Estimation and Correlation of Human Leukocyte Antigen-DR and Human Leukocyte Antigen-DQ in Children with and without Severe-early Childhood Caries: A Randomized Clinical Trial
ntroduction Early childhood caries (ECC) is a worldwide health concern affecting infants and preschoolers. It is the presence of one or more primary teeth that are decayed, missing, or filled in children younger than 6 years of age. Cariogenic microorganisms, poor eating habits, and societal factors all contribute to this complex illness. Streptococcus mutans breaks down carbohydrates, creating acids on teeth that are susceptible to caries over time, with bacteria like S. mutans and the immune system contributing to the cause. Research has indicated a robust correlation between caries susceptibility and major histocompatibility complex (MHC) or human leukocyte antigen (HLA) molecules. It is a group of genes structured within a DNA length, organized into two major regions: HLA class I and II. The immune defense system is activated when immune cells attach foreign peptides to class II molecules’ cell surfaces. Polymorphisms in the HLA molecules can affect an individual's vulnerability to ECC due to differences in the immune response against bacteria like S. mutans . Upon entering the body, extracellular organisms are processed by immune cells such as macrophages, attaching the processed antigen to HLA class II molecules. The immunological response is activated when this complex is delivered to the immune system's T-helper cells. Alterations in the immune response may lead to an increase in the incidence of caries. Recent studies indicate that HLA-DRB1*04 identification may serve as a biological marker for early caries detection in children. , Enzyme-linked immunosorbent assay (ELISA) is a vital analytical tool for quantifying biological molecules in complex samples, based on antigen-antibody binding in the immune system, and can be used as a qualitative or quantitative technique. The aim of the study is to estimate and correlate the association between HLA-DR and HLA-DQ levels in caries-active and caries-free children in the South Indian population using ELISA, as few such studies have been conducted in the Indian population. 

