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Acta Odontológica Latinoamericana

versión On-line ISSN 1852-4834

Acta odontol. latinoam. vol.27 no.2 Buenos Aires set. 2014

 

ARTÍCULOS ORIGINALES

Microbiological effects of periodontal therapy plus azithromycin in patients with diabetes: results from a randomized clinical trial

 

Juan P. Hincapié1, Cesar A. Castrillón1, Fanny L. Yepes1, Natalia Roldan1, María A. Becerra1, Sandra M. Moreno2, Jessika Consuegra2, Adolfo Contreras2, Javier E. Botero1

1 Faculty of Dentistry, University of Antioquia, Medellín, Colombia.
2 School of Dentistry, University del Valle, Cali, Colombia

CORRESPONDENCE Dr. Javier Enrique Botero, Facultad de Odontologia, Universidad de Antioquia, carrera 70 No. 52-21, Medellin, Colombia. e-mail: drjavo@yahoo.com


ABSTRACT

Current evidence suggests that periodontal infection may aggravate diabetes control. The aim of this study was to determine the changes in the frequency with which Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola and Aggregatibacter actinomycetemcomitans were detected in patients with diabetes with the use of non-surgical therapy plus azithromycin in a randomized clinical trial. One hundred and five (105) patients with diabetes and chronic periodontitis were randomly assigned to three treatment groups: subgingival mechanical therapy with azithromycin, subgingival mechanical therapy with placebo and supragingival prophylaxis with azithromycin. Complete periodontal clinical examinations and detection of periodontal pathogens using polymerase chain reaction were carried out at baseline, 3, 6 and 9 months after periodontal therapy. The frequency with which Porphyromonas gingivalis, Treponemadenticola and Aggregatibacter actinomycetemcomitans were detected decreased at 3 months in all groups. Tannerella forsythia increased after3 months in all groups. All organisms had similar frequencies at 9 months in all groups. Subgingival mechanical therapy with adjunctive azithromycin had no additional effect on the frequency with which the periodontal pathogens investigated were detected in patients with diabetes.

Key words: Diabetes mellitus; Chronic periodontitis; Microbiota; Azythromycin.

RESUMEN

Efectos microbiológicos de la terapia periodontal más azitromicina en pacientes diabéticos: resultados de un estudio clínico controlado aleatorizado

Evidencia actual sugiere que la infeccion periodontal puede agravar el control de la diabetes. El objetivo de este estudio fue determinar los cambios en la frecuencia de deteccion de Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola y Aggregatibacter actinomycetemcomitans en pa cientes con diabetes con el uso de terapia mecanica no quirurgica mas azitromicina en un estudio clinico controlado aleatorizado.Ciento cinco pacientes (105) con diabetes y perio - dontitis fueron asignados aleatoriamente en tres grupos: terapia mecanica mas azitromicina, terapia mecanica mas placebo y profilaxis supragingival mas azitromicina. Un analisis perio - dontal completo y deteccion de patogenos perio dontales por medio de reaccion en cadena de la polimerasa (PCR) se realizaron al inicio, 3, 6 y 9 meses despues de la terapia pe - riodontal. La frecuencia de deteccion de Porphyromonas gingivalis, Treponema denticola y Aggregatibacter actinomycetemcomitans disminuyo a los 3 meses en todos los grupos. La frecuencia de deteccion de Tannerella forsythia se incremento a los 3 meses en todos los grupos. Todos los patogenos periodontales mostraron una frecuencia de deteccion similar a los 9 meses en todos los grupos. La terapia periodontal mas azitromicina no tuvo efectos adicionales sobre la frecuencia de deteccion de los patogenos periodontales investigados en pacientes diabeticos.

Palabras claves: Diabetes mellitus; Periodontitis cronica; Microbiota; Azitromicina.


