Assessment of Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans in Down's syndrome subjects and systemically healthy subjects: A comparative clinical trial.

OBJECTIVES: To compare and quantify the presence of periodontal pathogens Aggregatibacter actinomycetemcomitans (Aac) and Porphyromonas gingivalis (Pg) in Down's syndrome (DS) and systemically healthy subjects with periodontitis and gingivitis.
MATERIALS AND METHODS: Fifty-nine age-matched subjects were categorized into four groups; Group I: DS subjects with gingivitis, Group II: DS subjects with periodontitis, Group III: Systemically healthy subjects with gingivitis and Group IV: Systemically healthy subjects with periodontitis. Plaque samples from all the four groups were collected and analyzed to evaluate the presence of Aac and Pg using real time polymerase chain reaction. The indices used were oral hygiene index simplified, community periodontal index for treatment needs and plaque index.
RESULTS: This study showed a statistically significant detection in the levels of both Aac (<0.001) and Pg (<0.001) in DS subjects with periodontitis when compared with the healthy controls with periodontitis; moreover, there was a statistically significant increase in Pg levels compared to Aac in DS gingivitis group.
CONCLUSION: There was a statistically significant presence of Pg and Aac in both DS gingivitis and DS periodontitis over control groups with increased levels of Pg compared with Aac in DS gingivitis group.

INTRODUCTION

Addressing the various needs and concerns faced by Down's syndrome (DS) subjects require the education of their caregivers by medical and dental professionals alike. Thus providing dental health care services for individuals with special health care needs continue to be an important aspect in these patients.[1]

Other than the periodontal disease DS individuals are characterized by[12] poor oral seal, poor tongue control, hypersalivation, cracked lips, angular cheilitis, fissured tongue,[34] submucous cleft and cleft palates,[5] reduction in salivary flow rate,[6] bruxism, midline deviation, developmental disturbances of the midfacial complex as well as the occlusal relationships.[7] DS individuals usually present with poor oral hygiene, which is manifested as marginal gingival inflammation, acute necrotizing gingivitis, suppuration or abscess formation and advanced chronic periodontitis including furcation involvement, increased tooth mobility, and loss of teeth.[8] DS individuals have a prevalence of 60-90% increased severity of periodontal disease compared with normal age-matched controls and subjects with other mental disabilities of similar age.[9]

Gingival inflammation is a characteristic feature seen in DS subjects where it persists predominantly in the lower dental arch and mixed dentition.[9] Severe periodontal inflammation is often seen in these individuals. Various periodontal bacteria colonize during early childhood of individuals with DS. Aggregatibacter actinomycetemcomitans (Aac), a key periodontal pathogen commonly found in aggressive periodontitis patients is often seen in individuals with DS.[1011] Porphyromonas gingivalis (Pg) also have a strong correlation to severe periodontal disease and have an increased prevalence with age, thus playing an important role in the onset and progression of periodontal disease in DS individuals.[10]

Agholme et al. in 1999 in his 7 years observational study found that the prevalence of bone loss increased from 35% to 74% among 33 DS individuals, however, the severity and progression of the disease was not as rapid as reported in the literature.[11] Though, the majority of DS children had poor oral hygiene the incidence of earlier onset periodontal disease could not be demonstrated in these subjects.[12] Simultaneously, there was only moderate relationship between dental plaque and periodontal disease severity in DS individuals[9] Thus, rapid and severe periodontal destruction in those DS individual affected could not be explained by poor oral hygiene alone.[11]

The current study hypothesis shows that, presence of elevated levels of Aac and Pg, predisposes Ds subjects to severe periodontal destruction. Hence, the study was designed to correlate the presence of Aac and Pg with the severity of periodontal disease in DS subjects when compared to systemically healthy controls with periodontal disease.

MATERIALS AND METHODS

Study subjects and setting

Twenty-nine DS subjects were considered as part of the experimental group and 30 age-matched systemically healthy individuals were included as controls in this study. DS subjects were recruited from Opportunity School, Vepery, Chennai, Tamil Nadu and DS Association of Tamil Nadu, Adyar, Chennai, Tamil Nadu, while systemically healthy age-matched controls with gingivitis and periodontitis were recruited from the patients attending the Department of Periodontology, S.R.M. Dental College, Ramapuram, Chennai. The institutional ethical committee approval was obtained for the study and informed consent was obtained from the heads of both the above-mentioned special schools and amongst the control subjects.

