Australian Dental Journal

The official journal of the Australian Dental Association

Australian Dental Journal 2014; 59: 490–496 doi: 10.1111/adj.12221

Full-mouth scaling and root planing in type 2 diabetic subjects: one-year microbiological outcomes LC Figueiredo,* DC Souza,* VR Santos,* TS Miranda,* M Feres,* M Faveri,* PM Duarte* *Department of Periodontology, Dental Research Division, Guarulhos University, S~ao Paulo, Brazil.

ABSTRACT Background: This study evaluated the microbiological effects of full-mouth (FM) and partial-mouth (PM) scaling and root planing (SRP) in type 2 diabetic subjects with chronic periodontitis (ChP), up to 12 months. Methods: Thirty-four type 2 diabetic subjects with ChP received either FMSRP (n = 17), in two sessions within two consecutive days, or PMSRP (n = 17) in four sessions within 21 days. Six subgingival biofilm samples per subject were analysed by checkerboard DNA–DNA hybridization for 40 bacterial species at baseline, 3 and 12 months. Results: Both therapies significantly reduced the levels of the red complex species up to 12 months (p < 0.05). The levels of three putative pathogens from the orange complex were significantly reduced in the FMSRP group, whereas a single orange complex species was significantly decreased in the PMSRP group (p < 0.05). Furthermore, the proportions of the host-compatible Actinomyces species were significantly increased in both groups at 3 and 12 months. No significant differences were observed between groups for the counts and proportions of the individual species and the proportions of microbial complexes at any time point (p > 0.05). Conclusions: There were no differences in the bacterial species evaluated after FMSRP and PMSRP in the treatment of type 2 diabetic subjects with ChP, up to 12 months. Keywords: Chronic periodontitis, diabetes mellitus, microbiology, periodontal disease. Abbreviations and acronyms: BoP = bleeding on probing; CAL = clinical attachment level; ChP = chronic periodontitis; DM = diabetes mellitus; FM = full-mouth; FMSRP = full-mouth scaling and root planing; PCR = polymerase chain reaction; PD = probing depth; PM = partial-mouth; PMSRP = partial-mouth scaling and root planing; SRP = scaling and root planing. (Accepted for publication 17 March 2014.)

INTRODUCTION Conventional periodontal therapy includes supra- and subgingival scaling by means of manual and ultrasonic devices to disturb the pathogenic biofilm that triggers and maintains periodontal inflammation and, consequently, tissue breakdown1,2 Therefore, a successful periodontal therapy should not only improve clinical parameters (e.g. reduction of inflammation and probing depth [PD] and attachment level gain), but also significantly reduce the counts and proportions of pathogenic species.3 Partial-mouth (PM) scaling and root planing (SRP), a quadrant- or sextant-wise SRP performed in multiple sessions with weekly intervals, is one of the most common treatment protocols for periodontitis. However, considering the hypothesis that previously treated areas could be reinfected by pathogenic species from not yet treated sites, some studies evaluated whether full-mouth scaling and root planing (FMSRP) performed within a short period could avoid this bacte490

rial reinfection and lead to better clinical outcomes.4–9 Nonetheless, three systematic reviews, compiling data from several investigations have failed to show that FMSRP provides statistically significant clinical and microbiological advantages over PMSRP for the treatment of periodontitis in non-diabetic subjects.10–12 Assuming that FMSRP and PMSRP have similar clinical effectiveness, it would still be important to consider some practical advantages of the FMSRP protocol. Completing periodontal treatment in a single or in fewer appointments might have benefits for both public and private dental practices, and expands the offer of treatment modalities based on patients’ needs and preferences. This is particularly relevant if this therapeutic approach could be safely applied in groups at risk for periodontal and other infections, such as diabetic subjects. Delivering periodontal treatment in the short term could be especially important in the case of diabetic subjects, which often require special care while receiving dental treatment. © 2014 Australian Dental Association

