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ORIGINAL RESEARCH  
Year : 2013  |  Volume : 24  |  Issue : 3  |  Page : 394
Effect of dental treatments on salivary immunoglobulin A of children with and without dental caries: A comparative study


1 Department of Pedodontics and Preventive Dentistry, K. S. R. Institute of Dental Science and Research, Thiruchengode, India
2 Department of Endodontics, K. S. R. Institute of Dental Science and Research, Thiruchengode, India
3 Department of Pedodontics and Preventive Dentistry, Rajah Muthiah Dental College and Hospital, Chidambaram, Tamil Nadu, India

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Date of Submission05-Jul-2012
Date of Decision18-Jan-2013
Date of Acceptance05-Mar-2013
Date of Web Publication12-Sep-2013
 

   Abstract 

Aim: To evaluate and compare the effect of dental treatment on the salivary immunoglobulin A (IgA) levels of children with and without dental caries.
Materials and Methods: The study involved 30 children, among which 15 had caries and the other 15 were without caries. Salivary sample collection was done for all the children before dental treatment, and for the children with caries, the sampling was repeated 3-4 weeks after the dental treatment. The salivary IgA quantitation was done by enzyme-linked immunosorbent assay (ELISA), using Human IgA ELISA Quantitation kit, and the results were statistically analyzed by independent sample "t" test.
Results:The salivary IgA level was significantly more in children with caries (13.07 ± 1.55 mg/100 ml) than in caries-free children (11.90 ± 1.58 mg/100 ml) in the pre-treatment phase. The salivary IgA level in children with caries was 13.52 ± 1.68 mg/100 ml in the post-treatment phase and it was not statistically different from the pre-treatment value.
Conclusion: Mere quantitation of salivary IgA levels might have no reflection on the functional antibodies involved in caries process, and successful dental treatment alone does not alter the salivary IgA levels, suggesting a multifaceted approach to combat the cariogenic challenge.

Keywords: Dental caries, enzyme-linked immunosorbent assay, salivary immunoglobulin A

How to cite this article:
Geetha Priya P R, Asokan S, Karthick K, Reddy N V, Rao V A. Effect of dental treatments on salivary immunoglobulin A of children with and without dental caries: A comparative study. Indian J Dent Res 2013;24:394

How to cite this URL:
Geetha Priya P R, Asokan S, Karthick K, Reddy N V, Rao V A. Effect of dental treatments on salivary immunoglobulin A of children with and without dental caries: A comparative study. Indian J Dent Res [serial online] 2013 [cited 2017 Mar 27];24:394. Available from: http://www.ijdr.in/text.asp?2013/24/3/394/118004
The principal means of treating dental caries involves removing the carious enamel and dentin, and replacing the defects with precious metals or synthetic materials. This constitutes what is known as conventional restorative treatment. [1] Research has shifted from this "surgical model" approach to the "medical model" approach of treating dental caries, which focuses on counteracting the causative factors. Saliva, a component of the oral fluid, is composed of several specific and nonspecific defense factors, and salivary immunoglobulin A (IgA) is the principal specific defense factor in it. [1],[2] The salivary IgA begins to appear as early as first week of life and reaches adult level in 1-2 years of life. [3] The salivary levels of IgA at birth were reported to be 2.5-fold higher in full-term children than in preterm children. [4] The specific immunoglobulin response is modulated by the amount of antigen load. [5] The intensities and specificities of salivary IgA antibody responses to antigens of Streptococcus mutans may influence colonization by these organisms during the first 1.5 years of life. [4] There are conflicting views regarding the relation between salivary IgA and dental caries. Caries-free children have been reported to have significantly higher levels of naturally occurring salivary IgA to S. mutans, the major causative agent of dental caries. [6] Rose et al. have shown that the caries-active children showed a significantly higher level of salivary IgA than the caries-free group. [7] However, a reduction in oral bacterial load following dental treatment has also been reported. [1] With this existing literature, a study was planned to evaluate and compare the effect of dental treatment on the salivary IgA levels of children with and without dental caries.


