Evaluation and association of periodontal status with levels of Porphyromonas gingivalis in chronic periodontitis with and without Type 2 diabetes mellitus following nonsurgical periodontal therapy using quantitative polymerase chain reaction: An interventional study.

BACKGROUND: The aim of the present study was to detect and correlate the levels of Porphyromonas gingivalis with clinical parameters after nonsurgical periodontal therapy (NSPT) in chronic periodontitis patients with or without Type 2 diabetes mellitus (T2DM), using quantitative polymerase chain reaction (Q-PCR) method.
MATERIALS AND METHODS: Sixty patients equally divided into three groups, i.e., periodontally healthy (Group I), chronic periodontitis (CP) (Group II), and CP with T2DM patients (Group III) were assessed through clinical parameters of probing pocket depth (PPD) and clinical attachment level (CAL) and were correlated for the presence of P. gingivalis in the respective groups. PPD, CAL, and saliva samples for microbiological evaluation were assessed at baseline, 1-, and 3-month post-NSPT.
RESULTS: Significant reduction of PPD was found 1.26 ± 0.22 versus 0.43 ± 0.33 mm in Group I, 4.62 ± 0.78 versus 2.58 ± 0.60 mm in Group II, and 6.28 ± 1.52 versus 4.01 ± 1.38 mm in Group III post-NSPT at 3 months. Similarly, a notable reduction of CAL was exhibited in both Group II (5.28 ± 0.80 vs. 3.12 ± 0.77 mm) and Group III (7.14 ± 1.59 vs. 4.51 ± 1.38 mm) patients after NSPT at 3 months. A greater reduction of P. gingivalis concentrations was observed in both Group II and Group III at 3-month post-NSPT.
CONCLUSION: The substantial improvement of clinical parameters was found to be in correlation with the load of P. gingivalis, which was reduced more in Group II than in Group III, emphasizing the applicability and sensitivity of Q-PCR method for its assessment. Copyright:

INTRODUCTION

As supported by the existing literature, nonsurgical periodontal therapy (NSPT) had been proved to be effective in reducing inflammation.[12] Traditional methods incorporating clinical and radiographic aids provide information about the existing periodontal diseases; however, prediction of early diagnosis or chances of progression of periodontal disease in lag phase is limited.[3] Therefore, a confirmatory method such as identification of biomarker using microbiological techniques for qualitative and quantitative diagnosis and risk assessment is required for successful management of periodontal diseases. Chronic periodontitis (CP) is a multifactorial disease which majoritively includes Gram-negative putative pathogens in dental biofilm.[4] A potential mechanistic link between Type 2 diabetes mellitus (T2DM) and CP involves the broad axis of inflammation.[5] NSPT was performed to evaluate the reduction in inflammatory status with an interval of 3 months in our previous study.[6] Although significantly positive results were obtained when evaluated clinically to know whether eradication of causative microbes and complete remission of disease could be achieved after 3 months of NSPT, led us to generate a further protocol involving microbiological analysis.

A comprehensive analysis of the oral microbiome in diabetic patients is required to better understand how the microbiota relates to both diabetes and periodontal disease status, which also is useful for diagnosis and risk assessment.[7] The red complex of periopathogens, including Tannerella forsythus, Treponema denticola, and Porphyromonas gingivalis (Socransky et al. 1998) particularly have shown a strong association with CP.[8] While at the disease-active site, the strongest evidences have been associated with the presence of a greater frequency of P. gingivalis.[9]

P. gingivalis is a black-pigmented anaerobic rod, rarely detected in periodontally healthy sites.[10] Thus, monitoring of microflora by qualitative and quantitative methods has been conducted by culture methods or immunological methods to define the specific pathogen involved.[11] However, none of these methods have been proven satisfactory in terms of reliability, sensitivity, and specificity and evaluating complete elimination of a disease.[12]

To establish causality, amplification of nucleotide sequences in hypervariable regions of 16s ribosomal nucleic acids, specially possessed by each bacterial species should be analyzed. The evidence should include fewer or no copies of RNAs in the inactive state of a disease.[13] Polymerase chain reaction-based assay with specific primers is considered to be a useful tool for precise quantification of individual species and detecting etiology of CP.[14] Many studies have been reported to detect microbial concentrations using subgingival plaque but very few have used saliva as a sample.[15] The number of studies incorporating quantitative polymerase chain reaction (Q-PCR) to investigate salivary bacteria are limited too.[16] While taking PCR into consideration, a comprehensive sampling and primer-based Q-PCR assay are sensitive than culture techniques, and it maximizes the likelihood of detection of P. gingivalis.[17] In case of detectable salivary pathogen even after single visit of NSPT, further therapy or changes in treatment plan is indicated until its complete eradication from the samples is achieved. Thus, the study was carried out to assess levels of P. gingivalis in saliva and correlate it with the clinical parameters following NSPT among periodontally healthy, CP with and without T2DM patients using Q-PCR method.