Periodontal therapy in diabetic subjects Two previous studies13,14 showed that FMSRP and PMSRP did not differ in improving clinical parameters up to 12 months in type 2 diabetic subjects with chronic periodontitis. In addition, FMSRP and PMSRP presented similar effects in modulating the levels of cytokines and osteoclastogenesis related factors in such diabetic subjects.14 Although the abovementioned studies provided important insights into the clinical and immunological impacts of FMSRP and PMSRP in diabetic subjects, to date there is no evidence of the microbiological effects of these therapies in this group at risk for periodontitis. In fact, to our knowledge, no previous studies have thoroughly compared the effects of these two treatment modalities on the subgingival microbial profile in diabetic subjects. Therefore, the aim of the present study was to evaluate and compare the microbiological effects of FMSRP and PMSRP in type 2 diabetic subjects with chronic periodontitis, up to 12 months. MATERIALS AND METHODS Sample size calculation This study presents the microbiological data of a RCT14 designed and powered to compare the clinical effects of FMSRP and PMSRP. Briefly, the required sample size to assure adequate power was calculated considering differences of at least 1 mm for clinical attachment level (CAL) and a standard deviation of 1 mm between groups in initially deep pockets (≥7 mm). Calculations determined that 17 subjects per group would provide an 80% power, at a significance level of 5%. Subject population During the clinical phase of the study (December 2007 to March 2009), 34 subjects (aged 42–67 years) with type 2 diabetes mellitus (DM) for at least the past five years and presenting generalized chronic periodontitis were selected from the population referred to Guarulhos University. Guarulhos University’s Ethics Committee in Clinical Research previously approved this study protocol. The ClinicalTrials.gov identifier is NCT02062047. Inclusion/exclusion criteria All subjects were >30 years old, had at least 15 teeth excluding third molars and teeth designated for extraction, and more than 30% of sites with PD and concomitant CAL ≥4 mm at baseline. Exclusion criteria were pregnancy, lactation, current smoking and smoking within the past five years, periodontal and/or antibiotic therapies in the previous six months, © 2014 Australian Dental Association

routine use of mouthrinses containing antimicrobials in the previous two months, other systemic conditions that could affect the periodontal tissues, long-term administration of anti-inflammatory and immunosuppressive medications, periapical pathologies, orthodontic appliances and multiple systemic complications of DM. Experimental design and treatment protocols In this prospective, blinded, randomized and controlled trial subjects were firstly submitted to supragingival plaque and calculus control, and then randomly allocated to one of the following treatment groups: FMSRP (n = 17) or PMSRP (n = 17). FMSRP was performed in two appointments within two consecutive days, in a maximum of 24 hours and PMSRP (n = 17) was completed in four appointments within 21 days. Both therapies were performed under local anaesthesia using periodontal curettes and ultrasonic device. Subjects did not receive systemic antibiotics or local antimicrobials during the treatment phase or during the 12-month period of observation. Supportive therapy was performed at 3, 6 and 9 months post-therapies. Subjects received clinical assessment at baseline, 3, 6 and 12 months,14 and microbiological monitoring at baseline, 3 and 12 months. Clinical assessment One examiner (VRS) carried out all clinical measurements while a second clinician (TSM) performed the treatments. Visible plaque (0/1), gingival bleeding (0/1), bleeding on probing (BoP) (0/1), suppuration (0/1), PD and CAL were measured at six sites per tooth (mesiobuccal, buccal, distobuccal, distolingual, lingual and mesiolingual) in all teeth, excluding third molars. Microbiologic assessment Sample collection Six subgingival plaque samples per subject, two from each PD category (shallow [PD 7 mm]), were collected at baseline and at 3 and 12 months post-therapies. The selected sites were distributed in different quadrants and presented no furcation involvement. After supragingival plaque removal, subgingival plaque was sampled using individual sterile mini-Gracey curettes and immediately placed in separate Eppendorf tubes containing 0.15 ml of TE (10 mM Tris-HCl, 1 mM EDTA, pH 7.6). One hundred microlitres of 0.5 M NaOH were added to each tube and the samples were dispersed using a vortex mixer. 491

