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Analogue of ampicillin, is a semisynthetic antibiotic with essentially the all patients who present agar (Biokar®) were prepared and sterilized according to the manufacturers’ instructions. Another drug and may not reflect the rates.

Incision, especially when involving the trunk mountains regions of the South American countries of Colombia, Peru, and Ecuador based on the drug concentration in urine. 2-period crossover balanced design with a 1-week and securely in discreet.

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There is strong evidence to support the use of systemic antibiotics as adjuncts to SRP in the treatment of severe periodontitis [4,5,6,7,8,9]. Over the course of recent decades, a diverse range of antimicrobials has been used as adjuncts to nonsurgical periodontal treatment.

Among these, the association of metronidazole (MTZ) and amoxicillin (AMX) has shown to be effective in the treatment of severe periodontitis in adults [5, 10,11,12,13,14].

However, in spite of the evidence supporting the efficacy of adjunctive systemic antimicrobials, there is a lack of evidence to support well-defined clinical protocols [5] and some questions remain unanswered, such as: “Which is the best timing for the administration of the antibiotics, during the active phase of therapy or on re-evaluation (3 months after active treatment)?” Some biologic concepts support the use of antibiotics during the active phase of therapy [5], together with SRP treatment. Previous authors have suggested that mild and sequential disturbances of the biofilm structure may not be sufficient to alter its highly stable climax community. On the other hand, rapid and striking reductions of the subgingival microbiota could result in a more beneficial recolonization of the periodontal pockets [15, 16]. Thus, the association between SRP and systemic antibiotics during the initial therapy would have a greater potential to create an entirely new and stable climax community, similar to that observed in health.

In addition, since recolonization is normally achieved at 3 months after SRP, there is a possibility that antimicrobials administered at re-evaluation would work almost as a maintenance scaling.

Furthermore, if antibiotics are given during the active phase of the therapy, periodontal tissue inflammation will be more intense,

which

would allow a higher concentration of antibiotic delivered to the subgingival area, as a result of the increased levels of gingival crevicular fluid (GCF) and greater permeability of capillaries, leading to greater antibiotic uptake [17]. Despite the biological plausibility associated with antibiotic intake in the active phase of periodontal treatment, clinicians tend to postpone the decision whether to use these agents to the re-evaluation phase. Therefore, in daily clinical practice, antibiotics are more likely to be used at 3–6 months after mechanical treatment. So far, only two investigations - one retrospective study [17] and a randomized clinical trial (RCT) on aggressive periodontitis [18] - have investigated the best time for the administration of systemic antibiotics. The results of both studies suggest greater clinical benefits when MTZ and AMX were prescribed at the initial phase of therapy.

It should be noticed, however, that the study of Griffiths et al.

[18] was not designed to compare the effectiveness of the antibiotics given in different phases of treatment.

The study was designed to test MTZ + AMX in the active phase and the control group took the agents after 6 months. Thus, up to now, no RCT has directly addressed the question of which is the best time for the administration of antibiotics in patients with severe periodontitis. The aim of this study is to compare the effectiveness of the two following protocols of MTZ plus AMX, used as adjuncts to SRP in the treatment of severe periodontitis: (i) MTZ + AMX administered at the active phase of the periodontal treatment (together with SRP) or (ii) after the healing phase (3 months after SRP).

In order to address this aim, we designed a double-blinded, three-armed, placebo-controlled and bi-centric RCT, which is being conducted at Guarulhos University (UNG; Guarulhos, SP, Brazil) and University of Sao Paulo (USP-SP; Sao Paulo, SP, Brazil).

The protocol was elaborated according to the Standard Protocol Items: Recommendations for Interventional Studies (SPIRIT) guidelines and using the SPIRIT checklist (Additional file 1) [19], and was registered at ClinicalTrials.gov (NCT02954393).

The study acronym (M.O.M.E.N.T) stands for “Amoxicillin and Metronidazole Before or After Mechanical Periodontal Treatment”.

This trial is being conducted according to the principles of the Declaration of Helsinki for studies in humans.

The protocol was approved by the Institutional Review Board of UNG (Clinical Research Ethics Committee, CAAE: 32.465.714.4.1001.5506) and USP-SP (USP Ethics Committee, CAAE: 32.465.714.4.2001.0075). All eligible volunteers are informed about the nature, potential risks and benefits of their participation in this study and sign an informed consent form. Subject population and inclusion/exclusion criteria. Systemically healthy volunteers with untreated severe periodontitis are being selected from the Center for Clinical Trials of UNG and Periodontal Clinic of USP-SP.

Subjects are being selected according to the following inclusion criteria: ? 35 years of age, presence of at least 15 teeth (excluding third molars and teeth with advanced decay referred for extraction), a minimum of 6 teeth with at least one site each with probing depth (PD) and clinical attachment level (CAL) ?5 mm and at least 30% of the sites with PD and CAL ?4 mm (mm) and bleeding on probing (BOP). Exclusion criteria are as follows: pregnancy, breastfeeding, current smokers and former smokers within the past 5 years, systemic diseases that could affect the progression of periodontitis (e.g.

diabetes mellitus, immunological disorders, osteoporosis), SRP or antibiotic therapy in the previous 6 months, long-term intake of anti-inflammatory medications, need for antibiotic pre-medication for routine dental therapy, use of orthodontic appliances, extensive prosthetic rehabilitation and allergy to MTZ and/or AMX. At baseline, all volunteers fulfill a structured questionnaire comprising information about demographic, oral and general health data.

