2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

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1 J Clin Periodontol 2014; 41: doi: /jcpe Metronidazole and amoxicillin as adjuncts to scaling and root planing for the treatment of type 2 diabetic subjects with periodontitis: 1-year outcomes of a randomized placebo-controlled clinical trial Tamires Szeremeske Miranda, Magda Feres, Paula Juliana Perez-Chaparro, Marcelo Faveri, Luciene Cristina Figueiredo, Neila Sumie Tamashiro, Marta Ferreira Bastos and Poliana Mendes Duarte Department of Periodontology, Dental Research Division, Guarulhos University, S~ao Paulo, Brazil Miranda TS, Feres M, Perez-Chaparro PJ, Faveri M, Figueiredo LC, Tamashiro NS, Bastos MF, Duarte PM. Metronidazole and amoxicillin as adjuncts to scaling and root planing for the treatment of type 2 diabetic subjects with periodontitis: 1- year outcomes of a randomized placebo-controlled clinical trial. J Clin Periodontol 2014; 41: doi: /jcpe Abstract Aim: To evaluate the clinical and microbiological effects of the use of metronidazole (MTZ) + amoxicillin (AMX) as adjuncts to scaling and root planing (SRP) for the treatment of chronic periodontitis (ChP) in type 2 diabetic subjects. Material and Methods: Fifty-eight type 2 diabetic subjects (n = 29/group) with generalized ChP were randomly assigned to receive SRP alone or with MTZ [400 mg/thrice a day (TID)]+AMX (500 mg/tid) for 14 days. Subgingival biofilm samples were analyzed by qpcr for the presence of seven periodontal pathogens. Subjects were monitored at baseline, 3, 6 and 12 months post-therapies. Results: The group receiving SRP+MTZ+AMX presented greater mean probing depth (PD) reduction and clinical attachment gain, a lower number of sites with PD 5 mm (primary outcome variable) and a reduced number of subjects with 9 of these residual pockets than the control group at 1-year post-therapy (p < 0.05). The antibiotic-treated group also presented reduced levels and greater decreases of the three red complex species, Eubacterium nodatum and Prevotella intermedia, compared to the control group at 1 year (p < 0.05). Conclusions: The adjunctive use of MTZ+AMX significantly improved the clinical and microbiological outcomes of SRP in the treatment of type 2 diabetic subjects with ChP. Key words: amoxicillin; chronic periodontitis; dental scaling; diabetes mellitus; metronidazole; real-time polymerase chain reaction; root planing Accepted for publication 9 June 2014 Conflict of interest and source of funding statement S~ao Paulo State Research Foundation (S~ao Paulo, S~ao Paulo, Brazil, # 2011/ ; 2013/ ). The authors declare that they have no conflict of interests. 890

2 Antibiotics for diabetic subjects 891 Diabetes mellitus (DM) is a widely prevalent disease associated with several major systemic and oral complications, such as periodontitis. It has been long demonstrated that DM is a risk factor for periodontal diseases, as diabetic subjects present higher prevalence and severity of periodontitis than non-diabetic subjects (Emrich et al. 1991, Novak et al. 2008, Jimenez et al. 2012,. In addition, periodontal infection/inflammation seems to aggravate hyperglycemia and impair an adequate glycemic control (Borgnakke et al. 2013, Chapple & Genco 2013), suggesting a bidirectional relationship between both diseases. The use of adjunctive local and/ or systemic antimicrobials has been proposed to improve the clinical and glycemic outcomes of the scaling and root planing (SRP) in diabetic subjects (Grossi et al. 1997, Promsudthi et al. 2005, Gilowski et al. 2012, Lin et al. 2012, Botero et al. 2013, Gaikwad et al. 2013, Santos et al. 2013). Doxycycline at antimicrobial and sub-antimicrobial doses has been long the most evaluated medication as adjunct to SRP for treatment of diabetic subjects (Grossi et al. 1997, Promsudthi et al. 2005, Gilowski et al. 2012, Gaikwad et al. 2013). Furthermore, Botero et al. (2013) recently suggested a trend towards better clinical and glycemic outcomes with the use of azithromycin as adjunct to SRP in diabetic subjects with moderate periodontitis. The combination of metronidazole (MTZ) and amoxicillin (AMX) has been largely recognized as an effective therapy for improving the clinical and microbiological outcomes of SRP in the treatment of non-smokers (Silva et al. 2011, Feres et al. 2012, Sgolastra et al. 2012, Zandbergen et al. 2013) and smokers (Matarazzo et al. 