Assessment of the quantity of microorganisms associated with bronchiectasis in saliva, sputum and nasal lavage after periodontal treatment: a study protocol of a randomised controlled trial.

INTRODUCTION: The association between periodontal disease (PD) and chronic obstructive pulmonary disease (COPD) has been widely studied, with aspiration of periodontal pathogens being one of the most accepted causal mechanisms for pulmonary exacerbation. Periodontal treatment (PT) was associated with a decrease in these exacerbations. Bronchiectasis is a pulmonary disease that has many similarities to COPD; however, there are no studies correlating this condition to PD thus far. This study will evaluate if PT reduces proinflammatory cytokines in serum and saliva, as well as halitosis and the amount of microorganisms associated with exacerbation of bronchiectasis in saliva, sputum and nasal lavage 3 months after PT. METHODS AND ANALYSIS: A total of 182 patients with PD and bronchiectasis will be randomly allocated to group 1 (positive control; scaling and root planing (SRP)+oral hygiene (OH)) or group 2 (experimental; SRP+photodynamic therapy+OH). After 3 months, samples of saliva, nasal lavage and sputum will be collected to determine the level of Pseudomonas aeruginosa, Staphylococcus aureus and Porphyromonas gingivalis by quantitative PCR. This protocol will determine the efficacy of PT in reducing the most likely niches of bronchiectasis exacerbation by comparing pre- and post-treatment microbiology samples. Furthermore, there will be assessment of oral halitosis and verification of inflammatory cytokines in serum and saliva. ETHICS AND DISSEMINATION: This protocol has been approved by the Research Ethics Committee of Universidade Nove de Julho. Data will be published in a peer-reviewed journal. TRIAL REGISTRATION NUMBER: NCT02514226. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

RCT Entities:   Population Interventions Outcomes

Although some studies have correlated chronic obstructive pulmonary disease (COPD) with periodontal disease, to date, bronchiectasis has not been studied in the dentistry field.

Other periodontopathogens, such as Tannerella forsythia and Treponema denticola, as well as other pathogens found in the oral biofilm, such as Haemophilus influenzae and Streptococcus pneumoniae, could be quantified, because they have been identified in tracheal aspirate of patients with COPD. However, since this is the first study, only the most representative microorganisms were chosen. Additional samples of this material will be collected for later analyses of these pathogens.

Despite the reduction in bacteria in periodontal pockets being followed by their decrease in saliva, it would be interesting to perform microbiological evaluation in this niche, because it could be a potential reservoir for lung bacteria. This material will also be collected for later analyses.

Introduction

Periodontal disease (PD) has an infectious origin and affects dental supporting tissue, impacting 19% of the adult Brazilian population.1 It has been associated with various systemic diseases, such as diabetes and cardiovascular disease.2 3 In recent years, an association has been suggested between PD and pulmonary diseases, such as pneumonia, asthma and chronic obstructive pulmonary disease (COPD), although the causal relationship between them is not clear.4 The most accepted causal mechanism is aspiration of resident bacteria from the periodontal pocket, which could cause or exacerbate lung infections,5 6 since there is association between COPD and the plaque index,7 and PD and COPD have pathophysiological processes in common: connective tissue destruction and exaggerated inflammatory reaction.8 Systemic inflammatory markers are amplified for both, especially tumour necrosis factor (TNF)α, interleukin (IL)-6 and IL-8.9 10 11 12 13 However, the levels of inflammatory markers are not similar among studies5 suggesting that more studies are necessary.9 Although some studies have correlated COPD and PD, bronchiectasis has not been studied in the dentistry field to date. It is a condition with irreversible and permanent abnormal dilation of the bronchi and bronchioles, high morbidity and decreased quality of life.14 Bronchiectasis is often misdiagnosed as asthma or COPD because of the similarities of the clinical manifestations, such as cough and sputum production.15 The clinical treatment for this condition is based on control and prevention of recurrent acute exacerbations, which, in turn, reduce public health spending. The aetiology of bronchiectasis can vary widely, including congenital genetic defect and idiopathic causes.16 The most common extrinsic factor of bronchiectasis is aspiration of infected mucus,6 which could be exacerbated by the presence of PD, but there are no previous studies investigating oral health in these patients. Inflammatory markers, such as IL-1, IL-6, IL-8 and TNFα are increased in bronchiectasis and in PD.17 18 19

