Study design
Case control study was performed on type 2 diabetics attending the Diabetes clinic of the Obafemi Awolowo University Teaching Hospitals’ Complex Ile-Ife, Osun State, Nigeria. Non diabetic patients attending the Dental Outpatient clinic of the hospital that met the inclusion criteria were recruited as controls. The study was conducted over a period of six months. From March 1st – August 31st 2018. Ethical approval was obtained from the Ethics and Research Committee of the Obafemi Awolowo University Teaching Hospitals Complex, Ile- Ife (IRB/IEC/0004553 and NHREC/27/02/2009a). Written informed consent was obtained from all participants during the study
Sample size estimation
The minimum sample size required for this study was estimated using the formula that compares two means. The parameters used for estimation were derived from a previous study. The mean value of IL-6 in the saliva of type 2 diabetes mellitus patients is 69.3 and 53 for healthy controls with a pooled standard deviation of 18.6. 6. With a power of 90% and alpha of 5%, a sample size of 67.7 was obtained. Seventy five participants were recruited, 36 type 2 diabetics and 39 apparently healthy controls.
Selection criteria
All type 2 diabetic consenting patients with a score of 5 or higher of the diabetes risk test at the time of diagnosis as proposed by the American Diabetes Association 2017 11 and aged 20 to 70 years were recruited. Diabetes risk test:
- How old are you? ……….
Less than 40 years (0 points)
40-49 years (1 point)
50-59 years (2 points)
60 years or older (3 points)
- Are you a man or woman?
Man (1 point) Woman (0 points)
- If you are woman, have you ever been diagnosed with gestational diabetes?
Yes 1(point) No (0 points)
- Do you have a mother, father, sister or brother with diabetes?
Yes 1(point) No (0 point)
- Have you ever been diagnosed with high blood pressure?
Yes 1(point) No (0 point)
- Are you physically active
Yes (1 point) No (0 points)
- What is your weight category?
Participants with other forms of diabetes mellitus besides type 2 with history of salivary gland surgery who were either pregnant or smokers or with any other known systemic diseases were excluded.
Consenting healthy controls with no known history of diabetes mellitus, other systemic diseases, who were not on any drug therapy, who were neither smokers nor pregnant and aged 20 to 70 years were also recruited.
Collection of samples
Before saliva and blood sample collection, participants were asked to rinse out their mouths with water to remove food debris, sit still in a comfortable position for six minutes and were not allowed to chew or speak until the saliva sample was collected. Unstimulated whole saliva was collected in the morning between the 8.00am and 11.00am from type 2 diabetics and healthy controls using the spit method every sixty seconds for 5 minutes. The saliva samples, stored in a sterile plain bottle were immediately taken to the laboratory in an ice pack at 4ºC and then frozen at or below – 20 ºC as immediately. Samples were defrosted at room temperature and then centrifuged at 6000 rpm for 10 minutes to remove contaminants such as oral epithelial cells, microorganisms and food debris. Venepuncture was done at the cubital fossa to collect blood sample from participants under sterile procedure using 5ml syringe and stored in EDTA bottle.
Assessment of periodontal status.
Basic periodontal examination (BPE) a simple and rapid screening tool used to indicate the level of examination needed and to provide basic guidance on treatment need was used to assess the periodontal status.12 A World health Organization (WHO) probe was used for this examination. This has ‘ball end’ 0.5mm in diameter and a black band from 3.5mm to 5.5mm. Light probing force was used (20-25g). The probe was walked round the teeth in each sextant. All sites were examined to ensure that the highest score was recorded before moving to the next. The participants were categorized using the scoring codes as:
Code 0: Given to the sextant if there were no pockets exceeding 3.5mm (black band entirely visible), no calculus or overhangs of fillings, no bleeding on gentle probing.
Code 1: Given to the sextant if there were no pocket exceeding 3.5mm (black band entirely visible), no calculus or overhang of fillings but bleeding on gentle probing.
Code 2: Given to the sextant if there were no pocket exceeding 3.5mm (black band entirely visible) but calculus and plaque retention factors are seen at or recognized underneath the gingival margin.
Code 3: Given to the sextant if the colour-coded area of the probe remains partially visible when inserted when inserted into the deepest pocket indicating pocket depth between 3.5-5.5mm
Code 4: Given to a sextant if at one or more teeth, the colour-coded are of the WHO probe disappears into the pocket indicating a pocket of 6mm or more.
Code *: Pocket+ recession (CAL) at least 7mm in total; Furcation involvement
Only the highest score was recorded for each sextant.
Estimation of salivary CRP: The salivary CRP levels was assessed using the ELISA test kit (Monobind Inc. Lake Forest, CA 52630 USA). Before proceeding with the assay, all reagents, serum references and control were brought to room temperature (20-27ºC).
Estimation of salivary IL-6: The saliva levels of IL-6 was assessed using the ELISA test kit (AVISCERA BIOSCIENCE INC, USA). All reagents and sample were brought to room temperature before use.
Estimation of saliva TNF-α: The levels of TNF-α in saliva was estimated using ELISA kit (AVISCERA BIOSCIENCE, INC, USA). All reagents and sample were brought to room temperature before use.
Estimation of serum glycated haemoglobin (HbA1C)
Commercial glycated haemoglobin kit (BIO QUANT DIAGNOSTICS) was used.
Statistical analysis
Data entry and analysis was done using STATA 14 statistical software (Statacorp, College station, Texas USA). Descriptive statistics, bivariate analysis such as paired t- test for IL-6 and Mann-Whitney U test were used as appropriate to compare the two groups. Spearman correlation coefficient was used to determine the relationship between salivary inflammatory biomarkers and serum levels of HbA1C in subjects with type 2 diabetes. Confidence level was set at 95% and p value of ≤ 0.05 was considered significant. Multivariate regression model was using the biomarkers CRP, IL-6 and TNF-α as outcomes and diabetic state, periodontal status and age as predictors. CRP and TNF-α were slightly skewed and log transformed, however the conclusion was not different without transformation and were fitted without transformation. Standard techniques were used for model checking.