The DS and controls were categorized into four groups.

Group I (DS subjects with gingivitis): Plaque-associated gingivitis with positive bleeding on probing, sulcus depth ≤3 mm, no clinical loss of attachment, gingival index score ≥2.0

Group II (DS subjects with periodontitis): Patients diagnosed with chronic periodontitis with probing depth of ≥4 mm and clinical attachment loss (CAL) ≥3 mm

Group III (control subjects with gingivitis): Plaque-associated gingivitis with positive bleeding on probing, Sulcus depth was ≤3 mm, No clinical loss of attachment and gingival index score ≥2.0

Group IV (control subjects with periodontitis): Patients diagnosed with chronic periodontitis with probing depth ≥4 mm and CAL ≥3 mm.

The experimental group (Group I and Group II) included 29 DS subjects and the control group (Group III and Group IV) included 30 systemically healthy subjects. Of the 29 DS subjects, 11 subjects had periodontitis and 18 had gingivitis. While, of the 30 control subjects, 15 had periodontitis and 15 had gingivitis according to community periodontal index for treatment needs (CPITN) scores. Subjects with mental retardation, endocrinal disease, coronary heart disease, smokers, pan chewers and other systemic diseases were excluded from the study.

Plaque sample collection

Oral hygiene status was assessed using simplified oral hygiene index. The presence or absence of calculus, bleeding on probing, shallow and deep pockets were assessed using CPITN index.

Subgingival pooled plaque samples were collected from the mesial and buccal sites of teeth with the deepest pocket by means of a sterile paper point (no. 35, DiaDent, Almere, The Netherlands) as shown in Figure 1. Samples were placed in 0.1 ml ethanol (99.9% pure, M.W. 46.08). The samples were stored at −80°C and processed at the Central Research Facility, Sri Ramachandra Medical College and Research Institute, Porur, Chennai. The samples were then analyzed for quantification of Aac and Pg using real time polymerase chain reaction (RtPCR). The processing reagent, PCR reagents and Master Mix Kit were obtained from Applied Biosystems, Warrington, UK.

Figure 1

Plaque collections

Primer design and selection

Species-specific primers (Inqaba Biotech Industries (Pty) Ltd.) were used to detect the presence of the Aac and Pg [Table 1]. The expected product lengths were 404 bp for Pg and (593 bp) for Aac and Pg. A pair of ubiquitous primers product length (602 bp), which matched most bacterial 16S rRNA genes at the same position was used as a positive control for the PCR reaction.

Table 1

PCR primer sequence

Processing of samples-(mRNA isolation)

Collected plaque samples were stored in Eppendorf tubes containing ethanol solution (99%) at −80°C and centrifuged at 7000 rpm for 5 min. The supernatant was discarded. Mixture of 1 g of lysosome powder was mixed with Tris hydrochloride (1 ml), ethylenediaminetetraacetic acid 200 μl, triton 600 μl and sterile water Milli Q was added to make the solution up to 40 ml and then vortexed for 5 min. 0.8 μl of this solution was added to each sample and incubated for 30 min at 37°C. 20 ml of sodium dodecyl sulfate was added to the sample, vortexed again and incubated for 30 min at 37°C.

To this, equal volume of phenol chloroform isoamyl was added and centrifuged at 10,000 rpm for 10 min and the supernatant was extracted and added to new Eppendorf tubes. 1 ml chloroform isoamyl alcohol was added to the new tubes and centrifuged at 10000 rpm for 10 min. This supernatant was discarded and added to new Eppendorf tubes. A volume of 1 ml sodium acetate and 1 ml ethyl alcohol was added and centrifuged under 10,000 rpm for 10 min. The supernatant obtained was discarded and 500 μl of ethanol was added to the remaining of the sample and centrifuged at 10,000 rpm for 5 min. The supernatant was discarded and the pellets that remained at the bottom of the tubes were dried for 2 h. A volume of 30 μl of sterile water was added to it, which was then frozen and sent for PCR analysis.

