Efficacy of Azithromycin as an Adjunct to Non-Surgical Periodontal Treatment
Efficacy of Azithromycin as an Adjunct to Non-Surgical Periodontal Treatment
This study evaluates the effect of the systemic administration of azithromycin as a therapy
 associated with non-surgical periodontal treatment, in the clinical and microbiological
 variables of patients with Severe Periodontitis with moderate rate of progression (Stage III
 grade B). A double-blind, randomized, controlled trial was conducted with 18 voluntary
 patients with a history of moderate to severe chronic periodontitis, who met the inclusion
 and exclusion criteria and signed an informed consent.

 The intervention group received Scaling and Root Planning (SRP) plus azithromycin, and the
 control group received SRP plus placebo. Probing Pocket Depth (PPD) Clinical Attachment Level
 (CAL), Plaque Index (PI) and Bleeding on Probing (BoP) were evaluated as clinical variables
 while Porphyromonas gingivalis (Pg), Tannerella forsythia (Tf), Treponema denticola (Td),
 Fusobacterium nucleatum (Fn) were the microbiological variables detected by conventional
 Polymerase chain reaction (PCR).
Definition of the sample: Patients over 18 years old admitted to the Diagnostic Unit of the
 Faculty of Dentistry (DUFD), Viña del Mar campus of the Universidad Nacional Andres Bello
 (UNAB) during 2016, with a diagnosis of severe periodontitis, meeting both the inclusion and
 exclusion criteria and voluntarily agree to participate of the present research work, signing
 an informed consent. The chosen participants were informed of the nature of the research,
 potential risks and compensation for participating in the study, obtaining informed consent
 from every patient.

 Sample Size Calculation: To calculate the minimum sample size necessary for the constitution
 of the control and intervention groups, the variance of the probing depth difference were
 considered as fixed values, which were based on the results described above.Considering the
 above and using a level of significance of 5%, a statistical power of 80% and an estimation
 error of 1 mm, at least 7 patients were obtained for each group, giving a total of 14 minimum
 subjects to study.

 Protocol and clinical exam: Out of a total of 32 patients who were selected, 20 of those who
 met the inclusion criteria were randomly assigned by the study coordinator to one of the two
 groups using a random generation sequence employing a software. Two subjects declined to
 participate in the research, so the study had 9 participants in each group. In this way, two
 groups were formed to investigate: an intervention group that received SRP plus 500 mg of
 azithromycin once a day for three days immediately after the SRP, and a control group that
 received SRP plus placebo under the same conditions as the intervention group.

 Randomization: Only the main investigator, who made the randomization by using the Epidat 4.0
 program, had knowledge about the content of the containers and the group to which each
 patient belonged in the study, therefore, was in charge of labeling the containers, achieving
 in this way that collaborators and participants in the investigation did not know their
 content until after the study was finished.

 Standardization and calibration: The data was collected using instruments and supplies with
 the same commercial brands, in a single chair, under the same lighting and by a single
 examiner in the case of clinical data and another examiner for microbiological variables.

 For standardization, measurements were performed under the same conditions and using the same
 type of instrument, in order to reduce the associated bias. The instruments used for data
 collection were all the same in design and brand: Basic Examination Kit: Mirror, caries
 probe, tweezers (Hu-Friedy® Manufacturing Inc., Chicago, IL, USA), manual periodontal probe
 (North Carolina Probe. Hu-Friedy® Manufacturing Inc., Chicago, IL, USA), plaque finder
 tablets (Curaprox © CURADEN AG, Switzerland) and number 40 sterile paper cones (Johnson &
 Johnson, Tokyo, Japan).As there was only one examiner (KL) involved in the study,
 intra-examiner reliability assessment was executed. This examiner (intra-class correlation
 coefficient of 0.96 and 0.95 for PPD and CAL), recorded the all stipulated variables, in a
 clinical file designed especially for the study. Microbiological sampling and NSPT were
 performed by a single clinician (PO).The complete process of PCR was performed by a
 biochemist belonging to the microbiology laboratory, Santiago campus of the UNAB.