   Materials and Methods Top


The study protocol was reviewed and approved by the institutional review board of Annamalai University, Chidambaram, Tamil Nadu, India. The procedures, possible discomforts or risks, as well as possible benefits were explained fully to the children and the parents involved in the study. Informed consent was obtained from parents/primary care givers to conduct the study. Survey form was prepared with the help of the WHO Oral Health Assessment Form 1997.

A total of 300 children were screened and 30 children of age between 7 and 12 years participated in this study. The age group was chosen in accordance with the study by D'Amelio et al., [8] which reported that the complete maturation of salivary IgA occurs after 6 years of age. Out of 30 children, 15 who had no carious teeth (DMFT/deft score of 0) belonged to the control group (Group I). The study group (Group II) included 15 children with caries (DMFT/deft score of ≥5). The study group was further subdivided into "A" (pre-dental treatment) and "B" (3-4 weeks post-dental treatment). The children included were physically and mentally healthy with the same economic status and no history of any allergic disorders or infections for past 3 months. Recent exposure to antibiotics may alter the saliva and gingival crevicular fluid, thereby leading to an imbalance in the oral microbiota. Chronic mouth breathers were excluded as they have decreased salivary secretion. Those children with any abscess, draining sinus, or cellulitis were excluded since their oral microbial levels are elevated. [9],[10]

Clinical procedure

Each day three to four children from the study group were selected randomly and were brought to the Department of Pedodontics, Rajah Muthiah Dental College and Hospital, Annamalai University. Patients were not allowed to eat or drink 1 h prior treatment to avoid food debris in sample collection. Approximately 2-3 ml of unstimulated whole saliva was collected immediately prior to the commencement of the dental treatment, in a wide-mouthed sterile container (uricup). The samples were then frozen and stored at -70°C until analyzed (Cryo Scientific Systems (Pvt.) Ltd., Chennai, India. The dental treatment included oral prophylaxis followed by restorative treatment and later topical fluoride application. Treatment procedures were performed in accordance with American Association of Pediatric Dentistry clinical guidelines. [11] The second saliva sample was collected 3-4 weeks after completion of the treatment procedure, to minimize the effect of antigenic stimulation produced by instrumentation. [12] These samples were frozen again and stored until analyzed.

Laboratory procedure

The saliva samples were thawed and centrifuged for 15 min at 12,000 g at 4°C to remove the mucin and debris. The supernatant was examined by enzyme-linked immunosorbent assay (ELISA) using Human IgA ELISA Quantitation kit (Catalog No: E80-102; Bethyl Laboratories, Montgomery, Texas, USA) in the Department of Biochemistry, Annamalai University.

Human salivary IgA quantitation method

All steps were performed at room temperature. The buffer preparations were done (according to the manual provided by the kit) prior to the ELISA procedure. The standards, blanks, study and control samples were analyzed in duplicate. Ninety-six microliters of capture antibody was diluted to 9600 μl coating buffer and coated on each well. The coated plate was incubated for 60 min. After incubation, the capture antibody was aspirated from the solution from each well with the Skan washer instrument. Each well was filled with wash solution and was removed by aspiration. This was repeated for three washes. Next 200 μl of blocking (postcoat) solution was added to each well and incubated for 30 min at room temperature. After incubation, the blocking solution was removed from each well and washed three times. Since our samples were expected to have high salivary IgA levels than the standard range given by the company, the samples were diluted in sample diluents. The standards which were supplied along the kit were also diluted in sample diluents. One microliter of saliva sample was diluted to 100 μl in sample diluents. One hundred microliter of the samples were transferred to assigned wells and incubated for 60 min at room temperature. After incubation, samples and standards were removed and each well was washed 5 times. Then, 100 μl of horseradish peroxidase (HRP) conjugate was transferred to each well and incubated for 60 min and each well was washed five times. Next, 100 μl of tetramethylbenzidine (TMB) peroxidase substrate solution was transferred to each well and incubated for 15 min at room temperature. Enzyme substrate reaction resulted in blue color formation. To stop the TMB reaction, 100 μl of 2 M sulfuric acid was added to each well and the blue color changed to yellow. Using a microtiter plate reader, Versamax molecular device (USA), the plate was read at a wavelength of 450 nm.