The most commonly isolated microorganism in infections is Pseudomonas aeruginosa, whereas Staphylococcus aureus, despite not being as common, is related to more complex infections.16 20 21 22 The presence of Ps. aeruginosa is associated with a higher number of hospital admissions.22 Periodontal treatment (PT) is not able to eliminate all subgingival pathogens because of inaccessible colonisation sites, and, furthermore, periodontopathogens such as Porphyromonas gingivalis can invade periodontal tissues,23 24 25 and it has been found in infected lung.26 27 Also, Ps. aeruginosa and S. aureus have been found in periodontal pockets of patients with COPD.28 Antibiotics are relegated to adjuvants in PT, since the risks outweigh the benefits.4 29 30 On the other hand, they are routinely administered in bronchiectasis, selecting resistant bacterial strains23 in the whole body,31 32 including, possibly, the oral microbiome. Macrolides, fluoroquinolones and azalides are long-term antibiotics used for bronchiectasis, and they are effective against oral bacteria.33 34 Photodynamic therapy (PDT) is an antimicrobial, atoxic alternative for reducing the quantity of microorganisms in inaccessible sites after PT,35 with no side effects and no bacterial resistance reported.36 PDT is the association of a photosensitiser with light in the presence of oxygen,29 producing reactive oxygen species, such as cytotoxic superoxide and hydroxyl radicals. This reaction generates energy, which is transferred to the bacterial oxygen molecules, leading to cell death.23

A common observation in patients with bronchiectasis is the presence of halitosis.37 38 It is expected that PT could partly solve this. To date it has not been tested if PT alone can eliminate halitosis in these patients or whether the pulmonary disease induces halitosis by itself.

Although bronchiectasis and COPD share very similar systemic clinical and immunological findings, there are no studies on the periodontal status of patients with bronchiectasis, which leaves a knowledge gap in this area. Therefore, the hypothesis to be tested is whether PT will decrease the number of microorganism species associated with bronchiectasis exacerbation in the saliva environment, sputum and nasal lavage. The primary objective of this study is to assess the decrease in the number of microorganisms associated with bronchiectasis exacerbation, as well as reduction of P. gingivalis (currently deemed a cornerstone in PD) in saliva, sputum and nasal lavage before PT and 3 months thereafter. The secondary objectives are to evaluate periodontal clinical parameters, halitosis and proinflammatory cytokines (IL-1, IL-6 and IL8, TNFα) in serum and saliva before PT and 3 months thereafter with and without PDT.

Methods

A randomised, controlled, 3-month, single-centre, parallel-group clinical trial was designed according to the CONSORT Statement and is registered at http://www.clinicaltrial.gov. After verbal and written explanation of the study, patients will sign the informed consent form approved by the Research Ethics Committee (1.057.901) of Nove de Julho University (UNINOVE), and a researcher involved in the study (EHP) will enrol the patients. The study will be performed in accordance with the Declaration of Helsinki (revised in Fortaleza, Brazil, 2013.) A total of 182 patients under medical care at InCor-FMUSP with generalised chronic periodontitis39 will receive PT at UNINOVE Dental Clinic, from September 2015 to September 2017. Patients with clinical bronchiectasis will be characterised by chronic sputum production and dilated bronchi confirmed by high-resolution thorax CT. The severity of bronchiectasis will be assessed using the FACED score.40

Calculation of sample size

Sample size calculation was performed for the microbiota (P. gingivalis) as the main outcome. A sample size of 91 participants per group was found to be required to detect a between-group difference of 0.741 in the amount of bacteria from baseline to 3-month follow-up (power=0.8, α=0.05). This predicted difference equates to an effect size of 0.42, considering an SD of 1.9 for group 2 (experimental group; PDT) and 1.4 for the root-planing group.41