2008) with chronic periodontitis (ChP). However, no previous clinical trials to date have evaluated the effects of this antibiotic combination in the treatment of diabetic subjects with periodontitis. Therefore, the aim of this randomized clinical trial (RCT) was to evaluate the clinical and microbiological effects of the use of MTZ+AMX as adjuncts to SRP for the treatment of type 2 diabetic subjects with generalized ChP. Material and Methods Sample size calculation The ideal sample size to assure adequate power for this study was calculated considering a difference of at least four residual pockets with probing depth (PD) 5 mm between groups and assuming a standard deviation of five sites, as previously described (Feres et al. 2012). Based on these values, it was defined that 24 subjects per group would be enough to provide an 80% power with an a of Considering an attrition of about 15%, it was established that at least 29 subjects should be included in each treatment group. Subject population Subjects with type 2 DM and generalized ChP (Armitage 1999) were selected from the population referred to the Periodontal Clinic of Guarulhos University (Guarulhos, SP, Brazil). Detailed medical and dental records were obtained. Subjects who fulfilled the inclusion criteria were invited to participate in the study. All eligible subjects were informed of the nature, potential risks and benefits of their participation in the study and signed an informed consent form. The study protocol was approved by Guarulhos University Clinical Research Ethics Committee. The ClinicalTrials.gov identifier of the present study is NCT Inclusion and exclusion criteria Inclusion criteria were: 35 years of age, diagnosis of type 2 DM for 5 years, DM treatment with diet and insulin supplementation or oral hypoglycemic agents, glycated hemoglobin (HbA1c) levels 6.5% 11%, at least 15 teeth (excluding third molars and teeth with advanced decay indicated for extraction), more than 30% of the sites with PD and clinical attachment level (CAL) 4 mm and a minimum of six teeth with at least one site with PD and CAL 5 mm and bleeding on probing (BoP) at baseline. Exclusion criteria were: pregnancy, lactation, current smoking and smoking within the past 5 years, SRP in the previous 12 months, antimicrobial therapies during the previous 6 months, medical conditions requiring prophylactic antibiotic coverage, continuous use of mouthrinses containing antimicrobials in the preceding 3 months, systemic conditions (except DM) that could affect the progression of periodontitis (e.g. immunological disorders, osteoporosis) and long-term administration of anti-inflammatory and immunosuppressive medications. Subjects presenting allergy to MTZ and/or AMX, periapical pathology, orthodontic appliances, extensive prosthetic rehabilitation and major complications of DM (i.e. cardiovascular and peripheral vascular diseases [ulcers, gangrene and amputation], neuropathy and nephropathy) were also excluded. Experimental design, allocation concealment and treatment protocol In this prospective, double-blinded, parallel-design, placebo-controlled RCT, firstly each selected subject received a code number and one of the study coordinators (M.Fe.) used a computer-generated table to randomly allocate them into one of the following treatment groups: SRP + placebos (control; n = 29) or SRP + MTZ (400 mg thrice a day [TID] for 14 days) + AMX (500 mg TID for 14 days) (test; n = 29) (Feres et al. 2012). Subjects in the control group received the placebo pills for 14 days. The administration of antibiotics/placebos started immediately after the first session of SRP. As two different operators (N.S.T. and V.R.S.) performed the SRP, the randomization was stratified to allow each operator to treat half of the subjects in each treatment group. Initially, all subjects received supragingival plaque and calculus removal, extraction of teeth with advanced decay, provisional restoration and filling overhang removal, as necessary. They were also instructed regarding the brushing technique and received the same brand of toothpaste during the course of the study (Colgate Total â, Colgate Palmolive Co., S~ao Paulo, SP, Brazil). Two trained periodontists (N.S.T. and V.R.S.) performed SRP in four to six appointments lasting approximately 1 h each, using manual curettes (Hu-Friedy, Chicago, IL, USA) and ultrasonic device

