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research-article2016

JDRXXX10.1177/0022034516639274Journal of Dental ResearchAntibiotics for Diabetic Subjects

Research Reports: Clinical

Amoxicillin Plus Metronidazole Therapy for Patients with Periodontitis and Type 2 Diabetes: A 2-year Randomized Controlled Trial

Journal of Dental Research 2016, Vol. 95(7) 829­–836 © International & American Associations for Dental Research 2016 Reprints and permissions: sagepub.com/journalsPermissions.nav DOI: 10.1177/0022034516639274 jdr.sagepub.com

N.S. Tamashiro1, P.M. Duarte1, T.S. Miranda1, S.S. Maciel1, L.C. Figueiredo1, M. Faveri1, and M. Feres1

Abstract The aim of this study was to assess the changes occurring in subgingival biofilm composition and in the periodontal clinical parameters of subjects with periodontitis and type 2 diabetes mellitus (DM) treated by means of scaling and root planing (SRP) only or combined with systemic metronidazole (MTZ) and amoxicillin (AMX). Fifty-eight subjects were randomly assigned to receive SRP only (n = 29) or with MTZ (400 mg/thrice a day [TID]) and AMX (500 mg/TID) (n = 29) for 14 d. Six subgingival plaque samples/subject were analyzed by checkerboard DNA–DNA hybridization for 40 bacterial species at baseline and 3 mo, 1 y, and 2 y posttherapy. At 2 y posttherapy, the antibiotic-treated group harbored lower mean proportions (5.5%) of red complex pathogens than the control group (12.1%) (P < 0.05). The proportions of the Actinomyces species remained stable in the antibiotic group but showed a statistically significant reduction in the control group from 1 to 2 y in subjects achieving a low risk clinical profile for future disease progression (i.e., ≤4 sites with probing depth [PD] ≥5 mm). The test group also had a lower mean number of sites with PD ≥5 mm (3.5 ± 3.4) and a higher percentage of subjects reaching the low risk clinical profile (76%) than the control group (14.7 ± 13.1 and 22%, respectively) (P < 0.05) at 2 y posttreatment. MTZ + AMX intake was the only significant predictor of subjects achieving the low risk at 2 y (odds ratio, 20.9; P = 0.0000). In conclusion, the results of this study showed that the adjunctive use of MTZ + AMX improves the microbiological and clinical outcomes of SRP in the treatment of subjects with generalized chronic periodontitis and type 2 DM up to 2 y (ClinicalTrials.gov NCT02135952). Keywords: periodontal diseases, chronic periodontitis, type 2 diabetes mellitus, microbiology, antibiotics, therapeutics

Introduction Diabetes mellitus (DM) is recognized as a major risk factor for periodontal diseases, as patients with DM present increased prevalence and severity of periodontal destruction compared with those without DM (Llambés et al. 2015). Although scaling and root planing (SRP) provides clinical benefits in the treatment of systemically healthy patients and those with DM, many of these individuals still present a high number of residual pockets as well as periodontal pathogens after mechanical treatment (Feres et al. 2012; Santos et al. 2013). Therefore, there is an increasing interest in studying adjunctive therapies that could improve the clinical and microbiological outcomes of SRP in these subjects. Previous investigations have tested the clinical and microbiological effects of certain systemic antibiotics as adjuncts to SRP in the periodontal treatment of subjects with DM, such as amoxicillin (AMX) (Rodrigues et al. 2003), azithromycin (Botero et al. 2013; Hincapié et al. 2014), and especially doxycycline (Grossi et al. 1997; O’Connell et al. 2008; Gaikwad et al. 2013; Tsalikis et al. 2014). Although these studies showed some degree of success with the use of these agents, the results were somewhat controversial and overall not remarkable.