We have also calculated the sample size for the secondary outcomes. A sample size of 42 participants per group was found to be required to detect a between-group difference of 0.8441 in TNFα from baseline to the 3-month follow-up (power=0.8, α=0.05). This predicted difference equates to an effect size of 0.62, considering an SD of 1.85 for group 2 and 0.49 for group 1 (positive control group; gold standard). A sample size of 22 participants per group was found to be required to detect a between-group difference of 4.9541 in IL-1β from baseline to the 3-month follow-up (power=0.8, α=0.05). This predicted difference equates to an effect size of 0.87, considering an SD of 5.37 for group 2 and 5.98 for group 1. A sample size of 61 participants per group was found to be required to detect a between-group difference of 2.141 in IL-6 from baseline to the 3-month follow-up (power=0.8, α=0.05). This predicted difference equates to an effect size of 0.51, considering an SD of 4.62 for group 2 and 3.48 for group 1. A sample size of 32 participants per group was found to be required to detect a between-group difference of 77.4841 in IL-8 from baseline to the 3-month follow-up (power=0.8, α=0.05). This predicted difference equates to an effect size of 0.72, considering an SD of 94.06 for group 2 and 120.33 for group 1. For halitosis, there is no study comparing scaling and root planing (SRP)+PDT with conventional treatment. However, a previous study comparing baseline with post-non-surgical PT (including oral hygiene (OH) instruction)42 demonstrated a high intragroup difference, determining an effect size of 0.77. Therefore, we believe that the sample size for the mean outcome (n=91) will cover the sample size required to detect intergroup difference for halitosis. The sample size calculation was performed on G*Power software (V.3.1.9).43

Calibration of clinical examiner

One researcher (gold standard) will perform periodontal clinical examinations to achieve maximum reproducibility of the measurements. Ten patients with PD will be evaluated at six sites per tooth (mesiovestibular, vestibular, distobuccal, mesial lingual, lingual and distal-lingual) in the whole mouth, except third molars, for probing depth and clinical attachment loss. Patients who undergo these clinical examinations will not be included in the study groups. The intraclass correlation coefficient (ICC) will be calculated to evaluate intraexaminer concordance, which must be higher than 0.9. Periodontal probes from University of North Carolina (UNC-15) by Hu-Friedy, Chicago, Illinois, USA will be used. Data will be analysed by ICC test.44 The results will be expressed in tables, and compliance with the standard examiner must be higher than 0.8.

Inclusion and exclusion criteria

Patients must be >35 years of age, of either gender, and have >10 teeth with chronic moderate PD—that is, more than 15% of teeth examined with ≥4 mm probing depth (PD). Patients with clinical bronchiectasis must be characterised by chronic sputum production and dilated bronchi confirmed by high-resolution thorax CT. All patients should be in a stable condition (no exacerbations requiring antibiotic use in the 3 months before and 3 months after PT). Exclusion criteria include smokers or former smokers who have quit less than 5 years previously, pregnant women, phenytoin or cyclosporin users (because they affect periodontal status), those with decompensated systemic diseases requiring prophylactic antibiotic therapy for PT, those who have used oral antiseptics or anti-inflammatory drugs in the past 3 months, or those who have had PT performed in the past 6 months. Patients with other pulmonary (eg, pulmonary cystic fibrosis, asthma) and nose or throat inflammatory diseases will also be excluded.

Randomisation of patients and allocation concealment

After SRP, a researcher not involved in the study will divide the patients into two groups by randomised order (Microsoft Excel, 2013 version). Then, opaque envelopes randomly containing information about application of PDT or not will be labelled with sequential numbers. The researcher responsible for PT will open the first envelope and perform the procedure written therein. The internal contents will be revealed only after statistical analysis.