3 892 Miranda et al. (Cavitron Select SPC, Dentsply professional, York, PA, USA) under local anesthesia. Periodontal therapy was completed in 14 days. The end point for each SRP appointment was the smoothness of the scaled roots, which was checked by one of the study coordinators (P.M.D). The same pharmacy (Proderma Farmacia de Manipulacß~ao Ltda, Piracicaba, SP, Brazil) prepared the antibiotics and placebos. Identical plastic bottles containing the antibiotics or placebos were sent to one of the study coordinators (P.M.D.), who marked the code number of each subject on each bottle, according to the therapy assigned. Allocation concealment was assured by means of sequentially numbered drug containers of identical appearance. Study personnel, including the examiner (T.S.M.), the two operators, the investigator responsible for the data analysis (M.Fa.) and the participants were blinded to treatment assignment. Code breaking was performed after final statistical analysis. Local antimicrobials were not used during the course of the study. Supportive therapy sessions were performed at 3, 6, and 9 months post-therapies. Compliance and adverse events monitoring An assistant monitored the compliance to antibiotics/placebos intake by calling the patients 3 times a week during the medication period. The assistant was neither examiner nor the therapist of the study. The subjects were asked to bring the bottles back at the end of each week, which were checked for any possible remaining pills of antibiotics/placebos. On the 14th day of the medication, subjects answered a questionnaire (administered by the assistant) about any self-perceived side effects. HbA1c (%) by high-performance liquid chromatography. The study examiner (T.M.S.) participated in a calibration exercise, and the standard error of measurement was calculated. Intra-examiner variability was 0.21 mm for PD and 0.24 mm for CAL. The agreement for categorical variables [e.g. BoP] was >85% (Kappa-light test). Visible plaque (presence/absence), marginal bleeding (presence/absence), BoP (presence/absence), suppuration (presence/absence), PD (mm) and CAL (mm) were assessed at six sites per tooth excluding third molars using the manual periodontal probe (North Carolina - Hu-Friedy, Chicago, IL, USA). Microbiological monitoring Microbiological monitoring was performed at baseline, 3 and 12 months post-therapies. Subgingival biofilm samples per patient were collected from six non-contiguous interproximal sites presenting no furcation involvement; two sites from each of the following baseline PD categories: shallow (PD 3 mm), moderate (PD = 4 6 mm) and deep (PD 7 mm). After supragingival plaque removal, the biofilm was collected using individual sterile mini-gracey curettes and placed in individual microtubes containing 0.15 ml of TE (10 mm Tris- HCl, 1 mm EDTA, ph 7.6). Quantitative Real-Time Polymerase Chain Reaction Test (qpcr) The samples were individually analyzed for the levels of seven bacterial species (Treponema denticola, Porphyromonas gingivalis, Tanerella forsythia, Eubacterium nodatum, Parvimona micra, Fusobacterium nucleatum ssp., and Prevotella intermedia) by qpcr, using the LightCycler 2.0 (Roche Diagnostics GmbH, Mannheim, Germany). Initially, the DNA was extracted from each biofilm sample using the MasterPure TM complete DNA and RNA purification kit (Epicentre, Madison, WI, USA). Amplification reactions were performed in a 10 ll final volume, containing 2.5 ll of the isolated DNA (20 ng/ll) and a reaction mixture containing the primer probe sets (2.5 lm each) and the FastStart DNA Master SYBR Green I (Roche Diagnostics GmbH). Absolute quantification of target species in each sample was performed using standard curves prepared with reference strains (Table S1). The determination of DNA content in controls was based on the genome size of each specie and the mean weight of one nucleotide pair (Dolezel et al. 2003). Based on standard curves, individual sample Ct scores were converted into the number of bacterial cells. The level of detection was set to 10 3 bacteria. PCR procedures were performed in Clinical and glycemic monitoring Subjects received clinical and glycemic monitoring at baseline, 3, 6, and 12 months post-therapies. The blood samples were examined at the Clinical Analysis Laboratory of Guarulhos University. The fasting plasma glucose (FPG, mg/dl) was measured by glucose oxidase method and the Fig. 1. Flow chart of the study design.