Several studies have pointed out metronidazole (MTZ) and AMX as adjuncts to SRP as the most promising treatment for chronic and aggressive periodontitis (Guerrero et al. 2005; Xajigeorgiou et al. 2006; Cionca et al. 2009; Mestnik et al. 2010; Sgolastra, Gatto, et al. 2012; Sgolastra, Petrucci, et al. 2012;  Feres et al. 2012; Goodson et al. 2012; Mestnik et al. 2012; Socransky et al. 2013; Zandbergen et al. 2013; Soares et al. 2014; Feres et al. 2015; Rabelo et al. 2015). This combination of antibiotics in the initial periodontal therapy leads to clinically significant benefits, over those observed by SRP alone, even at 1 or 2 y after intake of the agents (Ehmke et al. 2005; Feres et al. 2012; Goodson et al. 1

Department of Periodontology, Dental Research Division, Guarulhos University, Guarulhos, São Paulo, Brazil A supplemental appendix to this article is published electronically only at http://jdr.sagepub.com/supplemental. Corresponding Author: M. Feres, Centro de Pós-Graduação e Pesquisa-CEPPE, Universidade Guarulhos, Praça Tereza Cristina, 229 Centro, 07023-070 Guarulhos, SP, Brazil. Email: [email protected]

830 2012; Mestnik et al. 2012). We showed previously, for the first time, that subjects with periodontitis and type 2 DM exhibit advantages from the adjunctive use of 14 d of MTZ and AMX in the active phase of the periodontal treatment (Miranda et al. 2014). The antibiotic-treated group presented an average of 4 sites with probing depth (PD) ≥5 mm at 1 y after treatment, as opposed to 14.9 sites in the group treated by SRP only. There is also strong evidence indicating that the clinical benefits observed in systemically healthy subjects with chronic (Silva et al. 2011; Socransky et al. 2013; Soares et al. 2014; Feres et al. 2015) and aggressive periodontitis (Xajigeorgiou et al. 2006; Mestnik et al. 2010) treated with adjunctive MTZ and AMX are accompanied by a beneficial change in the composition of the subgingival biofilm. However, no studies to date have comprehensively evaluated the changes occurring in the subgingival microbial profile in subjects with DM receiving MTZ, AMX, and SRP. Therefore, the aim of this study was to assess the changes occurring in the levels and proportions of the 40 bacterial species described by Socransky et al. (1998) in subjects with periodontitis and type 2 DM treated by means of SRP only or combined with systemic MTZ and AMX. A secondary aim was to compare the clinical efficacy of these 2 treatment protocols at 2 y after therapies.

Materials and Methods Sample Size Calculation This study presents the microbiological data of a randomized controlled trial (RCT) (Miranda et al. 2014), designed and powered to compare the clinical effects of SRP alone or with MTZ + AMX in subjects with generalized ChP and type 2 DM, at 1 y after treatments. Therefore, a post hoc analysis was conducted to determine the actual power of the microbiological analysis presented in this article. Considering differences of at least 6.6 percentage points (pp) between groups for the proportion of the red complex species and a standard deviation of 8.9 pp, it was determined that 20 subjects per group would be necessary to provide 90% power with an α of 0.05.

Subject Population and Inclusion and Exclusion Criteria Subjects with type 2 DM and generalized chronic periodontitis (Armitage 1999) were selected from the Periodontal Clinic of Guarulhos University as previously described (Miranda et al. 2014). Briefly, the inclusion criteria were as follows: aged ≥35 y, diagnosis of type 2 DM during at least the past 5 y, glycated hemoglobin levels ≥6.5% to ≤11%, ≥15 teeth, >30% of the sites with PD and clinical attachment level (CAL) ≥4 mm, and ≥6 teeth with at least 1 site with PD and CAL ≥5 mm and bleeding on probing (BoP). Exclusion criteria were as follows: pregnancy, lactation, smoking, SRP in the previous 12 mo, systemic antibiotic treatment in the previous 6 mo, need of antibiotic prophylaxis, systemic conditions (except DM) that could affect the progression of periodontitis, long-term use of

Journal of Dental Research 95(7) anti-inflammatory or immunosuppressive medications, and allergy to MTZ and/or AMX. Subjects were informed of the nature, potential risks, and benefits of the study and signed a term of informed consent. Guarulhos University Clinical Research Ethics Committee approved the study protocol. The study was registered at the ClinicalTrials.gov database (NCT02135952).