 

INTRODUCTION

Patients with poor glycemic control present increased loss in the levels of periodontal attachment (CAL) and in consequence higher tooth loss 1-3. Furthermore, type 1 and 2 diabetes patients are equally susceptible to further periodontal attachment and tooth loss related to the degree of glucose control rather than to diabetes etiology 2, 3. On the other hand, periodontitis may impair glucose control in patients with diabetes by promoting systemic inflammation 4-6. It has been observed that periodontal treatment has beneficial effects on patients with diabetes7. Non- surgical periodontal treatment with or without the use of systemic antibiotics showed not only an improvement in periodontal parameters but also in the levels of glycated hemoglobin (HbA1C) in poorly controlled diabetes patients compared to placebo groups in recent randomized clinical trials 8, 9. A reduction of 0.40% in HbA1C has been reported, but further studies are necessary to confirm these results 10.
Studies have found that the presence of P. gingivalis after periodontal treatment was associated with poor glucose control11. In a previous study, we found that the frequency of P.gingivalis, Tannerella forsythia and Treponema denticola (red complex microorganisms) was lower in patients with diabetes while the frequency of A. actinomycetemcomitans was higher than in non-diabetic patients12. However, Yuan et al. 13 found no difference in the subgingival microbiota between patients with and without diabetes. Further analysis of the subgingival microbiota in patients with diabetes could help improve periodontal treatment and further the understanding of periodontalsystemic relationships.
Results of studies have shown improvement in periodontal parameters as well as in the counts of red and orange complex microorganisms with the combined use of non-surgical therapy and azithromycin14. Azithromycin is a macrolide that has anti-inflammatory effects, accumulation at inflamed sites and slow release through periodontal pockets that could add beneficial effects to non-surgical therapy 15, 16. To the extent of our knowledge there is no study addressing the use of azithromycin with periodontal therapy in subjects with diabetes.
The aim of this study was to determine changes in the frequency with which P. gingivalis, T. forsythia, T. denticola and A. actinomycetemcomitans were detected in patients with diabetes who were treated with non-surgical therapy plus azithromycin in a randomized clinical trial.

MATERIALS AND METHODS

Study Design
The microbiological results presented here are from a secondary data analysis of a randomized clinical trial (Clinical Trials NCT01271231) that was previously published 9. The impact of periodontal intervention on periodontal parameters (probing depth, clinical attachment loss) and glycemic control (reduction of glycated hemoglobin and glycemia) over a 9-month period have been analyzed else where 9.Briefly, the study was designed in accordance with the Declaration of Helsinki and approved by the Ethics Committee in Research of the Faculty of Dentistry (Uniersity of Antioquia 010-2011) and Hospital San Vicente Foundation (Medellin, Colombia). Potential candidates from the Hospital San Vicente Foundation were invited to participate in the study between January 2011 and July 2012. Volunteers were informed of the purpose of the study and screened for clinical parameters. Inclusion criteria included: ≥ 18 years of age, confirmed diagnosis of diabetes type I or II with duration of at least 2 years, ≥ 10 teeth present and chronic moderate periodontitis (ChP). ChP was defined as the presence of 2 or more interproximal sites with clinical attachment loss (CAL) ≥ 4 mm in different teeth, or, 2 or more interproximal sites with probing depth (PD) ≥ 5 mm in different teeth17. Exclusion criteria were administration of systemic antibiotics in the previous 3 months, any use of immunosuppressants or bisphosphonates, pregnancy, HIV, rheumatoid arthritis, cardiovascular disease or other systemic condition that contraindicated periodontal treatment. Patients were under treatment and control for diabetes at the hospital and received medications for diabetes (Insulin, Glibenclamide, Metformin) and other medications for hypertension (Enalapril, Captopril, Losartan) and cholesterol (Statins). The mean duration of diabetes in the volunteers was 13 years. Participants signed a written informed consent prior to the beginning of the study.

