3).

Multiple linear regression showed positive correlations between age and IgA and age and IgM in the Control group, and between age and IgA and gingival index and IgA in the DM1 group. However, a negative correlation between plaque index and IgA in the DM1 group was found (Table 1).

The ROC curve for diagnosing diabetes based on changes in IgA, IgM, and IgG levels showed a large area under the curve for IgG, but it was not sufficient to establish a diagnosis. However, with a cut-off point of 7 years old, a greater area under the curve was observed for children over 7 years old than in children up to 7 years old (Figure 4).

Finally, children over 7 years old with an IgA concentration of up to 43,000 ng/mL had 88.2% specificity, and children over 7 years old with an IgG concentration of up to 3,200 ng/mL had 78.6% sensitivity (Table 2).

Discussion

In the present study, association and correlation analyses were performed showing important results for the DM1 group, including high levels of IgA in saliva, higher plaque index and lower gingival index compared to the Control group, and significant correlations between IgA and age, IgM and IgG, and IgM and gingival index.

The salivary IgA concentration of children with DM1 in the present study was higher than that of children without the disease, although this difference was not significant between the groups. Siudikiene et al.²⁷27. Siudikiene J, Machiulskiene V, Nyvad B, Tenovuo J, Nedzelskiene I. Dental caries increments and related factors in children with type 1 diabetes mellitus. Caries Res. 2008;42(5):354-62. https://doi.org/10.1159/000151582
https://doi.org/10.1159/000151582... also observed high levels of this immunoglobulin when evaluating saliva of 63 pairs of children with and without DM1. Increased levels of salivary IgA in children with DM1 were also observed earlier by Belazi et al.¹⁹19. Belazi MA, Galli-Tsinopoulou A, Drakoulakos D, Fleva A, Papanayiotou PH. Salivary alterations in insulin-dependent diabetes mellitus. Int J Paediatr Dent. 1998 Mar;8(1):29-33. https://doi.org/10.1046/j.1365-263X.1998.00057.x
https://doi.org/10.1046/j.1365-263X.1998... . Salivary IgA (s-IgA) is produced by plasma cells and inhibits the adhesion of microorganisms to mucosal surfaces. Increased levels of s-IgA in the oral cavity may indicate periodontal disease or oral candidiasis.¹⁹19. Belazi MA, Galli-Tsinopoulou A, Drakoulakos D, Fleva A, Papanayiotou PH. Salivary alterations in insulin-dependent diabetes mellitus. Int J Paediatr Dent. 1998 Mar;8(1):29-33. https://doi.org/10.1046/j.1365-263X.1998.00057.x
https://doi.org/10.1046/j.1365-263X.1998... Furthermore, according to Belazi et al.¹⁹19. Belazi MA, Galli-Tsinopoulou A, Drakoulakos D, Fleva A, Papanayiotou PH. Salivary alterations in insulin-dependent diabetes mellitus. Int J Paediatr Dent. 1998 Mar;8(1):29-33. https://doi.org/10.1046/j.1365-263X.1998.00057.x
https://doi.org/10.1046/j.1365-263X.1998... , high levels of s-IgA combined with good oral health and a mild degree of dry mouth may indicate a prediabetic condition.

Although diabetes is recognized as a risk factor for periodontal diseases,²⁸28. Jensen E, Allen G, Bednarz J, Couper J, Peña A. Periodontal risk markers in children and adolescents with type 1 diabetes: A systematic review and meta-analysis. Diabetes Metab Res Rev. 2021 Jan;37(1):e3368. https://doi.org/10.1002/dmrr.3368
https://doi.org/10.1002/dmrr.3368...