The average of duplicate readings from each standard, control, and sample was taken. A standard curve was created by reducing the data using computer software (Softmax) capable of generating a four-parameter logistic (4-PL) curve fit. The results obtained were statistically analyzed by independent sample "t" test using SPSS software version 12.


   Results Top


The mean (±SD) salivary IgA level in children in no caries group (Group I) was 11.90 (±1.58) mg/100 ml. The mean salivary IgA level in children with caries, prior to dental treatments (Group IIA) was 13.07 (±1.55) mg/100 ml. The mean salivary IgA level in children with caries, 3-4 weeks after dental treatments (Group IIB) was 13.52 (±1.68) mg/100 ml. The salivary IgA levels were found to be statistically more in children with caries than in children with no caries [Table 1]. The effect of dental treatments on salivary IgA level was found to be statistically not significant [Table 2]. Furthermore, there was no significant correlation between salivary IgA levels and age or gender in the respective groups. The comparison of the salivary IgA levels in children with DMFT/deft score of 5 and >5 did not show a statistically significant difference.
Table 1: Comparison of salivary IgA levels in children with and without dental caries

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Table 2: Comparison of salivary IgA levels in pre‑ and post‑dental treatment samples

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   Discussion Top


Dental caries is not different from other diseases caused by microorganisms, in being dependent on the microbial attack on one hand and the resistance of the host on the other. Marcotte et al. reported that salivary IgA could help to maintain the integrity of the tooth surface by limiting the microbial adherence; by neutralizing enzymes, toxins, and virus; or by acting in synergy with other antibacterial factors such as lysozyme, lactoferrin, salivary peroxidase, and mucin. [9] The treatment of dental caries and its correlation with the change in salivary IgA levels has not been well established. [10] Hence, this study was planned with the null hypothesis that there were no differences in the levels of salivary IgA (a) in children with and without dental caries and (b) between the pre- and post-restoration scores in children with dental caries.

In the present study, the study group with dental caries had higher mean salivary IgA levels than the non-caries control group. The first part of our null hypothesis was hence rejected. These findings were similar to the results of Challacombe et al.[13] and Rose et al.[7] and contradictory to those of Camling et al. [6] The reason for an increase in salivary IgA levels might be the cumulative antigenic stimulation of repeated attacks of caries or more recent antigenic stimulation or both of the above factors. Thus, salivary IgA levels may be used as markers of incipient caries infection in children. However, repeated antigenic attacks on the gut-associated lymphoid tissue (GALT) by a pathogenic or commensal oral flora can lead to leveling out of the antibodies' production by the time the immune system reaches maturity. [12],[13],[14]

Najlaa Al Amoudi et al.[15] have shown that children with severe early childhood caries and their mothers have higher levels of salivary IgA than the caries-free children and their mothers. High concentration of salivary IgA in these children may be associated with an increased antigenic load, leading to high production of antibodies. Koga-Ito et al.[16] revealed that IgA levels in saliva were significantly higher in rampant caries children, suggesting a response reflecting the infectious nature of severe dental caries in contrast to the presence of small lesions. In contrast, Deoyani et al.[17] reported high salivary IgA levels in caries-free children than in caries-active children with DMFT >10. Kuriakose et al. [3] reported significant decrease in salivary IgA levels in rampant caries group. However, in their study, few caries-resistant children had low values of salivary IgA, compared to the rampant caries group. It might be due to the normal levels of non-immune defense factors which show a compensatory increase in other immunoglobulin isotypes.