MATERIALS AND METHODS

The study population comprised sixty patients of Caucasian population were equally divided into three groups; within the age range of 35–65 years visiting the outpatient department of periodontics and implantology in our institute. The study design was in accordance with the Helsinki Declaration and was approved by the institutional ethics committee (IEC/VSPMDCRC/20/2015), which was completed within a stipulated time period of 24 months. Before the initiation of the study, an informed consent was obtained from the patients who agreed to participate voluntarily. A sample size of twenty per group was needed to achieve the stringent effect size of salivary P. gingivalis count in the proposed study, with 90% power and 95% confidence level. Since it is a follow-up study, the sample size was considered with reference to the previous study.[6] A dental and medical history was recorded for the selected patients, and an intraoral examination was conducted by a single examiner (RK). The intrarater reliability in the measurements of periodontal parameters was evaluated before treatment in the study groups. Intraclass correlation (ICC) coefficient with two-way mixed effects model was obtained for each periodontal parameter. The ICC ranged between 0.92 and 0.99 in the groups (P < 0.0001), indicated excellent intrarater reliability.

The inclusion criteria were patients aged more than 35 years with the presence of at least twenty natural teeth with moderate-to-severe clinical attachment loss with >30% of sites involved.[18] The glycemic status of patients previously diagnosed with T2DM (having controlled T2DM since 5–10-year range) was confirmed by their glycated hemoglobin (HbA1c levels).[19] Pregnant women or lactating mothers, tobacco chewers and cigarette smokers, patients who had undergone any periodontal therapy in the past 6 months, those were on antibiotics, those had anti-inflammatory therapy, and those having any systemic diseases were excluded from the study. Patients were then categorized into following three groups:

Group I comprised twenty systemically and periodontally healthy individuals who presented teeth with periodontal pocket depth (PPD) ≤3 mm, clinical attachment loss (CAL) = 0, with no evidence of radiographic bone loss, and presenting HbA1c levels <6.5%.

Group II comprised twenty systemically healthy patients with generalized moderate-to-severe CP, having PPD of ≥5 mm and CAL ≥5 mm, presenting bleeding on probing (BOP),and radiographic evidence of bone loss with HbA1c levels <6.5%.

Group III comprised twenty generalized moderate-to-severe CP patients who were previously diagnosed with T2DM (as obtained from their medical history), with PPD of ≥5 mm and CAL ≥5 mm that were positive for BOP, presenting radiographic evidence of bone loss with HbA1c levels 6.5%–7%.

5 ml of nonstimulated saliva was collected in Eppendorf tubes by guiding patients to spit into the tubes. Samples were collected at baseline and after scaling and root planing (SRP) is done at the end of 1st and 3rd month. Collected samples were then stored at −80°C until assayed.

The analysis of levels of P. gingivalis by Q-PCR in saliva was done at baseline, post 1 and 3 months of NSPT. DNA extraction from saliva was done using the following protocol: 25 μl of blood was added to 500 μl phosphate-buffered solution and centrifuged at 10,000 rpm for 5 min (supernatant was discarded). To the remaining pellet, 0.5 M 50 μl NaoH was added and incubated at room temperature for 30 min. 50 μl of 1M Tris HCl was then added and vortexed to which 400 μl distilled water was added and stored at −20°C until further analysis.

Q-PCR for P. gingivalis was performed using following primers (5'-3').

Forward: 5 CATAGATATCACGAGGAACTCCGATT-3'.

Reverse: 5'AAACTGTTAGCAACTACCGATGTGG-3'.

The Q-PCR amplification was done in PCR Thermocycler machine with denaturation at 95°C for 5 min, annealing temperature of 60°C for 1 min, and polymerization temperature of 74°C for 2 min. Clinical parameters were recorded along with the detection of P. gingivalis in saliva samples at baseline, 1, and 3 months of NSPT.

Statistical analysis

The presence or absence of P. gingivalis in saliva samples was performed using Fisher's exact test and Z-test of proportion between independent samples, respectively. Anthropometric parameters such as random blood sugar (RBS) and HbA1c levels were compared across groups using one-way analysis of variance (ANOVA). The statistical comparison of parameters within groups before and after treatment was performed using paired t-test. The post hoc analysis was performed using Tukey's HSD test. Statistical significance was tested at 5% level, and all the analyses were performed using computer software (SPSS version 20.0, IBM Inc. Armonk, NY, USA).

RESULTS

In this interventional study, NSPT was carried out and its influence over clinical parameter and microbiological assessment of P. gingivalis load in saliva was conducted with the advanced and most sensitized Q-PCR technique. HbA1c was considered to be confirmatory test to validate the diabetic condition of the patient along with RBS estimation and was used to classify patients into different groups. To evaluate the inflammatory status among the groups, all the clinical parameters were assessed at baseline, 1, and 3 months post-NSPT.

