The Periodontal Disease and Diabetes Mellitus Interrelationship Among Adult Malaysians
Condition: Chronic Periodontitis; Type 2 Diabetes
MeSH terms: Periodontitis; Periodontal Diseases; Chronic Periodontitis
Study type: Interventional

Summary: 
      Periodontitis, a chronic inflammatory disease which results in irreversible attachment loss,
      bone destruction and tooth loss, is a major oral health problem affecting 90.2% of Malaysian
      population. It was initially demonstrated that Type 2 Diabetes (T2D) was a risk factor for
      periodontitis and subsequently a two-way relationship between diabetes and periodontitis was
      proposed. Diabetes has been shown to cause defects in neutrophil function by overproduction
      of pro-inflammatory mediators such as Tumour necrosis factor-α, Interleukin-1β and
      Prostaglandin E2 by macrophages. The inflammatory mediators released in response to plaque
      have been reported to be insulin antagonists that disturb binding of insulin to its receptors
      and further complicate hyperglycaemia in T2D. The hyperglycaemia in diabetics promotes more
      pathogenic bacteria into the subgingival microenvironment making them more susceptible to
      chronic periodontitis. Studies however differ in the types of periodontal pathogens present
      in these pockets. At the same time, very few studies have quantified them. This study
      proposes to investigate the effect that non-surgical periodontal therapy (NSPT) has on the
      periodontal parameters, HbA1c levels, microbiological profile and CRP levels of T2D patients
      with chronic periodontitis as compared to oral hygiene education (OHE)alone.
    

Detailed description: 
      Ethical approval was obtained from both the Medical Ethics Boards of University Malaya
      Medical Centre (MEC Ref No: 696.9) and University Malaya Dental Centre [DF PE1002/0045(P)].
      One hundred and twelve type 2 diabetic patients (diagnosed at least 1 year prior to the
      study) between ages of 30 to 70 were screened from the outpatient Diabetes Clinic of the
      University Malaya Medical Centre and a total of 40 patients who fulfilled the inclusion and
      exclusion criteria were recruited for the study. The recruited patients were then registered
      at the Periodontology Clinic at the Faculty of Dentistry, University of Malaya. Patient
      information sheets regarding the study and verbal explanations were given to all patients.
      Informed consent was obtained from all recruited patients with the understanding that they
      could withdraw from the study at any time. Screening and treatment of patients commenced from
      January 2010 till December 2011.

      The sample size calculation determined that 15 subjects per treatment arm would provide 80%
      power to detect a minimum difference of 1% (11.0 mmol/mol) change in HbA1c between test and
      control. Accordingly, a sample of 20 subjects per arm (40 in total) was recruited to
      compensate for possible drop-outs during the study period.

      All patients were randomly assigned via a coin toss to age matched NSPT and OHE groups.
      Investigator 1 screened and enrolled participants and assigned them to age-matched groups.
      Investigator 2 performed the random allocation sequence using coin toss method to assign
      participants to the different interventions while Investigator 1 performed all intervention.
      There was no blinding of participants or examiner.

      As only one examiner was involved in the study, intra-examiner reliability assessment was
      executed to validate the ability of the examiner to constantly reproduce the quantitative
      outcome measurements of the clinical parameters used. The Plaque Index (PI), Gingival
      Bleeding Index (GBI), Probing Pocket Depth (PPD) and Probing Attachment Loss (PAL) were
      measured in the time interval of about 3 hours. Utilizing Kappa statistics, good agreements
      (>0.8) were obtained for reproducibility of all recorded clinical parameters.

      All recruited patients underwent full periodontal assessment at baseline, 2 months after
      assigned treatment and 3 months after assigned treatment. The clinical examination included
      Plaque Index (PI), Gingival Bleeding Index (GBI), Probing Pocket Depth (PPD) and Probing
      Attachment Loss (PAL) measured with an electronic constant- force probe (Florida Probe®).