LC Figueiredo et al. Checkerboard DNA–DNA hybridization All microbiological analyses were performed at the Laboratory of Microbiology at Guarulhos University. Counts of 40 bacterial species were determined in each sample, using the checkerboard DNA–DNA hybridization technique.15 The samples were boiled for 10 minutes and neutralized using 0.8 ml of 5 M ammonium acetate. The DNA released was then placed into the extended slots (Minislot 30 apparatus; Immunetics, Cambridge, MA, USA) concentrated on a 15 9 15 cm positively-charged nylon membrane (Boehringer Mannheim, Indianapolis, IN, USA) and fixed to the membrane by baking it at 120 °C for 20 minutes. The membrane was placed in a Miniblotter 45 (Immunetics, Cambridge, MA, USA) with the lanes of DNA at 90° to the lanes of the device. Digoxigenin-labelled whole genomic DNA probes for 40 bacterial species were hybridized in individual lanes of the Miniblotter. After hybridization, the membranes were washed at high stringency and the DNA probes were detected using the antibody to digoxigenin, conjugated with alkaline phosphatase and chemiluminescence detection. The last two lanes in each run contained standards at concentrations of 105 and 106 cells of each species. Signals were evaluated visually by comparison with the standards at 105 and 106 bacterial cells for the test species on the same membrane by a calibrated examiner (k-test = 93%). The signals were recorded as: 0 = not detected; 1 = 106 cells. The sensitivity of this assay was adjusted to allow the detection of 104 cells of a given species by adjusting the concentration of each DNA probe. This procedure was carried out in order to provide the same sensitivity of detection for each species. Failure to detect a signal was recorded as 0, although conceivably, counts in the 1–1000 ranges could have been present. Statistical analysis Data were examined for normality by the Shapiro– Wilk test. As data did not present normality, nonparametric methods were used. The mean counts (9105) of individual bacterial species were averaged within each subject and then across subjects in both groups. The percentage of the total DNA probe counts was determined initially for each site, then per subject and averaged across subjects in each group. The significance of differences between groups for each microbiological parameter was analysed using the Mann–Whitney U-test. The Friedman test was used to test differences between the three time-points. The level of significance was set at 5%. Adjustments were made for multiple comparisons when the 40 bac492

terial species were evaluated.16 An overall p of 0.05 = 1-(1 k)40 was computed, where k is the desired individual p value. Therefore, p values of 0.05). The species found in highest mean counts at baseline were those from the red complex, followed by the orange complex species. Conversely, the two groups presented reduced levels of several host-compatible species at baseline. Figure 2 illustrates the mean counts of the 40 bacterial species from baseline to 3 and 12 months posttherapies. Both treatments significantly reduced the counts of the three red complex pathogens, Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia (p < 0.05). The levels of three putative pathogens from the orange complex (Eubacterium nodatum, Fusobacterium nucleatum ssp. nucleatum and Fusobacterium nucleatum ssp. polymorphum) were reduced in the FMSRP group, whereas E. nodatum was the only orange complex species that was significantly reduced in the PMSRP group (p < 0.05). The counts of Actinomyces oris and Actinomyces gerencseriae were significantly increased at 12 months in subjects of the FMSRP and PMSRP groups, respectively (p < 0.05). Figure 3 presents the mean proportions of the microbial complexes at baseline, 3 and 12 months. The proportion of red complex species was significantly reduced by the FMSRP and PMSRP therapies over time (p < 0.05). The proportions of the orange complex species were also reduced by both treatments; however, these reductions were not statistically significant (p > 0.05). In addition, the proportions of the host-compatible Actinomyces complex were significantly increased in both groups at 3 and 12 months (p < 0.05). © 2014 Australian Dental Association

Periodontal therapy in diabetic subjects DNA probe counts (%)

counts x 105 Baseline

1.9

8.3

12 months

14.7 –10.9

–4.5

A.gerencseriae A.israelii A.naeslundii A.oris A.odontolyticus V.parvulla S.gordonii S.intermedius S.mitis S.oralis S.sanguinis A.a C.gingivalis C.ochracea C.sputigena E.corrodens C.gracilis C.rectus C.showae E.nodatum F.nucleatum.ssp.nucleatum F .nucleatum.ssp.polymorphum F.nucleatum.ssp.vincentii F.periodonticum P.micra P.intermedia P.nigrescens S.constellatus T.forsythia P.gingivalis T.denticola E.saburreum G.morbillorum L.buccalis P.acnes P.melaninogenica N.mucosa S.anginosus S.noxia T.socranskii