29. Rapone B, Corsalini M, Converti I, Loverro MT, Gnoni A, Trerotoli P, et al. Does periodontal inflammation affect type 1 diabetes in childhood and adolescence? A meta-analysis. Front Endocrinol (Lausanne). 2020 May;11:278. https://doi.org/10.3389/fendo.2020.00278
https://doi.org/10.3389/fendo.2020.00278... -³⁰30. Zainal Abidin Z, Zainuren ZA, Noor E, Mohd Nor NS, Mohd Saffian S, Abdul Halim R. Periodontal health status of children and adolescents with diabetes mellitus: a systematic review and meta-analysis. Aust Dent J. 2021 Mar;66(S1 Suppl 1):S15-26. https://doi.org/10.1111/adj.12845
https://doi.org/10.1111/adj.12845... in the present study, plaque and gingival indices did not differ significantly between groups. According to the systematic review with meta-analysis by Jensen et al.²⁸28. Jensen E, Allen G, Bednarz J, Couper J, Peña A. Periodontal risk markers in children and adolescents with type 1 diabetes: A systematic review and meta-analysis. Diabetes Metab Res Rev. 2021 Jan;37(1):e3368. https://doi.org/10.1002/dmrr.3368
https://doi.org/10.1002/dmrr.3368... , children and adolescents with DM1 are more likely to have elevated risk markers for periodontal disease (plaque [standardized mean difference - SDM 0.45; 95%CI [0.21–0.70]; p < 0.001] and gingival [SDM 0.51 95%CI [0.28–0.74]; p < 0.001] indices, bleeding on probing [SDM 0.61 95%CI [0.40–0.82]; p < 0.001], pocket depth [SDM 0.55 95%CI [0.22–0.87]; p < 0.001], and clinical attachment loss [SMD 0.54; 95%CI [0.29–0.78]; p < .001]) compared to their healthy peers. Zainal Abidin et al.³⁰30. Zainal Abidin Z, Zainuren ZA, Noor E, Mohd Nor NS, Mohd Saffian S, Abdul Halim R. Periodontal health status of children and adolescents with diabetes mellitus: a systematic review and meta-analysis. Aust Dent J. 2021 Mar;66(S1 Suppl 1):S15-26. https://doi.org/10.1111/adj.12845
https://doi.org/10.1111/adj.12845... also found a worse periodontal status in children and adolescents with DM1, SMD pooled [95%CI] for plaque index of 0.54 [0.20–0.87], gingival index of 0.63 [0.39,0, 87], clinical attachment loss of 0.79 [0.52–.05], and periodontal probing depth of 0.67 [0.23–1.11]. This has also been attributed to the elevated and prolonged inflammation of the periodontal tissue due to impaired immune function, leading to tissue destruction.³⁰30. Zainal Abidin Z, Zainuren ZA, Noor E, Mohd Nor NS, Mohd Saffian S, Abdul Halim R. Periodontal health status of children and adolescents with diabetes mellitus: a systematic review and meta-analysis. Aust Dent J. 2021 Mar;66(S1 Suppl 1):S15-26. https://doi.org/10.1111/adj.12845
https://doi.org/10.1111/adj.12845... However, it is worth emphasizing that when comparing periodontal studies, differences may be due to the design of the registry, type/number of sites evaluated, and periodontal probe used.³¹31. Orbak R, Simsek S, Orbak Z, Kavrut F, Colak M. The influence of type-1 diabetes mellitus on dentition and oral health in children and adolescents. Yonsei Med J. 2008 Jun;49(3):357-65. https://doi.org/10.3349/ymj.2008.49.3.357
https://doi.org/10.3349/ymj.2008.49.3.35... Other factors such as diabetes duration and blood glucose may influence clinical outcomes.⁹9. Al-Khabbaz AK, Al-Shammari KF, Hasan A, Abdul-Rasoul M. Periodontal health of children with type 1 diabetes mellitus in Kuwait: a case-control study. Med Princ Pract. 2013;22(2):144-9. https://doi.org/10.1159/000342624
https://doi.org/10.1159/000342624...