Experimental Design, Allocation Concealment, and Treatment Protocol In this double-blinded, parallel-design, placebo-controlled RCT, the study coordinators (P.M.D. and M.Fe.) used a computer-generated table to randomly allocate subjects (1:1 ­ allocation ratio) into one of the following groups: SRP + placebo (control; n = 29) or SRP + MTZ (400 mg thrice a day [TID] for 14 d) + AMX (500 mg TID for 14 d) (test; n = 29). Randomization was stratified by operators (N.S.T. and V.R. Santos), and allocation concealment was ensured by means of sequentially numbered drug containers of identical appearance. Subjects in the control group received 2 placebo pills TID for 14 d, and antibiotic/placebo administration started at the day of the first SRP session. The protocol of antibiotic/placebo administration has been described previously (Mestnik et al. 2010; Feres et al. 2012; Feres et al. 2015). Initially, all subjects received supragingival plaque control and oral hygiene instructions. Two trained periodontists (N.S.T. and V.R.S.) performed SRP in 4 to 6 appointments lasting approximately 1 h each, using manual curettes and an ultrasonic device. An overall full-mouth SRP was performed during the first treatment visit to disrupt the subgingival biofilm and maximize the antibiotic effect from the beginning. Subsequently, 1 quadrant or sextant was treated per SRP session, depending on the number of deep pockets. Periodontal therapy was completed in 14 d. The coordinator (P.M.D.) checked the endpoint “smoothness of the scaled roots” of each SRP appointment. The same pharmacy prepared identical antibiotic and placebo pills (i.e., same aspect, taste, and color). Identical plastic bottles containing the antibiotics/placebos were sent to the study coordinator (P.M.D.), who marked the code number of each subject on each bottle, according to the therapy assigned. The coded bottles were given to the operators (N.S.T. and V.R.S.), who at no time during the study identified the contents of the bottle or subject assignment to therapies. The biostatistician (M.Fa.) and all participants were also blinded to treatment assignment. All subjects received microbiological and clinical monitoring at baseline and 3 mo, 1 y, and 2 y posttherapy. Clinical measurements were also performed at 6 mo. Periodontal maintenance was conducted at 3, 6, and 9 mo and 1 y and 2 y posttherapy and included oral hygiene instructions and supragingival/subgingival biofilm/calculus removal, as necessary.

Monitoring of Compliance and Adverse Events An assistant (S.S.M.) monitored the compliance with ­antibiotic/ placebo intake by calling the patients 3 times a week during the 14 d of medication. The subjects were asked to bring the empty

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Antibiotics for Diabetic Subjects bottles back at the end of each week, and these were checked for any possible remaining pills of antibiotics/placebos. On the 14th day, subjects answered a questionnaire about any selfperceived side effects of the medications.

Clinical and Microbiological Monitoring A single examiner (T.S.M.), calibrated as previously described (Miranda et al. 2014) (Appendix), performed all clinical examinations. Presence or absence of plaque, marginal bleeding, BoP, suppuration, and PD and CAL measurements were assessed at 6 sites per tooth excluding third molars using the manual periodontal probe (North Carolina–Hu-Friedy). The examiner was blinded to the treatment allocation of the subjects. After supragingival plaque removal, the subgingival biofilm samples were collected with individual sterile miniGracey curettes (#11–12) from 6 noncontiguous interproximal sites, 2 at each of the following baseline PD categories: shallow, PD ≤3 mm; intermediate, PD = 4 to 6 mm; and deep, PD ≥7 mm. These were evaluated for 40 bacterial species by checkerboard DNA–DNA hybridization (Socransky et al. 1994; Mestnik et al. 2010) (Appendix).

