This trial started in June 2014 and finished in August 2016. This triple-blind placebo-controlled parallel-arm randomized clinical trial was performed following guidelines recognized by the Declaration of Helsinki, as revised in 2013 for experimentation involving human participants. The study was approved by the Local Ethical Committee of the Faculty of Dentistry at the University of Chile (Decision number: 2012/ 08). The present report adheres to Consolidated Standards of Reporting Trials (CONSORT) guidelines and was registered in clinicaltrial.gov (identifier no. NCT02839408). The protocol of the study was explained to all patients, and informed consent was obtained after explanation of the purpose, nature, risks and benefits of participating in this study. The study details have been reported elsewhere .
Patient selection criteria
Ninety-six volunteers were initially examined in the Faculty of Dentistry, University of Chile, of which 47 were included in the present study. Eligibility criteria for participants were as follows: (a) Adult patients - aged 35 or older; (b) Self- reported ‘good health’, with an absence of a medical history of a chronic disease or taking medication known to affect periodontal disease; (c) Non- institutionalized male or female; (d) Presence of a minimum of 14 teeth (excluding third molars); (d) presence of at least 10 posterior teeth (e) Presence of at least 5 teeth with periodontal sites with PPD ≥5mm and clinical attachment loss (CAL) ≥3mm, BOP ≥20% and extensive radiographically determined bone loss.
The exclusion criteria were as follows: (a) Pregnancy or lactation; (b) Having received any periodontal treatment in the 6-month period before the study; (c) Having received non-steroidal anti-inflammatory medication, antibiotics or probiotics therapy in the past 6 months (prior to study).
Randomization of the study participants
Eligible individuals were randomly (simple randomization) allocated to groups according to gender, age, and smoking status after the basal examination using a computer-generated list. All participants were enrolled to one of the three groups: 1) placebo (SRP + azithromycin placebo + probiotic placebo), 2) probiotic (SRP + probiotic + azithromycin placebo) or antibiotic (SRP + azithromycin + probiotic placebo) group (Figure 1). Concealed allocation was performed using opaque, sealed envelopes containing probiotics or antibiotics arranged by an appointed research assistant. For the purpose of maintaining full blinding throughout the study period, the randomization code and details of the study groups were held by a research assistant only from the day of recruitment and were not revealed until all data had been collected and analyzed.
Periodontal clinical parameters including probing pocket depth (PPD), bleeding on probing (BOP), clinical attachment loss (CAL) and plaque index (PI) were evaluated at six sites in all teeth, excluding third molars using a manual probe (UNC probe; Hu-Friedy Mfg. Co. Inc., Chicago IL, U.S.A). All measurements were conducted by one calibrated examiner (AM). Clinical examinations were performed at baseline, 3, 6, 9 and 12 months after treatment.
After baseline examinations, all patients received non- surgical periodontal therapy (NSPT). Scaling and root planing (SRP) per quadrant (4-6 sessions, by PC, RC and NS) were performed using an ultrasonic scaler (Cavitron, Dentsply, York, PA, U.S.A) and Gracey curettes (Hu Friedy Mfg. Co. Inc., Chicago, IL, U.S.A). Treatment included oral hygiene instructions, using a manual toothbrush. The patients started taking the placebo, probiotic or antibiotics after the last session of SRP. The Placebo group received probiotic placebo sachets and antibiotic placebo capsules. Probiotic group received probiotic sachets and antibiotic placebo capsules. Antibiotic group received antibiotic capsules and probiotic placebo sachets. Identical sachets were presented to patients. Individuals were instructed to dissolve 1 sachet (Lactobacillus rhamnosus SP1 (2x107 colony forming units/day) (Macrofood S.A., Santiago, Chile ) or probiotic placebo sachets in 150 ml of water and ingest it once a day after brushing their teeth, for 3 months. Azithromycin 500 mg (capsules) or antibiotic placebo capsules were ingested once a day for 5 days. Placebo sachets (probiotic placebo) and placebo capsules (antibiotic placebo) were identical in taste, texture, and appearance to the probiotic sachets and antibiotic capsules. Periodontal supportive therapy was performed every 3 months (by PC). The therapy provided at the maintenance appointment included removal of plaque and calculus, utilizing curets, ultrasonic devices, and rubber cup low‐speed polish as suggested by the American Academy of Periodontology position paper . Behavior modification (tobacco cessation, oral hygiene instruction, and systemic factor counseling) was performed based on patient findings.
Compliance and adverse reactions
All patients returned capsules with antibiotics or placebo at 6-week visits and probiotics or placebo sachets at 1, 2 and 3-month visits. At every visit, patients received new sachets. In order to check the patient´s compliance, they were called by phone every week. In each control visit or phone call, the clinical examiner (AM) inquired after general health changes, use of mouth rinses, use of probiotic products and any adverse events.
The primary outcome was the change in CAL. Secondary outcome variables were changes in PPD, PI and BOP, percentages of patients, teeth and sites with PPD ³5mm, ³6mm, ³7mm. Sub-analyses were performed on CAL and PPD, taking into account the initial PPD. A pocket was considered moderate if its initial PPD was between 4-6mm and deep if ³7mm. Changes or delta (∆) in clinical parameters (at subject level) from baseline to 3, 6, 9 and 12 months were determined. “Pocket” closure was defined as mean and SD of percentage of sites going from PPD≥4 mm to PPD≤3mm at 3- and 12-months follow up . “Risk for disease progression” was defined at the patient level according to Lang and Tonetti . Low risk was defined as ≤4 sites with PPD ≥5mm, moderate risk was defined as 5-8 sites with PD ≥5 mm, and high risk was defined as ≥9 sites with PD ≥5 mm . The “need for additional periodontal treatment” was defined as persisting pockets ≥5 mm with BOP .
Sample size calculation was performed using CAL as the primary outcome variable. A significance level of a = 5% a and a power level of 80% was defined. Considering a difference ≥1mm between groups in CAL changes and a standard deviation of 0.8 mm , 14 participants per group were necessary to detect potential differences. For all statistical evaluations, the patient was the unit of measurement. The Shapiro–Wilk test was used to test the normality of the data sets. Quantitative data were recorded as the mean value ± standard deviation (SD) or percentage (%). The inter-group differences were determined using Fisher’s exact test, Kruskall Wallis test and ANOVA depending on the distribution of the data. Intra-group differences in clinical parameters over time were determined by the Related Samples Friedman's test (p<0.05) and. The Bonferroni-corrected Wilcoxon signed-rank test and Bonferroni-corrected Mc Nemar test were used to evaluate the intragroup multiple comparisons (p< 0.0125 or p<0.025). Effect Sizes (‘mean change’ divided by ‘standard deviation of the baseline values’) were calculated based on changes in clinical parameters from baseline to 3, 6, 9 and 12 months or from baseline to 3 and 12 months follow-up.The statistical analysis was performed using a statistical package (StataCorp, College Station, TX, USA).