Rappone et al.²⁹29. Rapone B, Corsalini M, Converti I, Loverro MT, Gnoni A, Trerotoli P, et al. Does periodontal inflammation affect type 1 diabetes in childhood and adolescence? A meta-analysis. Front Endocrinol (Lausanne). 2020 May;11:278. https://doi.org/10.3389/fendo.2020.00278
https://doi.org/10.3389/fendo.2020.00278... also reinforced that studies on the potential effects of periodontal inflammation in children and adolescents with DM1 are incomplete. Despite the recent exponential increase in the number of studies on the association between periodontitis and diabetes, no general consensus has yet emerged on a causal effect of periodontal inflammation in T1DM. The authors concluded that their meta-analysis did not provide strong evidence that periodontitis is a significant risk factor for DM1, and the link between periodontal disease and DM1 does not appear to be as solid as the link with DM2.

Pachoński et al.³²32. Pachonski M, Jarosz-Chobot P, Koczor-Rozmus A, Lanowy P, Mocny-Pachonska K. Dental caries and periodontal status in children with type 1 diabetes mellitus. Pediatr Endocrinol Diabetes Metab. 2020;26(1):39-44. https://doi.org/10.5114/pedm.2020.93249
https://doi.org/10.5114/pedm.2020.93249... observed similar resutls when comparing periodontal status (through plaque, proximal plaque, gingival, and modified sulcus bleeding indices) of healthy children, of children with compensated DM1, and of children with decompensated DM1, further suggesting the absence of an effect of metabolic control of diabetes on index values. However, a limitation of the study by Pachoński et al.³²32. Pachonski M, Jarosz-Chobot P, Koczor-Rozmus A, Lanowy P, Mocny-Pachonska K. Dental caries and periodontal status in children with type 1 diabetes mellitus. Pediatr Endocrinol Diabetes Metab. 2020;26(1):39-44. https://doi.org/10.5114/pedm.2020.93249
https://doi.org/10.5114/pedm.2020.93249... was the small sample of patients, which resulted in a small margin of error. In addition, the threshold value of A1C was set at 7.5%. Ismail et al.³³33. Ismail AF, McGrath CP, Yiu CK. Oral health status of children with type 1 diabetes: a comparative study. J Pediatr Endocrinol Metab. 2017 Oct;30(11):1155-9. https://doi.org/10.1515/jpem-2017-0053
https://doi.org/10.1515/jpem-2017-0053... also found a similar result for gingival index but observed that diabetic children had significantly higher plaque deposits and significantly higher mean plaque index when compared to healthy children.

In the DM1 group, an association between IgA and age was found. According to Romero et al.²¹21. Romero MR, Lozano ML, Posada C, Rueda PA, Roa NS, Rodríguez A. Immunoglobulin A, G and M levels in saliva in children between 3 - 12 years of age, healthy and with gingivitis. Acta Odontol Latinoam. 2011;24(2):176-82. PMID:22165316, in the general pediatric population, the changes due to the onset of the mixed dentition phase from the age of 6 years lead to changes in the gingival crevicular environment, facilitating the colonization of microorganisms associated with periodontal diseases, thus increasing the antigenic challenge, which is reflected in higher levels of this immunoglobulin. Furthermore, it is important to consider that hormonal changes that begin before puberty can also affect the subgingival microbiota with increasing age.³⁴34. Bimstein E, Ebersole JL. The age-dependent reaction of the periodontal tissues to dental plaque. ASDC J Dent Child. 1989;56(5):358-62.