Statistical Analysis The primary outcome variable was the difference between groups for the proportions of the red complex at 2 y. Secondary outcome variables are described in the Appendix. The significance of differences over the course of the study was determined by the Friedman and Dunn multiple-­ comparison tests for the microbiological parameters and by repeated-measures analysis of variance (ANOVA) and Tukey’s multiple-comparisons tests for the clinical parameters. The significance of differences between groups for the mean proportions of the microbial complexes, mean changes in PD and CAL, and mean number/percentage of sites with PD ≥5 mm and PD ≥6 mm was determined by analysis of covariance (ANCOVA), adjusting for the baseline mean values of each individual parameter. The significance of differences between groups for age, duration of DM, number of teeth, mean PD and CAL, and percentage of sites with plaque accumulation, gingival bleeding, BoP, and suppuration was determined by Student’s t test; for the frequency of both sexes and number of subjects with low, moderate, and high risk of disease progression by the χ2 test; and for the frequency of subjects with adverse events by the Fisher exact test. A stepwise forward logistic regression analysis explored the following variables: “presence of ≤4 sites with PD ≥5 mm at 2 y posttherapy (yes/no)” and “absence of sites with PD ≥6 mm at 2 y posttherapy (yes/no).” The predictor variables included age, sex, SRP, MTZ + AMX, and the following clinical characteristics at baseline: mean PD and CAL, number of sites with PD ≥5 mm and with PD ≥6 mm, and percentage of sites with plaque, gingival inflammation, suppuration, or BoP. The microbiological analyses were adjusted for multiple comparisons (Socransky et al. 1991). The clinical and

microbiological data were evaluated using intention-to-treat analysis with last observation carried forward, and the level of significance was set at 5%.

Results Subject Retention, Compliance, and Side Effects We conducted the study between September 2011 and March 2014. Patient compliance, adverse events, and the clinical effects of therapies up to 1 y posttreatment were presented in Miranda et al. (2014). Figure 1 presents the flow diagram of the study.

Microbiological Results Proportions of the microbial complexes.  The proportions of 5 (red, orange, and green complexes; Actinomyces species; and the “­others” group) and 3 microbial complexes (red and orange complexes and Actinomyces species) were statistically significantly changed in test and control, respectively (Fig. 2). Both treatments led to a significant reduction in the proportion of the red complex at 3 mo (SRP: from 16.3% to 7.6%; SRP + MTZ + AMX: from 17.8% to 5.3%) (P < 0.05). The proportions of red complex were maintained up to 2 y in the antibiotic-treated group (5.5%) but increased to 9.8% at 1 y and to 12.1% at 2 y in the control group. The difference between groups for the proportions of this complex at 2 y was statistically significant (primary outcome). Figure 3 presents the same type of data as Figure 2 but for subjects at low risk for future disease progression at 2 y posttreatment. SRP-only treatment significantly affected the mean proportions of the Actinomyces species, while SRP + MTZ + AMX affected the purple, orange, and red complexes (P < 0.05). At 2 y, the proportion of the red complex was lower in the antibiotic-treated group (5.2%) than in the control group (10.6%) (P < 0.05). The proportions of the Actinomyces species remained stable in the antibiotic group but showed a reduction in the control group from 1 y to 2 y (Figs. 2, 3). This reduction was statistically significant in subjects in the low risk category (from 32.3% to 24.7%) (Figure 3).

Mean levels of the individual species. Subjects with SRP-only treatment showed a significant reduction in the mean levels of Tannerella forsythia and Porphyromonas gingivalis (P < 0.05), while the levels of 9 species were altered in the test group, including a reduction in the 3 red complex pathogens (T. forsythia, P. gingivalis, and Treponema denticola). The reduction in the levels of T. forsythia and P. gingivalis from baseline to 2 y posttreatment was greater in the test than in the control group (P < 0.05) (Fig. 4).

Clinical Results We observed no statistically significant differences between groups for the demographic, glycemic, and clinical parameters at baseline or for the number of adverse events reported (Appendix Table 1).

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Figure 1.  Flowchart of the study design. AMX, amoxicillin; MTZ, metronidazole; SRP, scaling and root planing.