4 Antibiotics for diabetic subjects 893 a blinded fashion. The specific primers used for the detection of each bacterial species evaluated and the amplification profiles are described in Table S1. Primary and secondary outcome variables The primary outcome variable was the difference between groups for the mean number of sites with PD 5 mm post-treatment (Feres et al. 2012). Secondary outcome variables were differences between groups for: number of subjects with low, moderate or high risk for disease progression (Lang & Tonetti 2003), PD and CAL changes at moderate and deep sites, changes in number of sites with PD 5 and 6 mm, full-mouth PD and CAL, full-mouth percentages of sites with plaque, marginal bleeding, BoP, suppuration and PD 5 mm, levels of HbA1c and FPG, number of subjects presenting 0.5% of HbA1c change, levels and changes in bacterial species, and occurrence of adverse events. Statistical analysis Clinical, glycemic and microbiological parameters were computed per subject and across subjects in both groups. Changes in PD and CAL in moderate and deep sites, in bacterial levels and in number of sites with PD 5 and PD 6 mm were averaged per subject and then across subjects in each group. The significance of differences between groups for age, duration of DM, number of teeth, clinical, microbiological and glycemic parameters at each timepoint were compared by Student s t- test. Repeated measures ANOVA and the Tukey s tests were used to detect differences within each group over time for clinical, microbiological and glycemic parameters. Analysis of covariance (ANCOVA) with adjustments for baseline values was used to detect differences between groups in the mean changes in PD, CAL, in number of sites with PD 5 mm 6 mm and in the number of bacterial species. The chi-square test was used to compare the frequency of gender, number of subjects with low, moderate or high risk for disease progression and, subjects having HbA1c changes according to a cut off of 0.5%. Fisher s exact test compared Table 1. Demographic characteristics of the study population, number of subjects reporting adverse effects and means (SD) of full-mouth clinical parameters for both groups at baseline and at follow-up visits Variable Time point Treatment groups p-values SRP + placebo (control; n = 27) SRP + MTZ + AMX (test; n = 29) t-test Age (years) Baseline Duration of DM (years) Number of teeth Adverse effects Fisher s test Diarrhea (n) Reported Headache (n) at 14 days Metallic taste (n) Nausea/Vomiting (n) Irritability (n) 0 0 Clinical parameters t-test % of sites with Baseline a a 0.84 plaque 3 months b b 0.67 accumulation 6 months b b year b b 0.81 % of sites with marginal bleeding Baseline a a months b b months b b year b b 0.99 ANOVA p-values % of sites with bleeding on probing Baseline a a months b b 0.00* 6 months b b 0.00* 1 year b b 0.00* % of sites with suppuration Baseline a a months b b 0.00* 6 months b b 0.00* 1 year b b 0.00* Mean PD (mm) Baseline a a months b b 0.00* 6 months b b 0.00* 1 year b b 0.00* Mean CAL (mm) Baseline a a months b b months b b year b b 0.13 Number of sites with PD 5mm Baseline a a months b b 0.00* 6 months b b 0.00* 1 year b b 0.00* % of sites with PD 5 mm Baseline a a months b b 0.00* 6 months b b 0.00* 1 year b b 0.00* SRP, scaling root planing; MTZ, metronidazole; AMX, amoxicillin; DM, diabetes mellitus; PD, probing depth; CAL, clinical attachment level; SD, standard deviation. The symbol *indicate differences between treatment groups at each time point by the Student s t-test (p < 0.05). Different letters indicate significant differences over time by repeated measures ANOVA and Tukey s tests (p < 0.05). There were no differences between groups for adverse effects by Fisher s exact test (p > 0.05).

5 894 Miranda et al. the frequency of subjects reporting adverse effects between groups. The level of significance was set at 5%. The data were evaluated using intention-to-treat analysis with last observation carried forward. Results Subject retention, compliance and adverse effects The study was conducted between September 2011 and October Fig. 1 presents the flow diagram of the study design. Fifty-eight subjects (38 65 years old) entered the study and were randomly assigned to one of the two treatment groups (n = 29/ group). Two subjects from the control group did not attend the baseline monitoring visit. Therefore, 27 and 29 subjects were evaluated at baseline in the control and test groups respectively. Four subjects in the control group and two in the test group were lost during the follow up visits. Fifty-six subjects informed full adherence to the use of medications, and this information was confirmed by the empty bottles. Three and one subjects from the control and test groups, respectively, had one or two pills left in