 Periodontopathogens: The presence of periodontopathogens was considered when exposing the
 agarose gel of the electrophoresis to ultraviolet light, a band at the site of the 197 base
 pairs (bp) was observed in the lane possessing the patient's subgingival bioflora sample of
 Pg, 316 base pairs for Td, 745 for Tf and 167 for Fn.

 Data collection and instrument: Prior to the data collection, all subjects belonging to the
 study were asked for a panoramic radiograph for diagnostic purposes. Subsequently, the
 periodontal examination was performed registering the clinical variables described above in a
 clinical record.

 Obtaining microbiological samples: The microbiological examination was performed before SRP
 on both groups. The collection of microbiological samples was based on a protocol that is
 used at the University of Chile. Using sterile paper cones number 40, a sample of subgingival
 bioflora was taken from the site with the highest CAL, within those with PD ≥ 5mm, and an
 oral hygiene instruction was performed. Before the collection of subgingival bioflora, the
 area was cleaned with a sterile gauze to eliminate the supragingival biofilm. Then, with a
 tweezer, a paper cone was taken, and its tip was placed in the sulcus of the study site for
 20 seconds, to ensure the absorption of the crevicular fluid and subgingival bioflora. Each
 biological sample obtained was suspended in an Eppendorf tube with 1 ml of distilled water
 and transported in a refrigerator at a temperature of 4°C approximately, within a time not
 exceeding 3 hours, to be then temporarily stored at -80°C, until its transfer and subsequent
 extraction of deoxyribonucleic acid (DNA) and PCR technique processing. The time between
 sampling and DNA extraction did not exceed 48 hours, to avoid deterioration of the biological
 material.

 Periodontal Treatment: Once the preliminary collection of all the variables was completed,
 non-surgical periodontal treatment (NSPT) was carried out, beginning with supra and
 subgingival full-mouth decontamination, using an electric piezo (DTE®, Guilin Woodpecker
 Medical Instrument Co., Ltd., Guilin, Guangxi , People's Republic of China), followed by root
 planning at sites presenting PD ≥ 5mm and CAL≥ 4mm, using Gracey curettes (Hu-Friedy®
 Manufacturing Inc., Chicago, Illinois (IL), USA). This therapy was performed in all patients,
 both in the intervention and control group, under full mouth treatment protocol, in 1 or 2
 work sessions, lasting from 1 to 2 hours, achieving treatment within 24 hours.

 Azithromycin or placebo administration: At the end of the last treatment session, the
 antibiotic azithromycin (Azithromycin, Laboratory Chile) or a placebo (Lactose, Galenic
 Pharmacy) was administered, depending on the group, intervention or control respectively, to
 which the patient belonged. For both therapies, the administration regimen was 1 tablet of
 500 mg orally per day, for 3 consecutive days.To maintain the double-blind, the containers
 used for the azithromycin and placebo presented the same characteristics regarding size and
 color. About the azithromycin tablets and placebo, these were visually identical.

 Subsequently, at the first, third and sixth month after the end of the non-surgical
 treatment, a new collection of all the clinical variables measured at the beginning of the
 study was performed, while the microbiological variables were measured again at the third and
 sixth months after treatment, from the same site used in the first sampling.

 Statistical Analysis: The analysis of the data was carried out according to the specific
 objectives of the research, using the Statistical Package for the Social Sciences (SPSS)
 computer program. A descriptive analysis was performed in which the qualitative variables
 were studied by frequency, while the quantitative ones were analyzed through the average. The
 results obtained in both cases were expressed in frequency tables. For each variable, the
 data normality test was performed according to the Shapiro-Wilk test. Afterwards, an
 inferential analysis was carried out for the quantitative variables. The level of statistical
 significance was established according to the Student t and Chi-Square test, while for the
 qualitative variables, the non-parametric McNemar test was performed. Significant p-values
 <0.05 were considered, aiming to present a confidence level of 95%.