1.9

8.3

14.7

Baseline 6.6 2.1

–7.1

12 months –2.5 2.1

6.6

Actinomyces

Purple

Yellow

Green

Orange

Red

Others

PMSRP

FMSRP

Fig. 1 Mean counts (910 ) and mean percentage of DNA probe for the 40 test species at baseline and mean changes from baseline to 12 months posttherapies. The species were ordered according to the microbial complexes described by Socransky et al.17 There were no differences between groups, according to the Mann–Whitney test (p > 0.00127) (FMSRP: full-mouth scaling and root planing; PMSRP: partial-mouth scaling and root planing). 5

DISCUSSION Previous studies13,14 have shown that FMSRP and PMSRP did not differ significantly in terms of clinical and immunological outcomes for the treatment of diabetic subjects with chronic periodontitis. In order to supplement these previous data regarding the impact of FMSRP in diabetics, the current study evaluated the microbiological effects of FMSRP and PMSRP in type 2 diabetic subjects with generalized chronic periodontitis. Overall, the results indicated that both therapies were equally effective in reducing the subgingival pathogenic microbial load over a short period (three months). Subsequently, the microbiological benefits achieved at three months were sustained up to the 12-month in both groups. These findings suggest that the mechanical periodontal treatment performed within two consecutive days or in weekly appointments produced comparable microbiological benefits up to 12 months in the diabetic subjects, supporting our previous clinical and immunological results.13,14 © 2014 Australian Dental Association

The current study demonstrated that FMSRP and PMSRP significantly reduced the individual levels and the proportions of three well-recognized periodontal pathogens (T. forsythia, P. gingivalis and T. denticola) in type 2 diabetic subjects. In addition, both therapies also increased the proportion of the host-compatible Actinomyces species. The FMSRP was able to reduce the levels of three putative pathogens from the orange complex (E. nodatum, F. nucleatum ssp. Nucleatum and F. nucleatum ssp. polymorphum), whereas the PMSRP decreased the levels of a single orange complex species (E. nodatum). With the exception of these minor differences, overall both therapies did not differ significantly in altering the subgingival pathogenic microbial profile from baseline to 3 and 12 months. To the authors’ knowledge, this is the first study that has thoroughly compared the effects of FMSRP and PMSRP in changing the subgingival microbial composition in diabetic subjects with periodontitis. In fact, a systematic microbiological comparison between these two therapies, including the evaluation of 40 bacterial 493

LC Figueiredo et al.

Fig. 2 Mean counts of the 40 bacteria species from baseline to 3 and 12 months post-therapies. Each area of the graphs represent the mean counts of a single species over time. * Differences over time within the same group by Friedman test (p < 0.05). (FMSRP: full-mouth scaling and root planing; PMSRP: partial-mouth scaling and root planing).

species in a high number of plaque samples, is not available even for systemically healthy individuals. The current microbiological findings in diabetics are in line with previous studies that compared the microbiological effect of FMSRP with PMSRP in non-diabetic subjects by assessing a few bacterial species using polymerase chain reaction (PCR).5–9,11 Apatzidou et al.5 observed that FMSRP and PMSRP did not differ in terms of microbiological benefits at six months post-therapies. Koshy et al.6 also demonstrated no microbiological differences between quadrant-wise and single-visit full-mouth ultrasonic debridement at six months. Jervøe-Storm et al.7 showed similar levels of Aggregatibacter actinomycetemcomitans, F. nucleatum ssp., P. gingivalis, Prevotella intermedia, T. denticola and T. forsythia following the FMSRP and PMSRP approaches up to six months. Similar results were also demonstrated for the levels of A. actinomycetemcomitans, P. gingivalis, P. intermedia, Dialister pneumosintes, Campylobacter rectus and Parvimonas micra at eight months after both therapies.8 Similarly, Zijnge et al.9 showed no significant differences between FMSRP and PMSRP in the reduction of sites that were positive for A. actinomycetemcomitans, P. gingivalis, T. denticola, F. nucleatum and T. forsythia after three months. In contrast to the 494