Experimental groups and study design

Patients will be divided into two groups and they will be subjected to different treatments as follows: group 1, positive control group (gold standard; n=91; OH+SRP+simulation of PDT); group 2, experimental group (n=91; OH+SRP+PDT with methylene blue and the red laser diode (λ=660 nm)). All participants will receive PT from an experienced specialist with universal curettes (Hu-Friedy) and ultrasound (Ultra Sound Dabi Atlante–Profi Neo US, Ribeirão Preto, Brazil) in a full-mouth manner (figure 1).

Figure 1

Flow chart summarising the experimental design.

No antibiotics or oral antiseptics will be prescribed. The periodontal supportive therapy will be performed 3 months after the end of PT.45 After SRP, a researcher not involved in the study will divide the patients into two groups by randomised order (PDT use or not). In group 1, PT will be performed as described above with Therapy XT (see below) turned off as well. In group 2, the PT will be performed identically to that in group 1. PDT will be administered in periodontal pockets >4 mm. Methylene blue (Chimiolux 0.005%; DMC, São Carlos, São Paulo, Brazil) will be applied with a carpule syringe needle (with stop and no bisel) in the depth of periodontal pockets. After 5 min of application, the red laser diode (λ=660 nm) will be applied with an output power of 100 mW (Therapy XT; DMC) and 90 s of exposure—that is, 9 J in each point. The laser head will be placed in direct contact with the periodontal pocket. Applications will be held in six sites around the tooth in all teeth. To finalise, there will be 1 min of irradiation in scan around each tooth and rinsing with saline solution to remove the photosensitiser. All the proposed treatments in group 1 (OH+SRP+simulation of PDT) in each patient will be concluded within 1 week. The treatments for group 2 (OH+SRP+PDT) will be also concluded within 1 week. The data analysts who will assess outcome will be blinded during outcome analysis. All possible adverse effects will be noted and qualified during PT and during the maintenance period (3 months) through questionnaires developed for this protocol. The adverse events will be reported in the results section of the manuscript and will be discussed. The Research Ethics Committee of Nove de Julho University (UNINOVE) will be notified of any protocol alterations. In addition, the records at http://www.clinicaltrial.gov will be notified. Figure 2 summarises the principal points of this protocol.

Figure 2

Schematic diagram summarising time points of protocol. IL, interleukin; TNF, tumour necrosis factor.

Sputum, saliva and nasal lavage collection for microbiological evaluation

Fluid will be kept in the nasal cavities for 10 s and then washed out by allowing it to flow into a sterile container. The nasal wash will then be transferred to a graduated tube. The nasal lavage will be performed by asking the patient to breathe deeply and hold 10 mL sterile saline inserted through the nostrils. After collection, RNAlater (Qiagen, Valencia, California, USA) will be added to preserve the DNA.

The sputum will be collected in the morning before any food intake. Patients will be asked to breathe deeply several times and then cough deeply; the sample will be collected in a wide-mouth jar. In the laboratory, the sputum specimens will be decontaminated by N Acetyl-L-cysteine/NaOH centrifuged at 10 000×g for 5 min, and the pellet will be suspended in 50 μL buffer solution to extract DNA (1% Triton X-100, 0.5% Tween 20, 10 mM Tris/HC1, pH 8.0, and 1 mM EDTA). The material will be sent immediately to the laboratory.

The saliva will be collected and stored exactly as described above. The timeline in figure 3 shows the sequence of procedures and sample collection.

Figure 3

Timeline showing the sequence of procedures and sample collection.

Microbiological evaluation

Samples will be defrosted and vortexed. DNA extraction will be performed with Master DNA Extraction Kit Pure Kit (Epicentre Technologies, Chicago, Illinois, USA) according to the manufacturer's instructions. Quantitative PCR of the total amount of Ps. aeruginosa, S. aureus and P. gingivalis will be performed with the SYBR Green System (Applied Biosystems, Foster City, California, USA) in a thermal cycler Step One Plus Real-Time PCR System (Applied Biosystems). This quantification will be performed before treatment and 3 months thereafter to verify the effectiveness of PT and to evaluate if it causes reduction of microbial content of buccal, nasal and lung niches. The outcomes of this protocol will determine the efficacy of PT in reducing the total amount of microorganisms in saliva, sputum and nasal lavage. These outcomes will be classified as (i) better, (ii) no change or (iii) worse than baseline. These results will be compared with a possible reduction of systemic and local inflammation and halitosis after 3 months.