      All patients were instructed in oral hygiene methods using a soft bristled toothbrush, a
      compact-tuft toothbrush, inter-dental brushes and dental floss utilizing the modified Bass
      technique. Full mouth debridement, which consisted of scaling and root planing, was done in a
      single visit for all subjects in the NSPT group using an ultrasonic scaler and Gracey
      curettes. Additionally, all patients in the NSPT group were given a 0.12% Chlorhexidine
      mouthrinse (Hexipro®). They were instructed to rinse three times a day using 15ml each time
      for a period of 14 days commencing immediately after completion of full mouth debridement.
      Thereafter at each recall visit, all participants were re-motivated and professional
      prophylaxis was performed on those in the test group.

      15 ml of venous blood was collected from each patient at baseline, prior to treatment and at
      3 months after assigned treatments. Levels of glycosylated haemoglobin (HbA1c) and systemic
      hs-C-Reactive Protein (hs-CRP) were assessed. Hs-CRP levels were assessed using tests for
      high sensitivity CRP (hs-CRP). All blood investigations were done at a private laboratory
      (Gnosis Laboratories Sdn. Bhd) with no affiliation to the Department of Periodontology. The
      HbA1c testing is DCCT aligned and the Quality Assurance of the laboratory is certified under
      Bio-Rad Laboratories (USA), EQAS (External Quality Assurance Services).

      Deepest pockets were identified (minimum 5 pockets with PPD ≥ 5 mm) and isolated with cotton
      rolls. This was followed by careful removal of supragingival plaque using cotton pellets and
      curettes. Subgingival plaque samples were subsequently collected using sterile curettes and
      pooled into sterile DNAse-free and RNase-free polyethylene tube containing 1ml Phosphate
      Buffer Solution (PBS) and frozen at -800C until commencement of q-PCR analysis.

      Automated DNA extraction was performed in the lab using the QIAcube machine (Qiagen®). 100 µl
      of plaque sample was placed in a 1.5 ml centrifuge tube and was centrifuged at a speed of
      5,000 × g for 10 minutes on a tabletop centrifuge machine to obtain a pellet. The pellets
      were then placed on the QIAcube-shaker for automated DNA extraction by QIAcube machine using
      DNeasy® Blood & Tissue Mini Kit and QIAmp® DNA accessory set. All reagents provided in the
      kit were manually placed into the reagent bottle rack in the machine prior to the start of
      the process. Finally, the "Bacterial DNA" protocol was selected from the machine's protocol
      list. Briefly, the Qiacube machine underwent five consecutive steps which included lysis,
      incubate, bind, wash and elution processes. At the end of the process, 100µl of eluted DNA
      was available. The extracted DNA were then tested for concentration and purity using Nanodrop
      machine. The DNA was then stored in -80⁰C before bacterial identification using q-PCR
      machine.

      The concentration and purity of extracted DNA was determined using the spectrophotometer
      machine (Thermo Scientific NanoDrop™ 2000 Spectrophotometers) which was connected with a PC
      based software. 0.1 µl of eluted DNA was pipetted onto the lower pedestal arm. A fiber optic
      cable (the receiving fiber) is embedded within this pedestal. A second fiber optic cable (the
      source fiber) located in the upper pedestal arm is then brought into contact with the liquid
      sample causing the liquid to bridge the gap between the ends of the two fibers. A pulsed
      xenon flash lamp provides the light source and a spectrometer utilizing a linear CCD array
      analyzes the light passing through the sample. The DNA concentration from both plaque samples
      and reference strain were measured and recorded accordingly. The extracted DNAs were
      eventually stored in -20⁰C until the commencement of q-PCR procedure.

      The detection and quantification of P. gingivalis, A.actinomycetemcomitans, P.intermedia and
      T.forsythia from plaque samples were done using Applied Biosystems® 7500 Real-Time PCR
      Systems which was connected to PC based software. The quantification was done by projecting
      the cycle threshold (Ct) value of the samples on the standard curve of the known amount
      (concentration) of bacteria reference strain.

