The Periodontal Disease and Diabetes Mellitus Interrelationship Among Adult Malaysians

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.