the bottles on the 14th day. Six and fourteen subjects from the control and test groups, respectively, reported at least one adverse effect of the placebos/antibiotics use. However, no significant differences were observed between groups for the number of subjects reporting specific adverse effects (p > 0.05; Table 1). All subjects stated that the medications did not cause any major disturbance in their daily routine and, therefore, they would repeat the antibiotic treatment, if necessary. Clinical results The control group comprised 18 males and 9 females while the test group had 12 males and 17 females. There were no differences between groups for gender (Chi-square test; p = 0.06). There were no statistically significant differences between groups for age, duration of DM, number of teeth and for the clinical parameters evaluated at baseline (p > 0.05; Table 1). Both treatments yielded statistically significant Table 2. Mean ( SEM) PD reduction, CAL gain and reduction in the number of sites with PD 5 mm and PD 6 mm from baseline to 3 months, 6 months and 1 year Parameter PD category Time point Treatment groups p-value Reduction in PD Gain in CAL Reduction in the number of sites with: Table 3. Number and percentage of subjects presenting low ( 4 sites with PD 5 mm), moderate (5-8 sites with PD 5 mm) or high ( 9 sites with PD 5 mm) risk for disease progression, according to Lang & Tonetti (2003), at 1-year post-therapies Variable Categories Treatment groups p-value Risk for disease progression Initially moderate sites (PD 4 6 mm) Initially deep sites (PD 7 mm) Initially moderate sites (PD 4 6 mm) Initially deep sites (PD 7 mm) improvements in all clinical parameters at 3, 6 and 12 months posttreatments (p < 0.05; Table 1). The SRP+MTZ+AMX group presented lower mean PD and mean number of sites with PD 5 mm (primary outcome variable) as well as lower mean percentage of sites with BoP, suppuration or presenting PD 5 mm in comparison with the SRP group, at all follow-up evaluations (p < 0.05; Table 1). Subjects receiving MTZ+AMX had a statistically significant greater reduction in PD and gain in CAL than those receiving placebo from baseline to all follow-up visits for initially moderate and deep sites SRP + placebo (n = 27) SRP + placebo (control; n = 27) SRP + MTZ + AMX (test; n = 29) 0 3 months months year months months year months months year months months year PD 5 mm 0 3 months months year PD 6 mm 0 3 months months year SRP, scaling root planing; MTZ, metronidazole; AMX, amoxicillin; PD, probing depth; CAL, clinical attachment level; SEM, standard error of the mean. A p-value <0.05 indicates differences between treatment groups at each time point by ANCOVA. SRP + MTZ + AMX (n = 29) Low risk 6 (22.2%) 22 (75.9%) Moderate risk 6 (22.2%) 5 (17.2%) 0.00 High risk 15 (55.6%) 2 (6.8%) SRP, scaling root planing; MTZ, metronidazole; AMX, amoxicillin; PD, probing depth. The significance of differences between groups was assessed by Chi-square test (p < 0.05). (p < 0.05; Table 2). Furthermore, the reductions between baseline and all follow-up evaluations in the number of sites with PD 5 mm and 6 mm were also statistically significantly greater in the test than in the control group (p < 0.05; Table 2). According to Table 3, two (6.9%) subjects in the SRP+MTZ+AMX group and 15 subjects (55.6%) in the control group were at high risk for disease progression at 1-year post-therapies. Conversely, 22 subjects (75.9%) in the SRP+MTZ+AMX group and 6 subjects (22.2%) in the control group were at low risk for disease progression at 1-year (p < 0.05).

6 Antibiotics for diabetic subjects 895 Microbiological results Both therapies led to reduction in the mean number of all bacterial species evaluated at 3 months and 1 year, compared to baseline (p < 0.05; Table 4). The remaining mean numbers of E. nodatum were statistically significantly lower in the test than in the control group at 3 months posttherapy. At 1-year, the levels of P. intermedia were statistically lower in the test group than in the control group. In addition, the mean numbers of the three members of the red complex (T. denticola, P. gingivalis, T. forsythia) were significantly lower in the SRP+MTZ+AMX group than in the control group at 3 and 12 months post-therapies. (p < 0.05; Table 4). The levels of the three species of the red complex were statistically significantly more reduced in the test than in the control group at 3 and 12 months post-therapy, considering all sampled sites, and sites with initial PD 4and 5 mm(p < 0.05; Table 5). Furthermore, the test treatment led to a greater reduction in the levels of P. intermedia in all sampled sites than in the control treatment (p < 0.05). This same trend was observed in sites with initial PD 4 mm at 3 and 12 months and in sites with initial PD 5 mm at 12 