Sample, Allocation and Clinical Procedures
The original sample of 90 patients was calculated to detect changes of 0.7% in the values of glycated hemoglobin (HbA1C), with 0.05 significance and 85% power. To compensate for dropouts, the sample was increased by 10%. Patients were allocated to one of three groups by a block randomization process performed by one the investigators (JEB) independent of the clinical intervention. The following groups were formed: AZSRP group: azithromycin (MK, Cali, Colombia) 500mg/day x 3 days plus subgingival scaling using an ultrasonic device (Cavitron, Dentsply, York, PA, USA) at one session at medium intensity until the root surface was smooth. Patients were instructed to take one tablet (500 mg) per day during a threeday period. PBSRP group: Placebo 500mg /day x 3 days plus subgingival scaling using an ultrasonic device at one session at medium intensity until the root surface was smooth. Patients were instructed to take one tablet (500 mg) per day during a three-day period. AZPRO group: azithromycin 500mg/day x 3 days plus supragingival prophylaxis with polishing cups. Supragingival prophylaxis was repeated one week later. Patients were instructed to take one tablet (500 mg) per day during a three-day period. All patients assigned to AZPRO group received subgingival scaling at the end of study.
Azithromycin and placebo tablets were identical in shape and color and were given to patients in closed opaque coded envelopes with the corresponding instructions and contact number in the case of any adverse effects. Placebo tablets were manufactured by the Faculty of Pharmaceutical Chemistry (University of Antioquia, Medellin, Colombia). The type of antibiotic / placebo assigned was unknown by the patient and the clinicians who performed the interventions and clinical examinations.
A full periodontal examination was performed by 2 clinicians (JPH, CAC), who had been previously calibrated when clinical parameters were collected (κ>0.80). PD, CAL and bleeding on probing (BOP) were measured at six sites around all teeth excluding third molars using a periodontal probe (UNC- 15 USA Delta, Chicago, USA). Plaque index (PI) was measured with disclosing agent and establishing the percentage (%) of surfaces with plaque. HbA1C values (%) were also recorded. All parameters were measured at baseline 3, 6 and 9 months after periodontal interventions. Periodontal interventions were performed by 3 clinicians (FLY, NR, MAB). Any patient that presented worsening of periodontal or diabetes condition was removed from the study and treatment was initiated. Patients were contacted during the first week to ensure compliance with taking tablets and to check for any side effects: e.g. gastrointestinal problems, dizziness, etc.

Microbiological Analysis
Subgingival plaque samples were obtained from the 3 deepest sites by inserting sterile paper points to the bottom of the pocket for 30 seconds. The paper points were then transferred to Eppendorf vials and stored at -70°C until they were analyzed. DNA was extracted by the methods described by Botero et al. 18. P. gingivalis, T. forsythia, T. denticola and A. actinomycetemcomitans were detected by polymerase chain reaction (PCR) using primers and conditions described elsewhere19. Data were expressed as positive or negative and the frequency (%) of each microorganism was determined. Microbial samples were obtained at baseline and then at 3, 6 and 9 months after periodontal interventions.

Statistical Analysis
Demographic and clinical data are presented as mean SD unless otherwise indicated. To test for differences in clinical parameters, the paired and unpaired t test and Mann-Whitney test were used when appropriate. The primary outcome was the change in the detection of each microorganism and this was assessed with ANOVA and Chi2 where indicated. Statistical software (GraphPad Prism 5, GraphPad Software, San Diego, CA, USA) was used and statistical significance was assumed when P≤0.05.

RESULTS

Figure 1 shows the flowchart of patient inclusion in the study. Periodontal clinical parameters and HbA1C values were similar in all the allocated groups. There were more female patients participating in the study but they were balanced in all groups (Table 1). No side effects were reported in this study. Changes in the frequency of detection of red complex microorganisms and A. actinomycetemcomitans are presented in Table 2. The frequency of detection of T. forsythia tended to increase at 3 months in all groups. Nonetheless, the placebo group presented higher frequencies of T. forsythia at 9 months than the other groups. In contrast, P. gingivalis, T. denticola and A. actinomycetemcomitans were detected less frequently at 3 months and then increased progressively over the 9-month period. The frequency of detection of all periodontal pathogens studied was similar at 9 months. Table 3 depicts the changes in clinical attachment levels. An improvement in CAL was observed in the AZSRP and PBSRP groups, while no improvement was observed in the AZPRO group (Table 3). Changes in glycated hemoglobin values are presented in Table 4. A reduction in HbA1C was observed in the AZSRP and PBSRP groups over the 9-month follow-up. However, the reduction was greater in the AZSRP group. No improvement was observed in the AZPRO group.


Fig.1
: Flowchart of patient inclusion in the study.