In the present study, the difference in the salivary IgA levels between pre- and-post dental treatment samples was found to be statistically not significant, and hence the second part of our null hypothesis was accepted. Similar result was observed by Gregory et al. [5] in their study. They showed no significant overall difference in salivary IgA antibodies levels to S. mutans between pre- and post-restorative treatment, suggesting no whole mouth effect of restorative treatment on bacterial challenge. Wright et al. [1] reported significant reductions in salivary mutans streptococci and lactobacilli, immediately following restoration. However, mutans streptococci and lactobacilli levels returned to baseline values in 50% of their subjects in 1-199 days (mean 68 days) and 10-110 days (mean 50 days), respectively. Thus, they concluded that the dental treatment did not appear to have a prolonged effect on the salivary bacterial population. Kugler et al. [18] reported transient decrease of salivary IgA concentration following excavation of carious lesions. Axelsson et al. [19] reported that combination of professional mechanical tooth cleaning, tongue scraping, and chlorhexidine treatment was capable of reducing interproximal S. mutans, although with a transient effect. Fluoride can inhibit bacterial growth by reducing the sugar transport, glycolytic activity, and acid tolerance of many gram-positive species. [5] All treatment procedures done in our study were expected to reduce the bacterial challenge. But the salivary IgA levels had not significantly reduced, suggesting that additional emphasis has to be laid on both the professional and homecare preventive strategies.

In the present study, there was no statistically significant difference in salivary IgA levels between age, gender, and DMFT/deft status (score of 5 and >5) in the respective groups. Koga-Ito et al. [16] reported that there were no alternations related to age and salivary IgA. D'Amelio et al. [8] showed in their study that there was no difference in salivary IgA levels between males and females. Dental caries is a slowly progressing disease which is detected at a particular time. But clinical manifestation process may have been developing for long time. In contrast, the level of antibodies is usually determined on one occasion only, making it difficult to establish a correlation between the level of antibodies and the number of carious lesions. [12] In view of the multifactorial nature of the caries process, it is evident that a certain level of antibodies to S. mutans, protective in one individual, may not confer protection in another because of differences in oral hygiene, pattern of sugar consumption, plaque levels of cariogenic bacteria, and exposure to fluoride. The dental treatments are found to take part only in restoring the form and function of the infected caries tooth and temporarily eliminate the bacterial challenge. Hence, a periodic dental care providing permanent remedy has to be implemented. Regular use of fluorides, antimicrobials, appropriate nutrition, diet modification, vaccines, and dental materials that have antimicrobial properties are the methods that need to be addressed in identifying more permanent and effective therapies to manage this infectious multifactorial disease.

Mere quantitation of salivary IgA levels might have no reflection on the functional antibodies involved in caries process, and successful dental treatment alone does not alter the salivary IgA levels, suggesting a multifaceted approach to combat the cariogenic challenge. Comprehensive treatment of dental caries should address the infectious nature of this disease through modalities directed at controlling bacterial colonization as well as restoring the afflicted dentition. Within the limitations of the study, the following conclusions can be made:

  • The salivary IgA levels were significantly more in children with caries than in caries-free children.
  • The effect of dental treatments (pre- and post-treatment scores) on salivary IgA levels in children with dental caries was found to be statistically not significant, thereby concluding no whole mouth effect.
  • The salivary IgA levels between children with DMFT/deft score of 5 and >5 were statistically not significant.
  • There was no statistically significant difference in salivary IgA levels between age and gender in the respective groups.