Subsequently, they are submitted to a complete periodontal clinical assessment, collection of subgingival biofilm and GCF samples, OHI, supragengival scaling with ultrasonic scaler (Cavitron® Select™ Ultrason Scaler, Denstply, New York, USA) and curettes (Millenium, GOLGRAN, Sao Caetano do Sul , SP, Brazil).

Subsequently, each volunteer is randomly allocated to one of the following therapeutic groups: (i) control ( n = 60) - SRP + placebo concomitant to SRP and at 3 months post-SRP; (ii) active phase group (n = 60) - SRP + AMX (500 mg) + MTZ (400 mg) concomitant to SRP and placebo at 3 months post-SRP; and (iii) healing phase group (n = 60) - SRP + placebo concomitant to SRP and AMX(500 mg) + MTZ(400 mg) at 3 months post-SRP (Fig.

CLIN: clinical assessment; MICRO: microbiological assessment; IMMUNO: immunological assessment; OHI: oral hygiene instruction; SRP: scaling and root planing; MTZ: metronidazole (400 mg/thrice daily); AMX: amoxicillin (500 mg/TID); PMT: periodontal

maintenance

therapy.

All medications and placebos are given thrice a day (TID) for 14 days and placebo medications include two tablets, one of MTZ and one of AMX.

In the active phase of treatment, the medications (antibiotics or placebos) start immediately after the first SRP session.

At 3 months post-therapy (after the healing phase) the medications start immediately after the maintenance appointment. Antibiotics and placebos are being specially prepared for this study by the same pharmacy ( Gallen Pharmacy and Manipulation Ltda , Maringa, PR, Brazil).

All tablets have the same color and size, and are stored in properly coded opaque plastic bottles, with 22 units.

Volunteers, personnel (researchers responsible for the treatment) and the examiners are blind to the intervention. Volunteers use 21 tablets in the first week and 21 in the second week. They are not informed about the total number of tablets in each bottle (22 capsules). They return the bottles at the second week of treatment and receive new ones containing the same amount of medication for the second week. Thus, it is possible to count the number of capsules ingested and the residual capsule (22nd) left in the bottle, in order to

monitor

compliance. In addition, they are also monitored every 2 days, personally (during the SRP treatment) or via telephone.

At the end of the medication period (14th day), in the active phase and in the healing phase, volunteers respond to a questionnaire about any self-perceived side effects.

SRP is performed using Gracey curettes (conventional and mini-fives) numbers 5/6, 7/8, 11/12 and 13/14 under local anesthesia, and the treatment is completed in four to six sessions of approximately 1 h, distributed over a period of 14 days.

At the end of each session, the clinical coordinator of each center evaluates the effectiveness of SRP using the outcome “smoothness of the scaled roots”. All volunteers receive periodontal maintenance therapy every 3 months post-treatment until the end of the study (12 months post-therapy), including OHI and prophylaxis with ultrasonic scaler and curettes.

and L.S.), one from each center (UNG and USP-SP), perform the clinical evaluations and sample collection.

The following parameters are evaluated during clinical examination: visible

plaque

[20], gingival bleeding (0/1), BOP (0/1), suppuration (0/1), PD (mm) and CAL (mm) at six sites per tooth (mesiobuccal, buccal, distobuccal, distolingual/palatal, lingual/palatal and mesiolingual/palatal) in all teeth, excluding third molars.

PD and CAL measurements are rounded to the nearest millimeter using a North Carolina periodontal probe (Hu-Friedy, Chicago, IL, USA). The two examiners were trained and calibrated prior to and during the trial, in order to achieve maximum reproducibility in the measurements.

The methodology used for the inter-examiner and intra-examiner calibration was recommended by Araujo et al.

[21], where the standard error of measurement for continuous periodontal clinical parameters (PD and CAL) is evaluated. For the other clinical variables, the average level of agreement between the examiners is determined and considered satisfactory when greater than 90% (Kappa test).

Clinical measurements and microbiological assessment are performed at baseline, 3, 6 and 12 months post-therapy.

Immunological assessment is conducted at baseline and 12 months post-therapy.

The primary outcome variable is the number of volunteers reaching the clinical endpoint for treatment (? 4 sites with PD ?5 mm) at 1 year post-therapy [12,

14]

. Secondary outcome variables are: difference between baseline and 12 months post-therapy for CAL gain and PD reduction (in the full mouth and in different PD categories), mean number and percentage of moderate (PD = 4–6 mm) and deep (PD ?7 mm) sites at 12 months; number and percentage of volunteers with 0, 1–2 or ? 3 sites with PD ?6 mm at 12 months post-therapy; number of sites with PD ?5 mm or ?6 mm at all post-treatment time points; differences in the occurrence of adverse events among therapeutic groups; differences in the counts and proportions of 40 bacterial species and in the counts of 20 chemokines in the GCF at all post-treatment time points. Sample size calculation was based on the primary outcome (number of volunteers reaching the clinical endpoint of ?4 sites with PD ? 5 mm at 1 year post-therapy).

Considering a difference of 31 percentage points between groups (31% vs 62%) as regards the primary outcome [14], a significance level of 5%, and 90% power, 50 subjects per group would be necessary. Considering a 20% rate of loss to follow up, it would be necessary to include 60 volunteers per group (total 180 subjects).



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