Determination of systemic and saliva inflammatory markers

Sample collections will be performed through venous puncture by a trained technician at baseline and after 3 months. Sample collections will be centrifuged and serum will be stored. Unstimulated saliva samples will be obtained in sterile collecting tubes and will be kept in a container with ice. All samples will be stored in dry tubes at −80°C until use.

Commercial kits (Peprotech, Rocky Hill, New Jersey, USA) for measuring inflammatory markers (TNFα factor, IL-1, IL-6 and IL-8) by ELISA will be used according to the manufacturer's instructions.

Halitosis measurements

Air from inside the oral cavity will be collected with a syringe. With one movement it will be injected into an Oral Chroma (Abilit, Japan), a portable device connected to the computer which captures graph peaks of gas concentration, measuring the thresholds of volatile sulfur compounds (0–1000 parts per billion), after 8 min. To avoid bias, the examination will be conducted in the morning, and participants will be instructed just to brush with water during this day and not to eat food with garlic, onion and hot spices, not to consume alcohol and not to use antiseptic for 48 h before the oral assessment. They must not have eaten 2 h before the test and must not have had coffee, candies or chewing gum or used oral and personal care products with perfume (aftershave, deodorant, perfume and creams).

Data collection and management

Microbiological collection will always be performed by the same researcher (CCBdC) supervised by a professor (ACRTH), as described under ‘Sputum, saliva and nasal lavage collection for microbiological evaluation’. This material will be processed in the Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, Brazil supervised by two professors (MPAM and PLL). The data will be analysed in an Excel worksheet developed for this purpose.

The collection of saliva to analyse inflammatory markers will be performed by CCBdC supervised by ACRTH, and systemic inflammatory markers will be collected through venous puncture by a single trained technician, as described under ‘Determination of systemic and saliva inflammatory markers’. This material will be analysed by the same researcher (CCBdC) in the Laboratory of Biophotonics Applied to Health Sciences, Nove de Julho University, São Paulo, Brazil, supervised by two professors (KPSF and RAMF). The data will be analysed in an Excel worksheet developed for this purpose.

The collection of halitosis will be performed by the same researcher (CCBdC) supervised by SKB. The portable device, Oral Chroma, will be used for halitosis evaluation. Procedures will be performed as previously described.46 The air will be injected into the device with a single movement. The Oral Chroma will be connected to a computer (with specific software) allowing the capture of a chart with peaks of gas concentration after 8 min. The data will be analysed in an Excel worksheet developed for this purpose.

In order to improve adherence to the study, we will provide interested patients with information about the study results by mail.

Concerning data management, patients will be identified (ID) after randomisation. Every new datum or piece of material stored (records, documents, samples) will receive the same identifier (eg, ID-1A, related to patient 1 on baseline evaluation; ID-1B, related to patient 1 after 3 months of evaluation).

CT scan

A CT scan of patients' faces’ sinuses for those who have not had a recent one (over 6 months) will be performed. Acquisition of the images will take about 15 min. The CT scan will be used to confirm the diagnosis of the underlying disease (bronchiectasis) and as one of the components for calculating severity criteria (FACED).40

Variables of the protocol

The primary variable will be quantification of P. gingivalis, Ps. aeruginosa and S. aureus. The amount of microorganisms (measured as log) in saliva, sputum and nasal lavage at baseline will be compared with the amount of the same microorganisms after PT and between groups (group 1 and group 2). These outcomes will be classified as (i) better, (ii) no change or (iii) worse. All variables will be presented as mean±SD.

The secondary variables of the study will be quantification of inflammatory cytokines and halitosis. Cytokines will be quantified, (pg/mL) in saliva and serum. Results will be compared between groups (group 1 and group 2) and with baseline. We expect the reduction in local cytokines to be followed by a decrease in systemic ones.