aterials and M ethods Source of Data Fifty children, with as well as without ECC, aged 3–6 years, participated in this study. Allocation was done into caries-active and caries-free groups, 25 in each, visiting the Pediatric and Preventive Dentistry Department, RajaRajeswari Dental College and Hospital, Bengaluru. Method of Collection of Data The participants were selected using a nonrandom sampling method. Computer-based allocation was used to randomize the data. The sample size for the present research was assessed using G*Power Software v. 3.1.9.7, considering the effect at a 5% alpha error (α = 0.05), with an effect size of 80 and the study's power at 80%, which demonstrated that a minimum of 50 subjects would be required for the present study. Thus, there will be 25 samples in each group (25 × 2 = 50). The research employed a 1:1 allocation ratio with a parallel design ( ). A research proforma was created, which included children's age, gender, place of residence, ethnicity, oral hygiene habits, and dietary habits (including how often they are given sweets before meals), as well as history of bottle feeding, chief complaint, and a record of all the subjective and objective symptoms and clinical findings. Following the acquisition of informed consent from the parents or guardians of children aged 3–6 years, 25 patients exhibiting severe ECC were assigned to the experimental group (caries-active), while 25 patients without caries were designated to the control group (caries-free). For the current study, institutional ethical clearance was acquired with the number RRDCH/IEC/2024/044. Sample Collection A clinical examination was performed for active carious lesions with the aid of a dental mirror and explorer. Following patient inquiries, the principal investigator assigned 25 patients to the caries-active group and 25 to the caries-free group, who were blinded to group allocation. Inclusion/exclusion criteria consisted of: children between 3 and 6 years, children having 4 ≤ active carious lesions [severe early childhood caries (S-ECC)], children without caries, and parents who had given consent for the study. The exclusion criteria consisted of: children who had topically applied fluoride during the last month, children needing special health care, teeth with pulpal involvement, children who had received antibiotics in the last 3 months, those with hereditary diseases or syndromes, and children currently on medications. Caries in children were diagnosed visually and documented using a modified World Health Organization (WHO) oral health survey protocol. The deft index (decayed, missing teeth due to caries, filled teeth) was used to measure caries, and groups were divided based on the presence of caries (deft ≥ 4) or absence of caries (deft = 0). Teeth included in the caries-active group were enrolled according to the ICDAS criteria 4 [underlying dark shadow from dentin (with cavitation)] to criteria 6 [extensive distinct cavity with visible dentin]. To reduce daily variation, unstimulated saliva was collected in the morning, 1 hour after tooth brushing and at least 2 hours after fasting. The subjects were instructed to sit properly with their heads tilted forward to allow saliva to accumulate in their mouths. They were then directed to spit into a sterile 2 mL saliva-collecting vial ( ). The samples were delivered to the lab within an hour of collection and stored in an ice-filled, hermetically sealed box at –80°C. After assigning a number to each sample, the procedure was repeated until the laboratory analysis was complete. The laboratory investigator was blinded to avoid bias. All 50 samples were subjected to ELISA to determine HLA-DR and HLA-DQ levels ( ). Collection of Saliva Supernatant Sterile tubes were used for the collection of saliva supernatant. After 20 minutes of centrifuging saliva at 2000–3000 rpm, the resulting supernatant was meticulously gathered for analysis. Assay Procedure for Estimation of HLA-DQ The assay involved preparing reagents, standard solutions, and samples according to instructions and allowing them to reach room temperature. Excess unused microplate strips were preserved at 2–8°C. In the standard well, 50 μL of the standard solution was added (biotinylated antibody already present), followed by 40 μL of saliva sample, 10 μL of biotinylated anti-HLADQ antibody, and 50 μL of streptavidin-HRP. The contents were well combined, covered with a plate sealer, and incubated at 37°C for 60 minutes. After removing the sealer, wash buffer was used for five washes. The plate was then blotted onto absorbent material, and 50 μL of substrate solution A and substrate solution B were added to each well. The plate was incubated in the dark at 37°C for 10 minutes. As soon as 50 μL of Stop Solution was applied to each well, the blue color changed to yellow. A microplate reader set at 450 nm was used to measure each well's optical density (OD value) in 10 minutes ( ). Assay Procedure for Estimation of HLA-DR The assay involved preparing reagents, standard solutions, and samples according to instructions. Excess microplate strips were removed, stored at 4°C, and 50 μL of each saliva sample followed by 50 μL of the antibody cocktail was added to the appropriate wells. The plate was incubated for an hour at room temperature, washed three times with 350 μL of 1X Wash Buffer PT, and inverted to remove excess liquid. 100 μL of tetramethylbenzidine (TMB) development solution was added to every well, incubated for 10 minutes in the dark, and 100 μL of stop solution was added. The plate was shaken for 1 minute to mix. The OD was measured at 450 nm, considered an endpoint reading. Instead of recording the endpoint reading at 450 nm, the kinetic development of the TMB substrate can be recorded. The blue color development with elapsed time can be recorded in the microplate reader at a 600 nm wavelength ( ). Statistical Analysis Statistical Package for the Social Sciences (SPSS) for Windows, Version 22.0, released in 2013 (Armonk, NY: IBM Corp.), was used to perform statistical analyses. Descriptive analysis of all the explanatory and outcome parameters was done using frequency and proportions for categorical variables, and mean and standard deviation (SD) for continuous variables. Independent Student's t -test was used to compare the mean age, HLA-DR and DQ isotypes between two groups, also based on age-group and gender in the control and study groups, and based on caries scores in the study group. Pearson's correlation test was used to estimate the association between caries scores and HLA-DR and DQ isotypes in the study group, and linear regression was used to predict the HLA score of a child as the dependent variable and caries score as the independent variable. The level of significance was set at p < 0.05. 

Fifty children, with as well as without ECC, aged 3–6 years, participated in this study. Allocation was done into caries-active and caries-free groups, 25 in each, visiting the Pediatric and Preventive Dentistry Department, RajaRajeswari Dental College and Hospital, Bengaluru. 