Results thus indicated a significant reduction in the means of PPD and CAL after a comprehensive NSPT using one-way ANOVA [Table 1].

Table 1

Descriptive statistics for various parameters according to groups

Parameters	Group I (n=20)	Group II (n=20)	Group III (n=20)	P
PPD (mm)
Pretreatment	1.26±0.22a	4.62±0.78b	6.28±1.52c	<0.0001
Posttreatment	0.43±0.35a	2.58±0.60b	4.01±1.38c	<0.0001
CAL (mm)
Pretreatment	0a	5.28±0.80b	7.14±1.59c	<0.0001
Posttreatment	0a	3.12±0.77b	4.51±1.38c	<0.0001
HbA1C (%)	5.03±0.27a	5.14±0.41a	7.76±0.99b	<0.0001
Porphyromonas gingivalis
Pretreatment	-	19	20
Posttreatment 1 month	-	3	5
Posttreatment 3 months	-	1	3

P obtained using one-way ANOVA for continuous variables, statistically significant (P<0.0001). a,b,cSame alphabets in the superscript indicate statistically significant difference using Tukey’s post hoc test. PPD – Pocket probing depth; CAL – Clinical attachment level; HbA1C – Hemoglobin; n – Number of patients; P – Probability value

To evaluate the effect of NSPT on P. gingivalis, saliva samples from Group I, Group II, and Group III were analyzed to assess if complete elimination of P. gingivalis could be achieved at baseline, 1, and 3 months post-NSPT. None of the samples from Group I patients presented detectable amount of P. gingivalis at baseline. Among Group II, P. gingivalis was detected in 19 patients, which reduced to 3 after 1 month and only one patient was found to be positive for P. gingivalis detection, 3-month post-NSPT. In Group III, P. gingivalis was detected in all the twenty patients before SRP and the number of patients which retained P. gingivalis post 1- and 3-month recall were 5 and 3, respectively [Figure 1a-c]. Intragroup analysis showed a greater reduction of P. gingivalis in both Groups II and III, while no statistically significant reduction was observed between Group II and III at 3-month post-NSPT.

Figure 1

(a) Q-PCR for P. gingivalis, bp – base pair; L – LADDER; DNA marker of 1 Kb. PCR assay for the detection of P. gingivalis. Agarose gel showing the PCR amplification products of DNA obtained from strain of P. gingivalis (lanes 1–5) by using primers Pg 405. The representative image of Q-PCR assay of 5 samples from Group III patients showed the presence of P. gingivalis at baseline; (b) the representative image of Q-PCR assay of 5 samples from Group III patients of which 3 showed presence of P. gingivalis after 1 month of NSPT; (c) The representative image of Q-PCR assay of five samples from Group III patients, of which one sample showed the presence of P. gingivalis after 3 months of NSPT. DNA – Deoxyribonucleic acid; Q-PCR – Quantitative polymerase chain reaction; NSPT – Nonsurgical periodontal therapy; P. gingivalis – Porphyromonas gingivalis

The analysis of P. gingivalis levels by Q-PCR showed the following readings. According to densiometric analysis, the concentration ofDNA marker at the 450 bp position was 12 ng/μl and concentrations of P. gingivalis were 5 ng/μl pre-NSPT, 5 ng/μl post 1 month of NSPT, and 5 ng/μl post 3 months of NSPT with respect to saliva sample number 1. Similarly, for sample 2, concentration of DNA marker at the 450 bp position was 12 ng/μl and concentrations of P. gingivalis were 5 ng/μl pre-NSPT, 0 ng/μl post 1 month of NSPT, and 0 ng/μl post 3 months of NSPT. For sample 3, concentrations of P. gingivalis were 30 ng/μl pre-NSPT, 0 ng/μl post 1 month of NSPT, and 0 ng/μl post 3 months of NSPT. For sample 4, concentrations of P. gingivalis were 40 ng/μl pre-NSPT, 4 ng/μl post 1 month of NSPT, and 01 ng/μl post 3 months of NSPT. For sample 5, concentrations of P. gingivalis were 30 ng/μl pre-NSPT, 5 ng/μl post 1 month of NSPT, and 01 ng/μl post 3 months of NSPT [Figure 2].

Figure 2

Bar chart representing Porphyromonas gingivalis detection at baseline, post 1 month and post 3 months of nonsurgical periodontal therapy with respect to concentration of deoxyribonucleic acid marker. DNA - deoxyribonucleic acid; NSPTNon- surgical periodontal therapy

DISCUSSION

Pathogens causing periodontitis not only colonize subgingival and supragingival sites but adhere to mucosal cells too, thereby appearing in saliva.[2021] Considering saliva as one of the major sources of pathogens, we evaluated P. gingivalis in saliva samples of patients at baseline and after 1–3-month interval of NSPT by Q-PCR. Here, the influence of age and sex on clinical parameters was minimized by recruiting equal number of males and females in each group and confining age within a range of 35–65 years.