Quantification of Porphyromonas Gingivalis, Aggregatibacter Actinomycetemcomitans using polymerase chain reaction analysis

Commercially available Sybr green master mix (applied biosystems sybr green master mix, Life technologies 5791 van allen way carlsbad clifornia 92008 United States of America) 5 μl was added along with 0.5 μl of forward primer and reverse primer each, 2.0 μl of template DNA and 2.0 μl of sterile water. 10 μl of the prepared mix was dropped in micro wells and subjected to PCR analysis, while amplification was done using values obtained from cycle threshold (Ct) equation.

Statistical analysis

Mean and standard deviation were estimated from the samples for each study group. Mean values were compared among different study groups by using independent t-test with one-way ANOVA Kruskal-Wallis followed by post-hoc (Tukey HSD) test. The statistical software SPSS version 17.0.0 (SPSS manufacturer IBM Corporation, 1 New Orchard Road, Armonk, New York 10504-1722, United States) was used for the analysis of the data. P < 0.05 is considered level of significance.

RESULTS

Demographic data of this study are represented in Table 2.

Table 2

Demographic data

Real time polymerase chain reaction amplification plot

The samples are subjected to RtPCR and the results showed increased level of Pg and Aac in DS subjects in gingivitis and periodontitis group compared to systemically healthy subjects [Graphs 1 and 2].

Graph 1

Polymerase chain reaction amplification plot for Aggregatibacter actinomycetemcomitans

Graph 2

Real time polymerase chain reaction amplification plot for Porphyromonas gingivalis

The results revealed a statistical significant increase in the level of Aac in both gingivitis and periodontitis group in DS subjects with the P = 0.008 and 0.001, respectively compared with the control gingivitis and periodontitis groups as shown in Table 2. Similarly a statistically significant level of Pg were found in both DS gingivitis and periodontitis with the P = 0.001 and 0.001 respectively when compared to control gingivitis and periodontitis groups as shown in Tables 3 and 4 and represented as bar diagrams [Figures 2 and 3]. The inter group comparison of Ct values of Aac and Pg by one way ANOVA revealed a statistical significance in the level of Aac in DS subjects when compared to healthy controls with the P = 0.001.

Table 3

Independent t test for Aac among DS and systemically healthy subjects group statistics

Table 4

Independent t test for Pg among DS and systemically healthy subjects group statistics

Figure 2

Comparison of Aggregatibacter actinomycetemcomitans in Down's syndrome and control subjects with gingivitis and periodontitis

Figure 3

Comparison of Porphyromonas gingivalis in Down's syndrome and control subjects with gingivitis and periodontitis

In this study, Pg levels were elevated in DS subjects with gingivitis and periodontitis compared with the healthy controls. However the levels of Pg in DS gingivitis group were most significant when compared to all other groups in the present study as shown in Tables 5 and 6. The post-hoc test for multiple comparisons of Aac among the four groups showed a statistically significant levels of Aac in DS gingivitis group when compared to control gingivitis and control periodontitis groups with P = 0.011 and 0.003 respectively, Similarly, the test revealed a statistically significant levels of Aac in DS periodontitis group when compared to control gingivitis and control periodontitis groups with the P = 0.047 and 0.017, respectively as shown in Table 7.

Table 5

Intergroup comparisons for the levels of Aac using ANOVA between DS and systemically healthy subjects

Table 6

Intergroup comparisons for the levels of Pg using ANOVA between DS and systemically healthy subjects

Table 7

Multiple comparisons using post-hoc test for the levels of Aac between DS and systemically healthy subjects with gingivitis and periodontitis

The post-hoc test for multiple comparisons of Pg among the four groups showed a statistically significant levels of Pg in DS gingivitis group when compared to control gingivitis and control periodontitis groups with P = 0.001 and 0.001 respectively, similarly the test revealed a statistically significant levels of Pg in DS periodontitis group when compared to control gingivitis and control periodontitis groups with the P = 0.001 and 0.001, respectively as shown in Table 8.