The participants were selected using a nonrandom sampling method. Computer-based allocation was used to randomize the data. The sample size for the present research was assessed using G*Power Software v. 3.1.9.7, considering the effect at a 5% alpha error (α = 0.05), with an effect size of 80 and the study's power at 80%, which demonstrated that a minimum of 50 subjects would be required for the present study. Thus, there will be 25 samples in each group (25 × 2 = 50). The research employed a 1:1 allocation ratio with a parallel design ( ). A research proforma was created, which included children's age, gender, place of residence, ethnicity, oral hygiene habits, and dietary habits (including how often they are given sweets before meals), as well as history of bottle feeding, chief complaint, and a record of all the subjective and objective symptoms and clinical findings. Following the acquisition of informed consent from the parents or guardians of children aged 3–6 years, 25 patients exhibiting severe ECC were assigned to the experimental group (caries-active), while 25 patients without caries were designated to the control group (caries-free). For the current study, institutional ethical clearance was acquired with the number RRDCH/IEC/2024/044. 

A clinical examination was performed for active carious lesions with the aid of a dental mirror and explorer. Following patient inquiries, the principal investigator assigned 25 patients to the caries-active group and 25 to the caries-free group, who were blinded to group allocation. Inclusion/exclusion criteria consisted of: children between 3 and 6 years, children having 4 ≤ active carious lesions [severe early childhood caries (S-ECC)], children without caries, and parents who had given consent for the study. The exclusion criteria consisted of: children who had topically applied fluoride during the last month, children needing special health care, teeth with pulpal involvement, children who had received antibiotics in the last 3 months, those with hereditary diseases or syndromes, and children currently on medications. Caries in children were diagnosed visually and documented using a modified World Health Organization (WHO) oral health survey protocol. The deft index (decayed, missing teeth due to caries, filled teeth) was used to measure caries, and groups were divided based on the presence of caries (deft ≥ 4) or absence of caries (deft = 0). Teeth included in the caries-active group were enrolled according to the ICDAS criteria 4 [underlying dark shadow from dentin (with cavitation)] to criteria 6 [extensive distinct cavity with visible dentin]. To reduce daily variation, unstimulated saliva was collected in the morning, 1 hour after tooth brushing and at least 2 hours after fasting. The subjects were instructed to sit properly with their heads tilted forward to allow saliva to accumulate in their mouths. They were then directed to spit into a sterile 2 mL saliva-collecting vial ( ). The samples were delivered to the lab within an hour of collection and stored in an ice-filled, hermetically sealed box at –80°C. After assigning a number to each sample, the procedure was repeated until the laboratory analysis was complete. The laboratory investigator was blinded to avoid bias. All 50 samples were subjected to ELISA to determine HLA-DR and HLA-DQ levels ( ). Collection of Saliva Supernatant Sterile tubes were used for the collection of saliva supernatant. After 20 minutes of centrifuging saliva at 2000–3000 rpm, the resulting supernatant was meticulously gathered for analysis. Assay Procedure for Estimation of HLA-DQ The assay involved preparing reagents, standard solutions, and samples according to instructions and allowing them to reach room temperature. Excess unused microplate strips were preserved at 2–8°C. In the standard well, 50 μL of the standard solution was added (biotinylated antibody already present), followed by 40 μL of saliva sample, 10 μL of biotinylated anti-HLADQ antibody, and 50 μL of streptavidin-HRP. The contents were well combined, covered with a plate sealer, and incubated at 37°C for 60 minutes. After removing the sealer, wash buffer was used for five washes. The plate was then blotted onto absorbent material, and 50 μL of substrate solution A and substrate solution B were added to each well. The plate was incubated in the dark at 37°C for 10 minutes. As soon as 50 μL of Stop Solution was applied to each well, the blue color changed to yellow. A microplate reader set at 450 nm was used to measure each well's optical density (OD value) in 10 minutes ( ). Assay Procedure for Estimation of HLA-DR The assay involved preparing reagents, standard solutions, and samples according to instructions. Excess microplate strips were removed, stored at 4°C, and 50 μL of each saliva sample followed by 50 μL of the antibody cocktail was added to the appropriate wells. The plate was incubated for an hour at room temperature, washed three times with 350 μL of 1X Wash Buffer PT, and inverted to remove excess liquid. 100 μL of tetramethylbenzidine (TMB) development solution was added to every well, incubated for 10 minutes in the dark, and 100 μL of stop solution was added. The plate was shaken for 1 minute to mix. The OD was measured at 450 nm, considered an endpoint reading. Instead of recording the endpoint reading at 450 nm, the kinetic development of the TMB substrate can be recorded. The blue color development with elapsed time can be recorded in the microplate reader at a 600 nm wavelength ( ). 