Fig. 3 Pie charts of the mean proportion (%) of each microbial complex at baseline, 3 and 12 months post-therapies for the FMSRP and PMSRP groups. Each coloured sector of the pie charts represents the proportions of each microbial complex17 as well as Actinomyces (blue) and ‘other’ bacterial species (grey). Different letters represent significant differences over time within each treatment group by Friedman test (p < 0.05). There were no differences between groups, according to the Mann–Whitney test (p > 0.05) (FMSRP: full-mouth scaling and root planing; PMSRP: partial-mouth scaling and root planing). © 2014 Australian Dental Association

Periodontal therapy in diabetic subjects present and the above-mentioned studies, Quirynen et al.4, using dark-field microscopy and cultural technique, showed significantly greater reductions in subgingival pathogens up to eight months following FMSRP, compared to PMSRP. Likewise, Serrano et al.18 observed microbiological advantages of FMSRP over PMSRP. However, the authors provided only a short-term evaluation (4–6 weeks) after treatments.18 It should be noted that the current and all of the above-mentioned studies (Quirynen et al.,4 Apatzidou et al.,5 Koshy et al.,6 Jervøe-Storm et al.,7 Swierkot et al.,8 Zijnge et al.,9 Lang et al.,11 Serrano et al.18) verified that the subgingival microbiota improved significantly from baseline to post-therapy evaluations for both the FMSRP and PMSRP approaches. In fact, it is well documented that the proportions of periodontal pathogens are found at higher levels before than after SRP in subjects or at sites that responded clinically well, since positive microbiological changes must precede clinical improvements.3 Accordingly, our previous clinical study14 in these diabetic subjects demonstrated that both FMSRP and PMSRP promoted improvements in clinical parameters such as PD, CAL and BoP, and the therapies did not differ in terms of clinical outcomes up to 12 months. Other studies with similar experimental designs have also showed that positive microbiological changes after FMSRP and PMSRP led to significant clinical improvements in non-diabetic subjects, and the approaches did not differ in their clinical effects.5–9,19,20 The current study has limitations. It represents secondary analyses of microbiological data collected during a RCT designed based on clinical outcomes. In addition, since our first post-therapy biofilm sampling was performed at three months, it is not known whether any microbiological differences might have occurred between groups before three months, especially immediately after treatments. In spite of these limitations, this study presents the most comprehensive comparison of the microbiological effects of FMSRP and PMSRP. It was originally suggested that application of FMSRP in the short term would promote a better outcome than the application of PMSRP within week intervals by preventing the bacterial translocation from untreated to already treated periodontal sites.4 On the other hand, due to the restricted number of appointments required for FMSRP, one could argue that this approach would promote a poorer outcome, due to the risk of leaving greater amounts of residual calculus and/or the reduction in the number of sessions of plaque control reinforcement. However, the findings from the current and several previous studies5–14 have provided considerable evidence that the FMSRP approach does not result in a worse or better clinical or microbiological outcome than the PMSRP © 2014 Australian Dental Association