months (p < 0.05; Table 5). There was also a greater decrease in the levels of E. nodatum in the test than in the control group from baseline to 3 months in all sampled sites and in sites with initial PD 4 mm(p < 0.05; Table 5). Glycemic results Table 4. Mean number of periodontal pathogens ( SD), considering all sampled sites, for both groups at baseline and at follow-up visits Bacterial specie Time-points Mean number (log 10) There were no statistically significant differences between treatment groups for the mean levels of HbA1c and FPG at any time-point. Furthermore, there were no statistically significant changes in the HbA1c and FPG levels after both treatments (p > 0.05; Table 6). There were no differences between groups in the frequency of subjects showing decrease, increase or no change in the levels of HbA1c, according to a cut off of 0.5% (p > 0.05; Table 6). Regarding the hypoglycemic medications, all subjects included in this study reported to be under metformin or glibenclamide treatment. In addition, two subjects per group also reported to be under insulin supplementation. Discussion This was the first RCT evaluating the clinical and microbiological effects of MTZ+AMX as adjuncts to SRP for the treatment of type 2 diabetic subjects with ChP. Overall, the results demonstrated that the use of MTZ+AMX yielded considerable clinical and microbiological advantages over SRP alone at 3 months, which were sustained up to 1 year; providing the first evidence of the effectiveness of this therapeutic protocol for the treatment of diabetic subjects with periodontitis. Although both therapies significantly improved clinical parameters, the SRP+MTZ+AMX group exhibited the best clinical results up to 1 year for all the clinically relevant outcomes, such as the mean number SRP + placebo (n = 27) SRP + MTZ + AMX (n = 29) t-test p-values T. denticola Baseline a a months b b 0.00* 1 year b b 0.03* P. gingivalis Baseline a a months b b 0.00* 1 year b b 0.00* T. forsythia Baseline a a months b b 0.00* 1 year b b 0.04* E. nodatum Baseline a a months b b 0.01* 1 year b b 0.19 P. micra Baseline a a months b b year b b 0.07 Fusobacterium nucleatum ssp. Baseline a a months b b year b b 0.19 P. intermedia Baseline a a months b b year b b 0.03* SRP, scaling root planing; MTZ, metronidazole; AMX, amoxicillin; SD, standard deviation. Different letters indicate significant differences over time by the repeated measures ANOVA and Tukey s tests (p < 0.05). The symbol *indicate differences between treatment groups at each time point by the Student s t-test (p < 0.05). of residual sites with PD 5 mm (primary outcome variable), the number of these residual sites at subject level, percentage of sites with BoP and reductions in PD and gains in CAL in moderate and deep sites (Table 2). These findings are in agreement with previous studies in non-diabetic subjects that showed that the adjunctive use of MTZ+AMX provided shortand long-term additional clinical benefits over those achieved with SRP alone (Winkel et al. 2001, Cionca et al. 2009, Silva et al. 2011, Feres et al. 2012, Goodson et al. 2012, Sgolastra et al. 2012). Worthy of note, diabetic subjects seem to be benefited from this antibiotic therapy to a similar extent or even more than non-diabetic subjects. In the present study, the mean PD reductions in sites with PD 7 mm were 3.7 and 3.9 mm at 3 and 12 months, respectively, in the group taking MTZ+AMX. In this same category of deep sites, Winkel

7 896 Miranda et al. Table 5. Mean (SEM) reduction in the number of bacterial species (log 10) for all sampled sites, sites with initial PD 4 mm and sites with PD 5 mm from baseline to 3 months and 1-year post-therapies Parameter Bacterial specie Time point Treatment groups p-value SRP + placebo (n = 27) SRP + MTZ + AMX (n = 29) Reduction in all sampled sites Reduction in sites with initial PD 4mm Reduction in sites with initial PD 5mm T. denticola 0 3 months year P. gingivalis 0 3 months year T. forsythia 0 3 months year E. nodatum 0 3 months year P. micra 0 3 months year Fusobacterium nucleatum 0 3 months ssp. 0 1 year P. intermedia 0 3 months year T. denticola 0 3 months year P. gingivalis 0 3 months year T. forsythia 0 3 months year E. nodatum 0 3 months year P. micra 0 3 months year Fusobacterium nucleatum 0 3 months ssp. 0 1 year P. intermedia 0 3 months year T. denticola 0 3 months year P. gingivalis 0 3 months year T. forsythia 0 3 months year E. nodatum 0 3 months year P. micra 0 3 months year Fusobacterium nucleatum 0 3 months ssp. 0 1 year P. intermedia 0 3 months year SRP, scaling root planing; MTZ, metronidazole; AMX, amoxicillin; PD, probing depth; SEM, standard error of the mean. A p-value <0.05 indicates differences between treatment groups at each time point by ANCOVA. et al. (2001) reported a mean PD