Table 1: Demographic and clinical description of the study sample.

Table 2: Changes in the detection frequency (%) of periodontal pathogens before and after interventions.

Table 3: Changes in clinical attachment levels before and after interventions.

Table 4: Changes in HbA1C values (%) before and after interventions.

Discussion

The subgingival microbiota associated to the development of periodontitis is frequently composed of microorganisms of the red complex (T. forsythia, P. gingivalis and T. denticola) and A. actinomycetemcomitans19. While it is accepted that diabetes affects the progression of periodontitis, there is no consensus on how diabetes affects the subgingival microbiota. In previous studies, these microorganisms were detected in similar proportions as in systemically healthy patients with periodontitis11-14. The establishment of chronic subgingival infection in patients with diabetes and periodontitis may preclude the appropriate control of glycemia11. Hence, treatment of periodontitis and infection is an important objective in patients with diabetes. Azithromycin has been suggested for the treatment of periodontitis due to its antimicrobial activity, high compliance protocol, anti-inflammatory activity and persistence at low levels in macrophages and fibroblasts with good clinical results20, 21. To the extent of our knowledge, this is the first RCT to assess the microbiological effects of azithromycin as adjunct to periodontal mechanical therapy in patients with diabetes. All microorganisms analyzed in this study showed a marked reduction at 3 months except for T. forsythia. Overall, the reduction in the frequency detection of red complex microorganisms and A. actinomycetemcomitans was slightly better with the adjunctive use of azithromycin at 3 and 6 months. The frequency of detection of these microorganisms was similar at 9 months. Due to limited information regarding the use of azithromycin in patients with diabetes and periodontitis, the results observed in this study are comparable to other studies with chronic and aggressive periodontitis22-24. However, this study is limited since a non-quantitative PCR technique was used to detect the microorganisms and hence it is not possible to determine changes in the bacterial counts. It would be relevant to test whether small changes in the microbiota are related to changes in clinical parameters and in this case, glycemic control in patients with diabetes and periodontitis. The studies by Han et al. 23 and Emingil et al.24 showed that there is no significant difference in the counts of A. actinomycetemcomitans, P. gingivalis and T. forsythia and clinical parameters when compared to SRP and placebo in otherwise healthy subjects. It is not known whether the systemic condition of our patients may have influenced the results. In contrast, the use of the combination of amoxicillin and metronidazole appears to provide the best clinical and microbiological results available25. Differences in the pharmacological properties of different antibiotics may account for these differences. The microbiological adjunctive effects of azithromycin for the treatment of periodontitis remain to be elucidated. Although the detection of red complex and A. actinomycetemcomitans was similar in the AZSRP and PBSRP groups, clinical parameters and glycemic control showed an improvement when azithromycin was used as adjunct to mechanical therapy in patients with diabetes and this is analyzed and reported in a previous article 9. Despite the moderate beneficial effects, it is possible that the antiinflammatory effects of azithromycin helped to reduce periodontal inflammation independently of high microbial suppression and consequently improved the values in HbA1C21. Further studies are needed to understand the immuno-modulatory effects of azithromycin and its consequences on periodontal parameters. One shortcoming of this study is attrition of the study sample as results of patients lost to follow-up at 9 months. However, most beneficial effects are believed to occur within the first 6 months after therapy and results may have been maintained. In this study, variables other than the antimicrobial effects of azithromycin such as diabetes and medical control and periodontal maintenance may have an influence on the results. Long-term studies on larger samples are necessary to comprehend the results of periodontal therapy in patients with diabetes. The results from the present RCT indicates that SRP therapy with adjunctive azithromycin had no additional effect compared to SRP and placebo alone on the frequency of detection of the periodontal pathogens investigated in patients with diabetes.

ACKNOWLEDGEMENTS

The authors declare no conflict of interests. This study was supported by a grant from the Research Development Committee of the University of Antioquia (CODI 010-2011, Medellin Colombia). J.P.H., C.A.C., F.L.Y., N.R., M.A.B. researched data; J.C., S.M. molecular detection of pathogens and A.C. and J.E.B. data analysis and wrote the manuscript. All authors contributed to discussion and review/edited the manuscript.

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