Sterile tubes were used for the collection of saliva supernatant. After 20 minutes of centrifuging saliva at 2000–3000 rpm, the resulting supernatant was meticulously gathered for analysis. 

The assay involved preparing reagents, standard solutions, and samples according to instructions and allowing them to reach room temperature. Excess unused microplate strips were preserved at 2–8°C. In the standard well, 50 μL of the standard solution was added (biotinylated antibody already present), followed by 40 μL of saliva sample, 10 μL of biotinylated anti-HLADQ antibody, and 50 μL of streptavidin-HRP. The contents were well combined, covered with a plate sealer, and incubated at 37°C for 60 minutes. After removing the sealer, wash buffer was used for five washes. The plate was then blotted onto absorbent material, and 50 μL of substrate solution A and substrate solution B were added to each well. The plate was incubated in the dark at 37°C for 10 minutes. As soon as 50 μL of Stop Solution was applied to each well, the blue color changed to yellow. A microplate reader set at 450 nm was used to measure each well's optical density (OD value) in 10 minutes ( ). 

The assay involved preparing reagents, standard solutions, and samples according to instructions. Excess microplate strips were removed, stored at 4°C, and 50 μL of each saliva sample followed by 50 μL of the antibody cocktail was added to the appropriate wells. The plate was incubated for an hour at room temperature, washed three times with 350 μL of 1X Wash Buffer PT, and inverted to remove excess liquid. 100 μL of tetramethylbenzidine (TMB) development solution was added to every well, incubated for 10 minutes in the dark, and 100 μL of stop solution was added. The plate was shaken for 1 minute to mix. The OD was measured at 450 nm, considered an endpoint reading. Instead of recording the endpoint reading at 450 nm, the kinetic development of the TMB substrate can be recorded. The blue color development with elapsed time can be recorded in the microplate reader at a 600 nm wavelength ( ). 

Statistical Package for the Social Sciences (SPSS) for Windows, Version 22.0, released in 2013 (Armonk, NY: IBM Corp.), was used to perform statistical analyses. Descriptive analysis of all the explanatory and outcome parameters was done using frequency and proportions for categorical variables, and mean and standard deviation (SD) for continuous variables. Independent Student's t -test was used to compare the mean age, HLA-DR and DQ isotypes between two groups, also based on age-group and gender in the control and study groups, and based on caries scores in the study group. Pearson's correlation test was used to estimate the association between caries scores and HLA-DR and DQ isotypes in the study group, and linear regression was used to predict the HLA score of a child as the dependent variable and caries score as the independent variable. The level of significance was set at p < 0.05. 