reduction of 3.2 mm at 3 months after this combined treatment, and Feres et al. (2012) a decrease of 3.7 mm and 4.1 mm at 3 and 12 months, respectively, for non-diabetic subjects with ChP. A remarkable difference was observed for the number and percentage of residual sites between subjects treated or not with systemic antibiotics (Tables 1 and 2). At 1- year post-therapy, the group treated with MTZ+AMX had an averaged of 4 sites with PD 5 mm, against 14.9, detected in the group receiving SRP alone (Table 1). The analysis of residual sites at subject level showed that only 2 subjects in the test group were still at high-risk (i.e. 9 sites with PD 5 mm, Lang & Tonetti 2003) for disease progression (6.8%) versus 15 subjects (55.6%) in the control group. Previous investigations in non-diabetic subjects with ChP have confirmed the advantages of the adjunctive use of MTZ+AMX in reducing the number of sites and subjects with PD 5 mm over shortand long-term (Winkel et al. 2001, Cionca et al. 2009, Silva et al. 2011, Feres et al. 2012). Previous studies have suggested that subjects presenting residual pockets, especially those with PD 5 mm, are at greater risk for further attachment loss and may require additional treatments, such as periodontal surgeries (Lang & Tonetti 2003, Matuliene et al. 2008, 2010). In this regard, the findings of the present study are particularly important in the view of the fact that diabetic subjects present a poorer wound healing (Tsourdi et al. 2013); and therefore, surgical proce-

8 Antibiotics for diabetic subjects 897 Table 6. Mean levels (SD) of glycemic parameters and number (percentage) of subjects according to changes in HbA1c levels Parameter Time- points Treatment groups t-test p-value SRP + placebo (n = 27) SRP + MTZ + AMX (n = 29) HbA1c (%) Baseline months months year FPG (mg/dl) Baseline months months year Number (%) of subjects with at least 0.5% change in HbA1c Decreased Increased Stable Chi-square p-value Decreased Increased Stable Chi-square p-value 0 3 months 10 (37.0) 9 (33.3) 8 (29.6) 13 (44.8) 9 (31.0) 7 (24.1) 0 6 months 11 (40.7) 9 (33.3) 7 (25.9) > (41.4) 10 (34.5) 7 (24.1) > year 12 (44.4) 6 (22.2) 9 (33.3) 13 (44.8) 7 (24.1) 9 (31.0) SRP, scaling root planing; MTZ, metronidazole; AMX, amoxicillin; HbA1c, glycated hemoglobin; FPG, fasting plasma glucose; SD, standard deviation. There were no differences in the mean levels of HbA1c and FPG over time within groups by the repeated measures ANOVA (p > 0.05). There were no differences between groups for mean levels of HbA1c and FPG at any time point by Student s t-test (p > 0.05). An increase or decrease of 0.5% in HbA1c from baseline to the follow-up examinations was considered a change; otherwise the level of glycemic control was considered as stable. There were no differences between groups in the distribution of subjects according to the cut off of 0.5% of changes in HbA1c by the Chi-square test (p > 0.05). dures in these individuals might be associated with an increased morbidity. Furthermore, other disadvantages related to periodontal surgeries such as time and cost of treatment, anxiety, risk of root sensitivity and esthetic concerns must be considered while making a decision between surgical and non-surgical approaches for both diabetic and non-diabetic subjects. Previous investigations have assessed the effects of other systemic antibiotics as adjuncts to SRP in diabetic subjects, including doxycycline (Grossi et al. 1997, O Connell et al. 2008, Gaikwad et al. 2013), AMX/clavulanic acid (Rodrigues et al. 2003) and azithromycin (Botero et al. 2013). In contrast to the marked clinical advantages observed with the use of MTZ+AMX in the current study, these investigations demonstrated modest (Grossi et al. 1997, Botero et al. 2013, Gaikwad et al. 2013) or no (Rodrigues et al. 2003, O Connell et al. 2008) additional benefits of those adjunctive antimicrobials over SRP alone. It is important to mention that all these studies evaluated mild to moderate ChP while the population evaluated in the current study presented more advanced disease. Hence, further studies testing such antimicrobial protocols in diabetic subjects with advanced periodontitis and comparing them with the MTZ+AMX protocol combination would be of value to clinicians and patients. The adjunctive use of MTZ+AMX not only improved the clinical outcomes of SRP but also more predictably suppressed periodontal pathogens in type 2 diabetic subjects. The SRP+MTZ+AMX group exhibited lower levels and greater reductions of all red complex species and two putative pathogens from the orange complex (E. nodatum and P. intermedia) at 3 months, compared to the control group. The majority of these microbiological benefits were maintained up to 1 year. Clearly, these most favorable microbiological effects