approach. Therefore, as FMSRP and PMSRP produce similar beneficial clinical and microbiological results and are well tolerated by patients, both modalities seem to be equally adequate for the initial nonsurgical periodontal treatment in diabetics and non-diabetics and should be used according to patient’s needs and preferences and operator abilities. In conclusion, FMSRP and PMSRP did not differ in terms of microbiological benefits for the treatment of type 2 diabetic subjects with chronic periodontitis up to 12 months. REFERENCES 1. Garrett JS. Effects of nonsurgical periodontal therapy on periodontitis in humans. A review. J Clin Periodontol 1983;10:515– 523. 2. Drisko CH. Nonsurgical periodontal therapy. Periodontol 2000 2001;25:77–88. 3. Haffajee AD, Cugini MA, Dibart S, Smith C, Kent RL Jr, Socransky SS. The effect of SRP on the clinical and microbiological parameters of periodontal diseases. J Clin Periodontol 1997;24:324–334. 4. Quirynen M, Mongardini C, de Soete M, et al. The role of chlorhexidine in the one-stage full-mouth disinfection treatment of patients with advanced adult periodontitis. Long-term clinical and microbiological observations. J Clin Periodontol 2000;27:578–589. 5. Apatzidou DA, Riggio MP, Kinane DF. Quadrant root planing versus same-day full-mouth root planing. II. Microbiological findings. J Clin Periodontol 2004;31:141–148. 6. Koshy G, Kawashima Y, Kiji M, et al. Effects of single-visit full-mouth ultrasonic debridement versus quadrant-wise ultrasonic debridement. J Clin Periodontol 2005;32:734–743. 7. Jervøe-Storm PM, AlAhdab H, Semaan E, Fimmers R, Jepsen S. Microbiological outcomes of quadrant versus full-mouth root planing as monitored by real-time PCR. J Clin Periodontol 2007;34:156–163. 8. Swierkot K, Nonnenmacher CI, Mutters R, Flores-de-Jacoby L, Mengel R. One-stage full-mouth disinfection versus quadrant and full-mouth root planing. J Clin Periodontol 2009;36:240–249. 9. Zijnge V, Meijer HF, Lie MA, et al. The recolonization hypothesis in a full-mouth or multiple-session treatment protocol: a blinded, randomized clinical trial. J Clin Periodontol 2010;37: 518–525. 10. Eberhard J, Jervøe-Storm PM, Needleman I, Worthington H, Jepsen S. Full-mouth treatment concepts for chronic periodontitis: a systematic review. J Clin Periodontol 2008;35:591–604. 11. Lang NP, Tan WC, Kr€ahenmann MA, Zwahlen M. A systematic review of the effects of full-mouth debridement with and without antiseptics in patients with chronic periodontitis. J Clin Periodontol 2008;35(8 Suppl):8–21. 12. Farman M, Joshi RI. Full-mouth treatment versus quadrant root surface debridement in the treatment of chronic periodontitis: a systematic review. Br Dent J 2008;8;205:E18; discussion 496–497. 13. Santos VR, Lima JA, De Mendoncßa AC, Maximo MB, Faveri M, Duarte PM. Effectiveness of full-mouth and partial-mouth scaling and root planing in treating chronic periodontitis in subjects with type 2 diabetes. J Periodontol 2009;80:1237–1245. 14. Santos VR, Ribeiro FV, Lima JA, et al. Partial- and full-mouth scaling and root planing in type 2 diabetic subjects: a 12-month follow-up of clinical parameters and levels of cytokines and osteoclastogenesis-related factors. J Periodontal Res 2012;47:45–54. 495

LC Figueiredo et al. 15. Socransky SS, Smith C, Martin L, Paster BJ, Dewhirst FE, Levin AE. ‘Checkerboard’ DNA–DNA hybridization. Biotechniques 1994;17:788–792. 16. Socransky SS, Haffajee AD, Smith C, Dibart S. Relation of counts of microbial species to clinical status at the sampled site. J Clin Periodontol 1991;18:766–775. 17. Socransky SS, Haffajee AD, Cugini MA, Smith C, Kent RL Jr. Microbial complexes in subgingival plaque. J Clin Periodontol 1998;25:134–144. 18. Serrano C, Torres N, Bejarano A, Cavie M, Castellanos ME. Clinical and microbiological comparison of three non-surgical protocols for the initial treatment of chronic periodontitis. J Int Acad Periodontol 2011;13:17–26. 19. Apatzidou DA, Kinane DF. Quadrant root planing versus sameday full-mouth root planing. I. Clinical findings. J Clin Periodontol 2004;31:132–140.

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20. Jervøe-Storm PM, Semaan E, AlAhdab H, Engel S, Fimmers R, Jepsen S. Clinical outcomes of quadrant root planing versus full-mouth root planing. J Clin Periodontol 2006;33: 209–215.

Address for correspondence: Poliana Mendes Duarte Universidade Guarulhos Centro de P os-Graduacß~ao e Pesquisa Pracßa Tereza Cristina, 229 – Centro Guarulhos – SP CEP: 07.023-070 Brazil Email: [email protected]

© 2014 Australian Dental Association

Full-mouth scaling and root planing in type 2 diabetic subjects: one-year microbiological outcomes.

This study evaluated the microbiological effects of full-mouth (FM) and partial-mouth (PM) scaling and root planing (SRP) in type 2 diabetic subjects ...
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