 Ethical implications, biosecurity and others Each patient was informed in detail all that
 entails carrying out the research work, according to the level of understanding of each
 subject and was given an informed consent, in which the objective of the study is specified
 in writing along with the procedures to be performed for both,the control group and the
 intervention group, in addition to the costs and benefits of participating. The design,
 together with the protocol of the present study, was based on the Declaration of Helsinki and
 was approved by the Scientific Ethics Committee of the Faculty of Dentistry,Viña del Mar
 campus of the UNAB.On the other hand, for the correct development of the investigation,
 pertinent clinical examinations were carried out following the biosecurity regulations in
 force at that establishment. The sample taken from the subgingival bioflora of the patient
 consists of a totally innocuous, fast and simple technique, which was obtained only from a
 periodontal site. Once the investigation was completed, all the extracted material was
 destroyed, achieving its elimination by means of the international standards of handling of
 biological samples. All participants were given free of charge the necessary elements for
 proper oral hygiene, teaching them the correct way to sanitize their mouth. In addition, they
 were made aware of the diagnosis, treatment plan and results obtained. Regarding the patients
 examined who did not meet the inclusion and / or exclusion criteria of the study, they were
 referred to the subject of Clinical Periodontics for the fourth year, as a teaching assistant
 or Postgraduate of Periodontics at the UNAB, as appropriate, in order to be treated
 periodontally. Regarding the use of systemic antibiotics as adjuvant treatment, this has been
 shown to be clinically more effective than the periodontal treatment associated with placebo.
 Azithromycin, on the other hand, has an action spectrum that involves aerobic and anaerobic
 Gram negative bacteria, among which are the periodontopathogens, it has an excellent
 gastrointestinal absorption, long half-life and limited adverse effects. Due to its
 properties, it could become a viable alternative in periodontics to the current adjuvant
 antibiotic regimen of amoxicillin with metronidazole.Regarding the use of the placebo
 (lactose), it is characterized for being of the pure or inactive type, that is, what
 corresponds to an inert substance that has no pharmacological action in the patient, which is
 why it is usually the type of placebo of choice in studies of clinical trials. Lactose is a
 disaccharide resulting from the binding of glucose and galactose, and these placebos
 correspond to capsules with a crystalline powder, white, odorless and with a slightly sweet
 flavor, whose side effects to its use occur infrequently, mainly in patients who have
 ingested excessive amounts of lactose and / or have intolerance to it, generating symptoms
 such as abdominal pain, flatulence, diarrhea, bloating, nausea and vomiting.It should be
 noted that, despite the low prevalence of adverse reactions in both, lactose and azithromycin
 , patients were warned about the possible effects, indicating that in the face of any
 discomfort attributable to the consumption of the tablets they would go to the researchers to
 stop the administration of these and be eliminated from the study.
Inclusion criteria:

 (I) Patients ≥ 18 years; (II) classified by the American Society of Anesthesiologists (ASA)
 as ASA I or ASA II that are compatible with local anaesthesia procedures; (III) At least 10
 natural teeth present, excluding semi-erupted third molars; (IV) Presence of at least 6
 sites with a probing depth (PD) ≥ 5mm and clinical attachment loss (CAL) ≥ 4mm.

 Exclusion criteria: Patients with (I) Haemostasis disorders; (II) Medication associated
 with gingival disorders such as: Anticonvulsants (Phenytoin), Calcium channel blockers
 (Nifedipine), Immunosuppressive drugs (Cyclosporins); (III) Systemic diseases that affect
 the immunoinflammatory response; (IV) Treatment with antacids on a regular basis due to
 chronic gastritis and/or self-medication with antacids; (V) Treatment with drugs such as:
 warfarin, digoxin and acetylsalicylic acid; (VI) History of allergy to local anesthetics;
 (VII) Orthodontic appliances; (VIII) Antibiotic treatment in the previous 3 months; (IX)
 History periodontal treatment; (X) Pregnancy; (XI) Valvular prostheses or failures in heart
 valves, with risk of endocarditis; (XII) Psychic and Intellectual Disability, in accordance
 with Chilean law number 20,584, title II, paragraph 8, article 28; (XIII) Consumption of
 more than 10 cigarettes per day; (XIV) Allergy to AZM; (XV) Lactose intolerance.