were responsible for the best long-term clinical outcomes observed in the SRP+MTZ+AMX group. Accordingly, previous studies have shown greater benefits of the adjunctive use of MTZ+AMX than SRP alone for reducing subgingival periodontal pathogens in non-diabetic subjects with ChP (Winkel et al. 2001, Cionca et al. 2010, Silva et al. 2011, Soares et al. 2014). As the combination of MTZ+AMX yields concentrations in healthy and inflamed gingival tissues that are normally higher than the minimum inhibitory concentration of the drug necessary for the inhibition of most oral pathogens (Amid et al. 2012), this antibiotic combination probably achieves pathogenic species invading the periodontal tissues. Furthermore, MTZ+AMZ might also reduce putative pathogens in the non-periodontal niches such as tonsils, saliva, tongue, and mucosa. In the present study, bacterial reductions were analyzed at sites with initial PD 4 mm (shallow sites) and PD 5 mm (deep sites), separately. Shallow sites, which are not often the main therapeutic concern, are likely to dilute bacterial changes observed at the deeper sites, which are expected to experience the greatest benefits after therapy. Interestingly, in the present study, the microbiological benefits of MTZ+AMX were not only restricted to deep, but were also extended to shallow sites, which exhibited a statistically significant greater reduction in five pathogens in the test than in the control group. In fact, it has been proposed that an optimum periodontal therapy should be able to achieve shallow sites that might also harbor high levels and proportions of periodontal pathogens (Socransky & Haffajee 2005, Soares et al. 2014). These benefits of MTZ+AMX in sites with PD 4 mm are also important to be interpreted in the light of the knowledge that diabetic subjects present a hyper-immunoinflammatory response even in shallow sites (Duarte et al. 2014). Despite the evidence that periodontal therapy may improve the

9 898 Miranda et al. levels of HbA1c, the precise significance of the adjunctive use of systemic antibiotics on glycemic control is still unknown (Teeuw et al. 2010, Chapple & Genco 2013). Some studies have shown that adjunctive doxycycline (Grossi et al. 1997, O Connell et al. 2008) or azithromycin (Botero et al. 2013) may improve the glycemic control of diabetic subjects. Conversely, other investigations did not find significant benefits with the use of antibiotics as adjunct to SRP in reducing the levels of HbA1c (Rodrigues et al. 2003, Promsudthi et al. 2005, Gaikwad et al. 2013). In this study, despite the notable clinical and microbiological improvements obtained with the adjunctive use of MTZ+AMX, none of the therapies yielded statistically significant improvement in the glycemic parameters at any time point. However, it is important to consider that our sample size calculation was not based on changes in glycemic parameters, which were secondary outcome variables. It is likely that larger sample sizes would be necessary to find possible significant changes in the HbA1c levels after these periodontal treatments. This study has some limitations that should be taken into consideration. Firstly, the microbiological analysis was restricted to seven periodontal pathogens. As the target of an ideal periodontal therapy should be not only a reduction in pathogens, but also an increase in the proportions of host-compatible species (Teles et al. 2006, Feres 2008), a broader microbiological evaluation, including the host-compatible species, would provide a better view of the actual impact of MTZ+AMX on the subgingival biofilm of these diabetic subjects. Secondly, the high sensitivity and exponential nature of the real-time PCR and the variations of the 16S rrna gene target sequences may increase risk of false negative results (Klein 2002). Furthermore, it important to highlight that subjects with major complications of DM, like nephropathy, were excluded in this study. Since the decline of renal function in diabetic nephropathy may alter the pharmacokinetics and pharmacodynamics of drugs (Dostalek et al. 2012), diabetic subjects with renal dysfunction would require medical evaluation prior to the antibiotic intake for dose adjustments, in order to assure the efficacy of the treatment and patient safety. In conclusion, the adjunctive use of MTZ+AMX significantly improved the clinical and microbiological outcomes of the SRP alone in the treatment of ChP in type 2 diabetic subjects up to 1 year. References Amid, R., Tabeie, M. B., Kadkhodazadeh, M., Mehdizadeh, A. R. & Youssefi, N. (2012) Local concentration of systemic amoxicillin and metronidazole in healthy and inflamed gingiva: a comparative in vivo study. Drug Metabolism and Drug Interactions 10, Armitage, G. C. 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