Figure 2.  Cumulative mean proportions of microbial complexes in subgingival biofilm samples taken from subjects at baseline and at 3 mo, 1 y, and 2 y posttreatment. The samples were analyzed separately to determine their content of 40 subgingival bacterial species described in Figure 4. The colors represent the different complexes described by Socransky et al. (1998). The gray color represents species that did not fall into any complex, and Actinomyces species are represented in blue. The significance of differences among time points was determined using Friedman (***P < 0.0001) and Dunn multiple-comparison tests (different letters represent significant differences between time points, P < 0.05). The significance of differences between the 2 groups at 2 y was determined by using analysis of covariance adjusted for baseline values of each microbial complex (&P < 0.001). AMX, amoxicillin; MTZ, metronidazole; SRP, scaling and root planing.

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Figure 3.  Cumulative mean proportions of microbial complexes in subgingival biofilm samples taken from subjects in the low risk profile category for future disease progression (Lang and Tonetti 2003) at 2 y posttreatment (i.e., with ≤4 sites with PD ≥5 mm). The samples were analyzed separately to determine their content of 40 subgingival bacterial species described in Figure 4. The colors represent the different complexes described by Socransky et al. (1998). The gray color represents species that did not fall into any complex, and Actinomyces species are represented in blue. The significance of differences among time points was determined using Friedman (***P < 0.0001) and Dunn multiple-comparison tests (different letters represent significant differences between time points, P < 0.05). The significance of differences between the 2 groups at 2 y was determined by using analysis of covariance adjusted for baseline values of each microbial complex. AMX, amoxicillin; MTZ, metronidazole; SRP, scaling and root planing.

The percentage of sites with BoP and suppuration and fullmouth mean PD were significantly lower in the test group at 1 y and 2 y (P < 0.05; Appendix Table 1). At 1 y, the antibiotic group had significantly fewer sites with PD ≥5 mm (primary outcome variable) than the control group, and this benefit was maintained up to 2 y (SRP = 14.7 ± 13.1, SRP + MTZ + AMX = 3.5 ± 3.4, P < 0.05) (Table). The Table presents the number and percentage of subjects achieving the clinical endpoint for treatment according to Feres et al. (2012)—that is, low risk for future disease progression (≤4 sites with PD ≥5 mm); 75.8% of the subjects treated by SRP + MTZ + AMX and 22.3% who had SRP-only treatment were at low risk at 2 y. The antibiotictreated group showed a greater reduction in mean PD and gain in mean clinical attachment at initially moderate and deep sites (P < 0.05) than the control group at 1 and 2 y posttreatment (Appendix Table 2). Stepwise forward logistic regression analysis showed that, of all predictor variables included in the model, the treatment with MTZ + AMX was the only variable that significantly increased the probability of a subject reaching the low risk profile for future disease progression and not having any site with PD ≥6 mm (odds ratio [OR], 14.3; P = 0.0000) at the 2-year posttreatment (OR, 20.9; P = 0.0000).

Discussion This study indicated that SRP + MTZ + AMX was more effective than SRP-only treatment in changing the composition of

the subgingival biofilm of subjects with type 2 DM toward that of a community more compatible with periodontal health. This therapeutic protocol was also more effective than SRP-only treatment in maintaining the clinical benefits observed at 1 y (Miranda et al. 2014), up to 2 y posttreatment. The adjunctive systemic antibiotics yielded the most beneficial change in the composition of the subgingival microbiota, including a more striking reduction in the proportions of the red complex (primary outcome) at 2 y posttreatment. The proportions of the red complex pathogens remained totally stable in the test group from 3 mo to 2 y (~5%) but rose from 7% to 12% in the control group. Conversely, the proportions of the host-compatible Actinomyces species remained stable from 1 to 2 y in the test group but showed a tendency to decrease in the control group. Interestingly, we observed this same unfavorable trend of recolonization even in patients from the control group that showed the best clinical response to treatment (low risk profile), who still had 10.6% of red complex at 2 y. In previous studies of nondiabetic subjects with chronic or aggressive periodontitis, we detected instability of periodontal attachment when the red complex accounted for more than 10% of the 40 bacterial species from the checkerboard panel (Mestnik et al. 2010; Silva et al. 2011; Soares et al. 2014; Feres et al. 2015). Taken together, these data indicate that even those subjects who respond very well to SRP-only treatment clinically might be at greater risk for future attachment loss if they had not been treated with adjunctive MTZ + AMX in the initial therapy.