When prevalence and severity of periodontal disease in Type 2 DM patients were evaluated, it was found that the increased HbA1c value increased the severity of periodontal disease. On the other hand, the clinical improvements in parameters were found to correlate with the good glycemic control and the microbial burden, especially of the periodontopathic microbes. There was a significant improvement in clinical parameters such as PPD and CAL after SRP in both the groups, but the Group II showed better improvement as compared to Group III. Many studies have reported that DM patients have an inherent increased risk for initiation of inflammation and delayed tissue healing due to accumulated glycation end products.[2223] Hence, it was probable to find lesser improvement in clinical parameters after therapy in patients with DM as compared to absence of DM.[24]

A strong association of the putative pathogens, especially P. gingivalis, P. intermedia, and A. actinomycetemcomitans with that of the disease has been confirmed in the previous study.[25] The results of our study were in accordance with He et al.[26] and Saygun et al.,[16] where P. gingivalis count was significantly higher in CP patients than periodontally healthy participants. NSPT is capable of significantly reducing the number of these subgingival microorganisms and therefore considered as a cornerstone of periodontal therapy. McNabb et al. reported as a significant change in the composition of subgingival microflora including a decrease of P. gingivalis and spirochetes after a supragingival tooth cleaning.[27] Furthermore, Haffajee et al. found reduction in gingival redness along with the reduction of fourty bacterial species including A. actinomycetemcomitans, P. gingivalis, P. intermedia, and T. denticola and mean gain of clinical attachment level after NSPT.[28] Periodontal pathogen reduction along with a significant improvement in clinical parameters, thus necessitates microbial monitoring along with clinical monitoring to provide guidance for planning treatment strategies.

Scanning through the literature showed limited studies that have incorporated detection of salivary pathogens and their correlation with clinical parameters in CP patients. Advantages of utilizing saliva as a biomarker over plaque have been observed by Yamanaka et al. as the microbial diversity and richness were found to be reduced in plaque samples but not in saliva before as well as after surgical periodontal therapy.[29] Taking into account higher detection rates of P. gingivalis in saliva samples and possibilities of amplification of nonviable bacterial cells by PCR as compared to culture methods, Q-PCR method was used for analysis of saliva samples in our study.[3031] When the reduction in P. gingivalis count among Group II and Group III patients was compared, the difference was negligible. It has been emphasized in the literature that the patients presenting significantly a higher number of P. gingivalis (target organisms) are indicated as diseased, while mere presence of these organisms will not suffice. A threshold of 6 × 105 P. gingivalis cells was considered to be associated with a risk of new attachment loss.[8] In such a compromised periodontal status, these concealing P. gingivalis strain could serve as a potential pathogen carrier. Therefore, further treatment is indicated in cases with the presence of P. gingivalis even after 1 month of NSPT and continuing attachment loss.

In Type 2 DM patients with periodontitis, the pro-inflammatory cytokines enhance insulin resistance by inhibiting insulin signaling and thus inflammatory burden is increased as compared to nondiabetic condition. Hence, the chances of elimination in P. gingivalis after therapy are comparatively lesser in CP patients with T2DM than in patients of CP without T2DM. Thus, patients found positive for target pathogen; P. gingivalis even after 3 months of NSPT was considered for further continuation of therapy and should be kept under observation.

Although mechanical therapy effectively has been proved to reduce microbial load[3233] when evaluated using DNA probes, its complete elimination is difficult. Thus, if chairside diagnostic test is included to monitor changes in microbial levels, it would help in estimating type and duration of therapy provided, need of any adjunctive as well as prediction of outcome of a planned therapeutic strategy. Limitations were smaller sample size and analysis of P. gingivalis load alone. However, a larger sample size with longitudinal evaluation and complete microbial array of PCR would have given a proactive approach for diagnosis, prevention, and treatment planning of periodontal disease.

CONCLUSION

Although NSPT works in reducing inflammation, its efficiency can be a mere false positive response if results are produced relying over clinical parameters only. In contrast, PCR was found to be valuable for accurately detecting the presence or absence of causative pathogen before and after the therapy to counter check the diagnosis, prognosis, and treatment provided. In the present study, the microbial load of P. gingivalis at baseline and at various intervals post-NSPT was found to positively correlate with clinical parameters in diseased periodontal condition and on remission of the disease. Thus, within the limitations of the present study, it can be concluded that higher sensitivity and specificity of PCR play a potential role in diagnosis and effective management of CP with greater advantages over clinical assessment alone.

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Conflicts of interest

There are no conflicts of interest.