      For this purpose, a known amount of extracted DNA of the bacteria was serially diluted (10
      fold dilution) and was subjected to RT-PCR amplification. This was done to provide a standard
      curve prior to amplification of DNA from plaque samples using q-PCR. Optimization was carried
      out in order to get the optimum standard curve for quantification. A final 10 fold dilution
      standard curve of reference strains (range from 10ng/µl- 0.0001 ng/µl) with R2 value of 0.956
      was used as a base for quantification.

      To determine the sensitivity of the q-PCR technique, serial samples containing known
      concentrations of individual microorganisms were processed for q-PCR analysis. The lowest
      concentration that resulted in a positive PCR product was regarded as the sensitivity of the
      assay.

      q-PCR amplification was done in a total reaction mixture volume of 20µl. This mixture
      contained 2µl of purified DNA from plaque sample/ reference strain to act as PCR template, 10
      µl of 2x TaqMan® Fast Advanced Master Mix (PCR buffer, dNTP's, Amplitaq Gold, reference
      signal, uracil N-glycosylase, MgCl2; Applied Bio systems, Foster City, CA, USA), 1µl Custom
      Taqman® Gene Expression Assay (20x) and 7µl nuclease-free water. The procedures were
      conducted using 96-well reaction plates.

      All the components of the reaction mixture (except DNA sample) were added onto a 1.5mL micro
      centrifuge tube. The tube was capped and vortexed briefly. The tubes were then centrifuged to
      spin down the contents and eliminate air bubbles. The mixture was then transferred to a 96
      well reaction plate and followed by addition of 2µl DNA sample. The reaction plate was then
      covered with optical adhesive cover after the entire well had been occupied. This reaction
      plate was again briefly centrifuged to spin down the content and to ensure all bubbles were
      eliminated.

      This mixture was then subjected to an initial amplification cycle of 50ºC for 2 minutes and
      95ºC for 10 minutes, followed by 45 cycles at 95ºC for 15s and 60ºC for 1 minute. The q-PCR
      amplification plot for each sample was analyzed to determine the Ct value. The Ct value was
      then projected to the standard curve of reference strain for the quantification.

      Comparisons of changes in PI, GBI, PPD(%) and PAL (%) both within and between the groups were
      performed using the chi-square test. Intragroup comparison for mean PPD, mean PAL, mean
      HbA1c, mean CRP and frequency of detection of P. gingivalis, A.actinomycetemcomitans,
      P.intermedia and T.forsythia were assessed with the paired sample t-test whereas intergroup
      comparisons for the same variables was accomplished using an independent sample t-test.
    

Intervention name(s): Nonsurgical periodontal therapy; Oral hygiene instructions
Intervention type(s): Procedure; Procedure
Intervention description(s): Periodontal intervention All patients were instructed in oral hygiene methods using a soft bristled toothbrush, a compact-tuft toothbrush, interdental brushes and dental floss utilizing the modified Bass technique. Full mouth debridement, which consisted of scaling and root planing, was done in a single visit for all subjects using an ultrasonic scaler and gracey curettes. Additionally, all patients were given a 0.12% Chlorhexidine mouthrinse (Hexipro®). They were instructed to rinse three times a day using 15ml each time for a period of 14 days commencing immediately after completion of full mouth debridement. Thereafter at each recall visit, all participants were re-motivated and professional prophylaxis was performed.; All patients were instructed in oral hygiene methods using a soft bristled toothbrush, a compact-tuft toothbrush, interdental brushes and dental floss utilizing the modified Bass technique.
Allocation: Randomized
Intervention model: Parallel Assignment
Masking: None (Open Label)
Primary purpose: Treatment
Keywords: HbA1c; hs-CRP; non-surgical periodontal therapy; oral health education; probing pocket depth; plaque index; gingival bleeding index.