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Figure 4.  Profiles of the mean counts (×105) of 40 taxa in subgingival biofilm samples, at baseline and at 3 mo, 1 y, and 2 y posttreatment. The panel at the far right presents the reductions in the mean counts of each species between baseline and 2 y posttreatment. Subgingival biofilm samples were taken from each subject at each time point and were analyzed separately to determine their content of 40 species of bacteria, ordered and grouped according to the microbial complexes described by Socransky et al. (1998). The mean values for each species were averaged within a subject and then across subjects in each group at each time point. The significance of differences among time points was determined using Friedman test (*P < 0.05, **P < 0.001, ***P < 0.0001) and between groups at 2 y using analysis of covariance adjusted for baseline values of each individual bacterial species (#P < 0.05, ###P < 0.0001). All the analyses were adjusted for multiple comparisons (Socransky et al. 1991). AMX, amoxicillin; MTZ, metronidazole; SRP, scaling and root planing.

The benefits of the antibiotics in the composition of the subgingival biofilm were accompanied by a greater improvement in the clinical parameters. The major advantages of the antibiotic therapy were the reduction in the number of residual pockets. At 2 y, the antibiotic-treated subjects presented an average of 10 more residual sites with PD ≥5 mm than those who received mechanical treatment alone. Moreover, 76% of the subjects in the antibiotic-treated group reached the clinical endpoint for treatment according to Feres et al. (2012)—that is, low risk for future disease progression at 2 y posttherapy— while only 22% of the subjects with SRP-only treatment achieved/maintained this clinical endpoint. Furthermore, MTZ + AMX intake was the only significant predictor for subjects achieving the low risk clinical profile for future disease progression at 2 y (OR, 20.9; P = 0.0000). The overall long-term clinical and microbiological benefits observed with the adjunctive use of MTZ + AMX in this investigation were similar or even better than those observed in nondiabetic individuals (Rooney et al. 2002; Cionca et al. 2009; Cionca et al. 2010; Feres et al. 2012; Goodson et al. 2012; Socransky et al. 2013; Soares et al. 2014), smokers (Matarazzo

et al. 2008) with chronic periodontitis, and subjects with aggressive periodontitis (Guerrero et al. 2005; Xajigeorgiou et al. 2006; Mestnik et al. 2010; Mestnik et al. 2012). No previous RCTs have assessed the effects of MTZ + AMX or have compared its effects with those obtained with the use of other antibiotics in the treatment of subjects with periodontitis and DM. Nevertheless, the clinical and microbiological benefits achieved with MTZ + AMX in this study do not seem to be attainable with other systemic antibiotic protocols assessed by previous investigations, such as doxycycline (Grossi et al. 1997; O’Connell et al. 2008; Gaikwad et al. 2013; Tsalikis et al. 2014), AMX (Rodrigues et al. 2003), and azithromycin (Botero et al. 2013; Hincapié et al. 2014). Overall, the results of these studies showed only minor benefits in the clinical outcomes and no additional benefit in the microbial outcomes (Tsalikis et al. 2014; Hincapié et al. 2014). In the present study, we clearly demonstrated that MTZ + AMX led to a longterm stable biofilm climax community more compatible with periodontal health, which allowed greater stability of the periodontal tissues up to 2 y. It is probable that this antibiotic combination led to ecological advantages over other antibiotic

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Antibiotics for Diabetic Subjects Table.  Mean Number ± SD and Mean Reduction ± SEM of Sites with PD ≥5 mm and PD ≥6 mm over the Course of the Study and Number (%) 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 at 2 y Posttherapy. Treatment Groups Variable 

Time Point or Category

SRP + Placebo (n = 27)

SRP + MTZ + AMX (n = 29)