Halitosis will be quantified by measuring the thresholds of volatile sulfur compounds (hydrogen sulfide, methyl mercaptan and dimethyl sulfide). The gas concentrations will be given in standard units of ppd and ng/10 mL. Halitosis will be compared with baseline after PT and between groups (group 1 and group 2). We expect PT to reduce halitosis.

Statistical analysis

The primary variable will be the quantification of periodontal pathogens and pulmonary microorganisms, and the secondary variables of the study will be quantification of inflammatory cytokines and halitosis. Dental clinical parameters will be assessed at baseline and 3 months after treatment. The Lilliefors test will be used to test the normality of the data. If the data behave normally, the Student t test (Bioestat 5.3, Pará, Brazil) will be used to compare continuous and dependent variables of group 1 and group 2 (quantification of microorganisms, inflammatory cytokines and halitosis). If the data do not behave normally, the Friedman test will be used. p<0.05 will be considered significant. The χ2 test will be used to compare categorical variables between the groups (demographic data) in the comparison between baseline and 3 months. The results will be expressed as mean±SD.

Strategies for achieving adequate participant enrolment

Participants will be recruited in InCor-FMUSP, the hospital where they undergo semi-annual medical care. A researcher (EHP) will perform the periodontal examination in an appropriate room (inside the hospital) and will invite them for PT (after 1 week) in the Dental Clinic of Nove de Julho University, right after periodontal diagnosis. They will be instructed about oral health and be given a set of instructions for performing daily OH.

Discussion

Bronchiectasis is characterised as a serious lung disease with severe exacerbations, recurrent infections and frequent hospital admissions.14 The association between oral biofilm control and severity of lung diseases such as COPD has been demonstrated.7 Clinical trials are necessary to confirm whether this premise applies to bronchiectasis. In this study, the conventional PT (gold standard) will be compared with treatment with PDT. This alternative treatment was chosen because antibiotics are used more frequently in patients with bronchiectasis than in the general population.47 The PDT would provide an additional action, which does not select resistant microorganisms and does not have contraindications.36

Several microorganisms, such as T. forsythia, Tr. denticola, P. gingivalis, H. influenza, Ps. aeruginosa, S. aureus and Strep. pneumonia, have been identified in the tracheal aspirate.28 Thus, any of them could have been selected as a microorganism indicator of bacterial contamination of saliva, nasal lavage and sputum. We chose to quantify three microorganisms of medical interest. S. aureus was chosen because of its high resistance to antimicrobials,48 and Ps. aeruginosa was chosen because of its biofilm-forming ability, high resistance to antimicrobials, and high prevalence in bronchiectasis exacerbation.48 49 The periodontal pathogen P. gingivalis was selected because of its well-established relationship with periodontitis and its association with extra oral infections.24 The outcomes of this protocol will determine the efficacy of PT in reducing the total amount of microorganisms in saliva, sputum and nasal lavage. These outcomes will be classified as (i) better, (ii) no change or (iii) worse than baseline. These results will be confronted with a possible reduction in systemic and local inflammation and halitosis after 3 months. Greater improvement in all parameters measured over 3 months is expected for group 1 and group 2. It is expected that group 2 (PDT group) will have better results for periodontal parameters than PT alone after 3 months.50

Regarding the other outcomes, such as local and systemic inflammatory cytokines, a slight decrease in local cytokines is expected after PT. Since these patients have a systemic inflammation per se, the reduction in local inflammation is expected to be followed by a slight systemic inflammation. In relation to the halitosis outcome, this is expected to be partly solved after PT, since PD is one of the most probable causes.37 38 As mentioned above, to date, it has not been tested if PT alone can eliminate halitosis in these patients or if the pulmonary disease itself induces halitosis.

Maintaining all these outcomes for a 1–2 year follow-up will probably improve lung disease, as demonstrated in patients with COPD.51 These patients will be followed (periodontal maintenance) for 2 years and future analyses will be possible.