IgM and IgG antibodies work together in immediate and long-term protection against infections.³⁵35. Tiller KE, Tessier PM. Advances in antibody design. Annu Rev Biomed Eng. 2015;17(1):191-216. https://doi.org/10.1146/annurev-bioeng-071114-040733
https://doi.org/10.1146/annurev-bioeng-0... Although the present study has demonstrated a direct relationship between such immunoglobulins in the DM1 group, there are no other studies on this association in children to date. In adults, Tenovuo et al.¹⁶16. Tenovuo J, Lehtonen OP, Viikari J, Larjava H, Vilja P, Tuohimaa P. Immunoglobulins and innate antimicrobial factors in whole saliva of patients with insulin-dependent diabetes mellitus. J Dent Res. 1986 Jan;65(1):62-6. https://doi.org/10.1177/00220345860650011101
https://doi.org/10.1177/0022034586065001... observed significantly elevated levels of IgG (p < 0.05) but not of IgM (p > 0.05). IgG and IgM are produced locally, for example, in the inflamed gingiva³⁶36. Mackler BF, Frostad KB, Robertson PB, Levy BM. Immunoglobulin bearing lymphocytes and plasma cells in human periodontal disease. J Periodontal Res. 1977 Jan;12(1):37-45. https://doi.org/10.1111/j.1600-0765.1977.tb00107.x
https://doi.org/10.1111/j.1600-0765.1977... or are derived from serum via gingival clefts and/or salivary glands.³⁷37. Brandtzaeg P. Evaluation of immunofluorescence with artificial sections of selected antigenicity. Immunology. 1972 Jan;22(1):177-83. PMID:4552229,³⁸38. Challacombe SJ, Russell MW, Hawkes J. Passage of intact IgG from plasma to the oral cavity via crevicular fluid. Clin Exp Immunol. 1978 Dec;34(3):417-22. In whole saliva, increased levels of immunoglobulins were detected in patients with periodontal disease.³⁹39. Ranney RR, Ruddy S, Tew JG, Welshimer HJ, Palcanis KG, Segreti A. Immunological studies of young adults with severe periodontitis. I. Medical evaluation and humoral factors. J Periodontal Res. 1981 Jul;16(4):390-402. https://doi.org/10.1111/j.1600-0765.1981.tb00990.x
https://doi.org/10.1111/j.1600-0765.1981...

The significance of the positive correlation between IgM and gingival index in diabetics in the present study is still unclear. Romero et al.²¹21. Romero MR, Lozano ML, Posada C, Rueda PA, Roa NS, Rodríguez A. Immunoglobulin A, G and M levels in saliva in children between 3 - 12 years of age, healthy and with gingivitis. Acta Odontol Latinoam. 2011;24(2):176-82. PMID:22165316, who evaluated levels of IgA, IgG, and IgM in saliva of healthy children aged 3 to 12 years with gingivitis, found a direct relationship between IgM and gingival index, and suggested two possible interpretations for this finding: the activation of IgM production as a response to an antigenic challenge leading to tissue inflammation or the increased exudate as a result of inflammation generates more IgM in saliva. Such hypotheses could eventually also apply to diabetic children.

Given the above, in the DM1 group, multiple linear regression showed that age, plaque, and gingival indices were good predictors of s-IgA levels. However, these variables were not good predictors of IgM or IgG levels in this population.

The present study included assessment of important risk markers for periodontal diseases that were validated as predictive for the amount of biofilm and the degree of gingival inflammation in a child.⁴⁰40. Goldberg P, Matsson L, Anderson H. Partial recording of gingivitis and dental plaque in children of different ages and in young adults. Community Dent Oral Epidemiol. 1985 Feb;13(1):44-6. https://doi.org/10.1111/j.1600-0528.1985.tb00419.x
https://doi.org/10.1111/j.1600-0528.1985... Furthermore, all measurements were performed by a single experienced examiner and complete data on periodontal markers and immunoglobulin analysis were obtained from all study participants. However, the present study also has some limitations. The cross-sectional design prevented the determination of causality between the variables evaluated. Also, the small sample size increases the margin of error.

Conclusions

IgM showed a direct correlation with gingival index and IgA showed a significant association with plaque and gingival indices only in diabetic children.

Acknowledgments

The authors thank the SBPqO (Brazilian Society for Dental Research - the International Association for Dental Research [IADR] Brazilian Division) for the opportunity to present this study at the 35th SBPqO annual meeting, where it was awarded first place during the IADR Joseph Lister Award New Investigators Competition. Also, this work was supported by the Brazilian National Council for Scientific and Technological Development (CNPq) process # 445591/2014-5.

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