Table 8

Multiple comparisons using post-hoc test for the levels of Pg between DS and systemically subjects with gingivitis and periodontitis

DISCUSSION

Down's syndrome individuals usually present with poor oral hygiene which manifests as marginal gingival inflammation, acute necrotizing gingivitis, suppuration or abscess formation and advanced chronic periodontitis.[8] The severity of periodontal disease among DS individuals has improved in the recent years owing to better dental care and awareness. Sakellari et al.[13] evaluated the severity of periodontal disease in DS individuals and compared with healthy individuals or cerebral palsy patients and concluded that periodontal inflammation and treatment needs were significantly higher in DS individuals.[13] Oral health in individuals with DS were similar to that of early onset periodontitis and lower incisors reportedly exhibited early signs of alveolar bone loss in approximately 35% of DS adolescents.[14]

As the amount of plaque and calculus alone cannot explain the severity of periodontal disease in DS individuals, abnormal capillary morphology, disorders in connective tissue and anatomical aspects of teeth might also contribute to the pathology.[15] Alteration in immunological response may also play a role in the progression of the disease process in DS subjects.[16] Disorders in the polymorphonuclear leucocytes and monocytes function have been reported in these individuals. Other immune cell defects like decline in T-cell functioning after the first 10 years of life, decrease in T-cell counts and other immune defects have been reported increasing plasma levels of interleukin-6 and defective neutrophil migration.[17] However, the basis of these immune defects is still unclear in DS subjects. It has been proposed that when compared to DS children living at home, institutionalized DS children live in conditions of relative stress hence explaining the difference in severity of periodontal disease.[14]

In this study, the gingivitis group in DS subjects showed a statistically significant increase in the levels of both Aac (P < 0.008) and Pg (P < 0.001) when compared with healthy controls, however Pg was more significantly increased when compared to Aac. The results of this study is in accordance with Amano et al.[18] who stated that increase in Pg levels in DS gingivitis subjects played an important role in the initiation of gingival inflammation and plaque maturation.

In the periodontitis group, increased levels of Aac (P < 0.001) and Pg (P < 0.001) were seen in DS group when compared to healthy controls. The results were similar to the study done by Amano et al. and Agholme et al. The reason for the prevalence of these organisms in DS could be due to early colonization of these organisms in their childhood.[1018]

Multiple comparisons within the four groups using post-hoc test showed that the Ct levels of Aac and Pg were statistically significant in DS gingivitis and DS periodontitis groups. The results of this study correlated with the study done by Sakellari et al.[13] However Agholme et al.[10] and Amano et al.[1819] in their studies on DS subjects without controls had shown elevated levels of Aac and Pg respectively. Elevated levels of Pg in DS subjects resulting in early onset periodontitis could be due to the more susceptible host for the causative microbial agents including Pg with type II Fimbrea.[19]

In the present study both Pg and Aac were elevated in DS subjects with gingivitis and periodontitis compared to healthy controls, further there is a highly significant increase in the level of Pg than Aac in DS gingivitis group. The probable explanation for the above results can be due to the fact that institutionalized DS subjects are exposed to periodic oral prophylaxis programs limiting the progression of gingivitis to periodontitis. This statement is further validated by Yoshihara et al.,[20] where the author evaluated the effect of periodic preventive care on the progression of periodontal disease in young adults with DS and concluded that institutionalized DS subjects have less periodontal destruction and high prevalence of gingival inflammation with Pg being the predominant microflora.

This study is one of the first studies to evaluate and quantify specific periodontal pathogens in DS subjects among the Indian subpopulation comparing the results with healthy controls. The limitations of the current study could be that it is a single centered trial, with uneven distribution of samples in the DS and healthy control groups and nonevaluation of posttreatment changes in microflora.

Within the limits of the study, it can be concluded that the presence of Aac and Pg with a probable contributory compromised immune response exists in DS subjects, predisposing them to gingival inflammation and possible early onset periodontitis. Further studies evaluating multiple periodontal pathogens and assessing the immune cell functions could widen our understanding on the etiopathogenesis of periodontal diseases in DS subjects.

CONCLUSION

The present study showed a significant presence of potent periodontal pathogens Aac and Pg in DS subjects compared to healthy controls. Additionally Pg was more significantly detected than Aac in DS gingivitis group.