esults The study included high-risk participants with S-ECC, considering common confounding factors like history of bottle feeding, sugary intake frequency, snacking intervals, plaque amount, and enamel defects with cavitation when enrolling them. A comparison of HLA-DR levels in the caries-active and caries-free groups using an independent t -test was done. The HLA-DR isotype level was 33.032 ± 2.869 pg/mL in the caries-active group and 5.288 ± 0.960 pg/mL in the caries-free group, and was statistically significant at p < 0.001 ( ). In a comparison of HLA-DQ levels in the caries-active and caries-free groups, we found the HLA-DQ isotype level was 5.603 ± 1.264 ng/L in the caries-active group and 4.596 ± 0.748 ng/L in the caries-free group, and was statistically significant at p < 0.001 ( ). Caries scores and HLA-DR and HLA-DQ levels were compared using Pearson's correlation test. The HLA-DR levels showed a significant positive moderate correlation with caries score, with rho = 0.43. Therefore, as caries increase, HLA-DR levels also increase, and the result was statistically significant at p < 0.001, whereas the HLA-DQ levels had a significant positive very weak correlation with caries scores, with rho = 0.08, and were not statistically significant ( ). Linear regression was used to evaluate the predictability of the HLA level from the caries score ( ). Thus, HLA predictability can be determined from the equation derived from the above results as follows: HLA-DR = (0.426 × caries score) + 30.052 This shows that for every 1 score increase in deft, there is a significant increase in the HLA-DR isotype by 0.426 pg/mL, respectively. Age-wise comparison of HLA levels showed that both HLA-DR and HLA-DQ findings showed no statistically significant results ( and ). Comparison of HLA-DR levels with caries score in the caries-active group was 31.976 ± 2.996 pg/mL at caries score <6 and 34.376 ± 2.128 pg/mL at caries score >6, and was statistically significant at p < 0.001. This shows that the number of caries rises along with the HLA-DR levels. The HLA-DQ levels in the caries score showed not much difference and were not statistically significant ( ). Mean HLA-DR levels in the caries-active group were 33.066 ± 2.908 and 32.980 ± 2.965, whereas in the caries-free group, they were 5.634 ± 1.000 and 4.770 ± 0.636 in males and females, respectively, and were not statistically significant. Mean HLA-DQ levels in the caries-active group were 5.382 ± 1.249 and 5.934 ± 1.277 in males and females, respectively, and were statistically significant, whereas in the caries-free group, they were 5.634 ± 1.000 and 4.770 ± 0.636, which were not statistically significant ( and ). 