P Value 

Number of sites with PD ≥5 mm     Reduction in the number of sites with PD ≥5 mm   Number of sites with PD ≥6 mm     Reduction in the number of sites with PD ≥6 mm   Risk of disease progression    

Baseline 1y 2y Δ 0–1 y Δ 0–2 y Baseline 1y 2y Δ 0–1 y Δ 0–2 y Low risk (≤4 sites PD ≥5 mm) Moderate risk (5–8 sites PD ≥5 mm) High risk (≥9 sites PD ≥5 mm)

33.37 ± 16.81a 14.92 ± 13.20b 14.74 ± 13.18b 17.97 ± 1.77b 18.69 ± 1.74b 16.9 ± 12.39a 7.44 ± 9.24b 7.40 ± 9.19b 8.69 ± 1.19 10.49 ± 1.16 6 (22.3) 5 (18.5) 16 (59.2)

32.24 ± 18.68a 4.00 ± 3.39b 3.51 ± 3.39b 28.68 ± 1.70b 29.14 ± 1.68b 15.03 ± 12.5a 0.89 ± 1.5b 0.82 ± 1.6b 14.84 ± 1.15 14.85 ± 1.12 22 (75.8) 6 (20.6) 1 (3.6)

0.81* 0.00* 0.00* 0.00# 0.00# 0.57* 0.00* 0.00* 0.00# 0.00# 0.00    

The significance of differences between groups at baseline, 1 y, and 2 y was assessed by the Student’s t test (*P value) and over time by repeated measures analysis of variance and Tukey’s tests (different superscript letters indicate P < 0.05). The significance of differences between groups for the reduction of mean number of sites with PD ≥5 mm and PD ≥6 mm was assessed by analysis of covariance adjusted for baseline values (#P value). The significance of differences between groups for risk of disease progression was assessed by χ2 test. AMX, amoxicillin; MTZ, metronidazole; PD, probing depth; SD, standard deviation, SEM, standard error of the mean; SRP, scaling and root planing.

protocols, as MTZ is quite effective in inhibiting certain strict anaerobes keystone pathogens and AMX in reducing total bacterial load. Another possible benefit of the combination MTZ + AMX is suppression of the overgrowth of proteolytic pathogens that could benefit from tissue damage during SRP. This might diminish inflammation, providing a second mechanism of controlling strict anaerobe and proteolytic pathogens. The association of these effects might allow the initial host-compatible colonizers to recolonize the recently scaled pockets, preventing pathogens from recolonizing during healing (Feres et al. 2015). All these benefits are particularly important for patients with DM who present an increased risk of general infections and progression of periodontitis (Llambés et al. 2015; Magliano et al. 2015; Zhang et al. 2015). This study has some limitations: it presents secondary microbiological and clinical analyses from RCTs designed and powered to observe clinical differences between groups at 1 y, and one-third of the subjects were lost between 1 and 2 y. However, its main strength lies in being the first RCT to provide microbiological and clinical data up to 2 y of follow-up for subjects with chronic periodontitis and type 2 DM treated with SRP + MTZ + AMX. In conclusion, the adjunctive use of MTZ + AMX in the active phase of periodontal treatment improves the microbiological and clinical outcomes of SRP in subjects with generalized chronic periodontitis and type 2 DM, up to 2 y posttreatment.

Author Contributions N.S. Tamashiro, T.S. Miranda, S.S. Maciel, contributed to data acquisition, critically revised the manuscript; P.M. Duarte, M. Feres, contributed to conception, design, and data interpretation, drafted and critically revised the manuscript; L.C. Figueiredo, contributed to

data interpretation, critically revised the manuscript; M. Faveri, contributed to data analysis, critically revised the manuscript. All authors gave final approval and agree to be accountable for all aspects of the work.

Acknowledgments This study was supported by research grants 2011/14872-4 and 2013/01072-5 from São Paulo State Research Foundation (FAPESP, São Paulo, SP, Brazil). The authors thank Vanessa Renata Santos for carrying out the periodontal treatment of the patients. The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.

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