 
   References Top

1.Wright JT, Cutter GR, Caufield PW, Ananda P, Dasanayake, Mac Stiles H. Effect of conventional dental restorative treatment on bacteria in saliva. Community Dent Oral Epidemiol 1992;20:138-43.  Back to cited text no. 1
    
2.Available from: http://en.wikipedia.org/wiki/Saliva. [Accessed on 2012 Jun 14].  Back to cited text no. 2
    
3.Kuriakose S, Chintu S. Salivary Immunoglobulin A and its relationship with rampant dental caries in South Indian children. J Indian Dent Assoc 2002;73:312-6.  Back to cited text no. 3
    
4.Nogueira RD, Sesso ML, Borges MC, Mattos-Graner RO, Smith DJ, Ferriani VP. Salivary IgA antibody responses to Streptococcus mitis and Streptococcus mutans in preterm and full term newborn children. Arch Oral Biol 2012;57:647-53.  Back to cited text no. 4
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5.Gregory RL, Amina MA, El-Rahman, Avery DR. Effect of restorative treatment on mutans streptococci and IgA antibodies. Pediatr Dent 1998;20:273-7.  Back to cited text no. 5
    
6.Camling E, Gahenberg L, Krasse B. The relationship between IgA antibodies to streptococcus mutans antigens in human saliva and breast milk and the numbers of indigenous oral streptococcus mutans. Arch Oral Biol 1987;32;21-5.  Back to cited text no. 6
    
7.Rose PT, Gregory RL, Gfell LE, Hughes CV. IgA antibodies to streptococcus mutans in caries-resistant and susceptible children. Pediatr Dent 1994;16;272-5.  Back to cited text no. 7
    
8.D'Amelio R, Bonomo R, D'Offizi GP, Mezzaroma I, Pontesilli O, Le Moli S, et al. Salivary IgA levels in normal children. Diagn Immunol 1986;4:145-8.  Back to cited text no. 8
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9.Marcotte H, Lavoie MC. Oral microbial ecology and the role of salivary immunoglobulin A. Microbiol Mol Biol Rev 1998;62:71-109.  Back to cited text no. 9
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10.Reifft RL. Serum and salivary IgG and IgA response to initial preparation therapy. J Periodontol 1984;299-305.  Back to cited text no. 10
    
11.American Academy of Pediatric Dentistry: Reference manual 10/11. Pediatr Dent 2010;32.  Back to cited text no. 11
    
12.Bhatia S, Tewari A, Chawla HS, Ganguly NK. Naturally occurring S-IgA in saliva of adults and children-correlation with dental caries activity. J Indian Soc Pedod Prev Dent 1986;4:1-7.  Back to cited text no. 12
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13.Challacombe SJ. Serum and salivary antibodies to streptococcus mutans in relation to the development and treatment of human dental caries. Arch Oral Biol 1980;25:495-502.  Back to cited text no. 13
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14.Everhart DL, Grigsby WR, Carter JR. Evaluation of dental caries experience and salivary immunoglobulins in whole saliva. J Dent Res 1972;51:1487-92.  Back to cited text no. 14
    
15.Najlaa Al Amoudi, Haneen Al Shukairy, Azza Hanno. A comparative study of the secretory IgA immunoglobulins in mothers and children with SECC versus a caries free group children and their mothers. J Clin Pediatr Dent 2007;32:53-6.  Back to cited text no. 15
    
16.Koga-Ito CY, Martins CA, Balducci I, Jorge AO. Correlation among mutans streptococci counts, dental caries and IgA to streptococcus mutans in saliva. Braz Oral Res 2004;18:350-5.  Back to cited text no. 16
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17.Doifode D, Damle SG. Comparison of Salivary IgA levels in caries free and caries active children. Int J Clin Dent Sci 2011;2:10-4.  Back to cited text no. 17
    
18.Kugler J, Breitfeld I, Tewes U, Schedlowski M. Excavation of caries lesions induce transient decrease of total salivary immunoglobulin A concentration. Eur J Oral Sci 1996;104:17-20.  Back to cited text no. 18
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19.Axelsson P, Kristoffersson K, Karlsson R, Bratthall D. Six month longitudinal study of the effects of some oral hygiene measures on streptococcus mutans and approximal dental caries. J Dent Res 1987;66:761-5.  Back to cited text no. 19
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Correspondence Address:
P R Geetha Priya
Department of Pedodontics and Preventive Dentistry, K. S. R. Institute of Dental Science and Research, Thiruchengode
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0970-9290.118004

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