iscussion Looking at the most effective techniques currently in use to forecast the occurrence of dental caries is dependent on whether or not the patient has previously experienced dental decay. Because dental caries can be predicted before they occur, it is necessary to focus on preventive measures. Preventive measures are crucial, especially for children, as untreated dental caries can have severe public health and quality of life consequences. Against this backdrop, the study's objective was to investigate potential genetic associations with dental caries infection. Caries development is known to be influenced by several variables. Genetic variables, such as genetic alterations in the immune response, have been shown to play a substantial role in the etiology of caries, in addition to environmental factors (sugar intake, dental hygiene, etc.) and other aspects of the host's oral health. HLA class II molecules are crucial in the immunological reaction, as the antigen-presenting cells digest cariogenic bacteria upon their entry into the body and attach them to HLA class II molecules. Fifty healthy children between the ages of 3 and 6 years made up the study sample because ECC affects primary teeth in children under 6 years. The degree of caries severity was recorded according to ICDAS criteria and the deft index for primary teeth. It stands out for being straightforward to use, and it is the first standard worldwide to offer a consistent research methodology for determining the frequency and caries severity across various age-groups. The sample was split into two equal groups, with the control group having deft = 0 and the experimental group having deft ≥4, so that there was a noticeable difference between the two samples, which can produce more definite and clearer outcomes. The study found that saliva samples from caries-active individuals had higher levels of HLA-DR activity, with a statistically significant positive moderate correlation between the HLA-DR isotype and an increased risk of developing S-ECC. This aligns with a previous study that found DR4-positive subjects younger than 5 years had a tenfold increased risk of developing ECC compared to those who did not. Also, a median level of S. mutans in saliva showed higher levels for the DRB1*04-positive group. Other studies also indicate an association between dental caries and HLA-DR. , A study examining the association between HLA types and oral diseases showed increased HLA-DR in periodontitis patients. Our results contrast with previous studies in which it was concluded that HLA-DR either showed decreased frequency in the caries-active group, or the frequency of HLA alleles in individuals with high-caries and caries-free groups did not differ significantly. , , This study found that saliva samples from caries-active individuals had higher levels of HLA-DQ activity, with a slight positive, very weak correlation between HLA-DQ and caries development. Previously conducted studies have reported an increased HLA-DQ level in severe caries. Additionally, HLA-DQB1*0601 was linked to the lactobacilli count of S. mutans , indicating a possible association between HLA class II alleles and oral microorganism populations found in saliva. Contrastingly, certain studies observed either a decreased frequency or no association between any of the HLA-DQ alleles and caries; also, certain HLA-DQ alleles might serve as a barrier against dental caries. , , A study conducted on autistic adolescents showed that HLA-DQ2, -DQ6, and DR4 were more prevalent in the caries-free subgroup than in the high-caries subgroup. Because HLA-DR and HLA-DQ affect T-cell receptor recognition and interaction with HLA molecules, there is a positive correlation between HLA molecules and immune responses to microorganisms. Different HLA alleles may encode different molecules with different affinities for bacterial peptides. A theory suggests that HLA class II gene alleles could contribute to the build-up of S. mutans and lactobacilli in the mouth and affect dental caries vulnerability. , In our study, a comparison of HLA levels with caries scores in the caries-active group revealed a statistically significant association between HLA-DR and the number of caries. The results of this study were in line with previous studies where there was an increase in HLA-DR level with an increase in the caries score. , Contrastingly, a few studies showed either a decrease in the levels or no association between HLA levels and caries scores. , , , , A study found a positive correlation between S. mutans levels and DR3 and DR4 alleles, but no significant correlation was found between DRB1 or DQB1 alleles and the DMFS index. Human leukocyte antigens-DR and HLA-DQ levels were compared across age and gender groups, and only HLA-DQ levels in the caries-active group showed significant differences between both genders. Furthermore, there is scarce literature correlating the HLA alleles with different ages and genders. It must be borne in mind that findings tend to be influenced by the methods used, age-group differences (since the study was done on children), as well as race differences examined and the differences in the studied community. The current novel study provides a comparison and correlation of HLA isotypes with age, gender, and caries score in Bengaluru children, which comprises people from different ethnicities who have varying dietary habits that affect their oral health. Limitation The limitation of this study is that it did not examine the specific alleles seen in caries-active patients. Furthermore, future research will confirm these findings with a larger sample size. 

The limitation of this study is that it did not examine the specific alleles seen in caries-active patients. Furthermore, future research will confirm these findings with a larger sample size. 

onclusion In the present study, estimation and correlation of HLA-DR and HLA-DQ in the saliva of children with and without S-ECC using ELISA was done, and it was found that HLA-DR and HLA-DQ levels increased in the caries-active group, with the results being statistically significant. HLA-DR showed a moderate positive correlation, whereas HLA-DQ showed a very weak positive correlation in the S-ECC group. Additionally, HLA-DR was found to increase as the caries score increased in a patient. Comparison of salivary HLA isotypes with different age groups was not statistically significant. A comparison of salivary HLA-DQ levels showed a statistically significant difference between males and females in the caries-active group. Clinical Significance The study indicates a potential link between HLA alleles and caries susceptibility, suggesting that HLA-DR could aid in the early detection and prevention of dental caries, and the development of user-friendly devices could further enhance patient benefits. 

The study indicates a potential link between HLA alleles and caries susceptibility, suggesting that HLA-DR could aid in the early detection and prevention of dental caries, and the development of user-friendly devices could further enhance patient benefits. 

rcid Umapathy Thimmegowda https://orcid.org/0000-0003-2426-5057 Pradnya Dhamnekar https://orcid.org/0009-0008-4081-5593 

