Clinical Trial Details
— Status: Completed
Administrative data
| NCT number |
NCT02633345 |
| Other study ID # |
H-8-2014-014 |
| Secondary ID |
|
| Status |
Completed |
| Phase |
N/A
|
| First received |
December 10, 2015 |
| Last updated |
June 14, 2016 |
| Start date |
September 2015 |
| Est. completion date |
June 2016 |
Study information
| Verified date |
June 2016 |
| Source |
University of Copenhagen |
| Contact |
n/a |
| Is FDA regulated |
No |
| Health authority |
Denmark: The Danish National Committee on Biomedical Research Ethics |
| Study type |
Interventional
|
Clinical Trial Summary
The aim of the study is to investigate the effect of tablets containing probiotic candidate
strains, on gingival inflammation and the levels of selected pro- and anti-inflammatory
cytokines in gingival crevicular fluid (GCF). A secondary aim is to describe the effect of
the tablets on the salivary microbiome. The null hypothesis is that the clinical
measurements, levels of cytokines and microbial composition of saliva will not differ from
baseline or between the study and control group.
The study is planned as a double-blind; placebo controlled randomized trial with two
parallel arms. The intervention period is planned to be 4 weeks.
80 participants are planned to be enrolled after informed consent and then randomly
allocated to either the probiotic group or the placebo group.
The participants are randomly assigned to one of the study groups and given supply of either
probiotic tablets or placebo tablets.
The probiotic tablets contain Lactobacillus rhamnosus PB01 and Lactobacillus curvatus P2-2
at a dose of 1 * 109 CFU(Colony Forming Units)/tablet. The participants will take two
tablets a day for four weeks. Follow up registrations are conducted after two weeks, four
weeks and six weeks.
At each visit, saliva and GCF will be collected and then plaque index (PI)) and bleeding on
probing (BOP) will be registered.
All data will be processed with SPSS software (v 22.0; Chicago, Ill, USA). The follow-up
values are compared to baseline within each group by Wilcoxon paired two-sided test and
differences between groups are analyzed by Wilcoxon unpaired test. A p-value < 0.05 is
considered statistical significant.
The participants are fully covered by the patient insurance of The School of Dentistry which
cover any damage to the trial participant during the study .There is no expected side
effects to the treatment with probiotics. Probiotics are generally considered safe (GRAS:
Generally Recognized As Safe) by The European Food Safety Authority (EFSA).
The participants will not benefit directly from the treatment, but the knowledge gained from
the study might contribute to the prevention of microbial derived illnesses in the mouth,
e.g. caries and periodontitis, in the future. The sample collection does not cause any
discomfort to the participants and the tablets have no known side effects. Therefore, the
personal benefit of the participants corresponds to the minimal risk and discomfort to the
participants.
Description:
Background:
The human saliva offers an easy accessible and non-invasive opportunity to study bacterial
interactions as well as biomarkers of health and disease. The Human Microbiome Project has
provided insights that the biofilms inside and outside our bodies have co-evolved with
mankind and play an important role for health. This is also true for the oral cavity in
which the microbiota does not play a passive role but actively contributes to the
maintenance of oral health. However, certain ecological shifts in the microbiome allow
pathogens to manifest and cause oral diseases. According to the ecological plaque
hypothesis, caries and periodontitis are the result of environmentally induced overgrowth of
aciduric and proteolytic species, respectively.
Bacteriotherapy is the term used when a harmless effector strain is implanted in the host's
microflora to maintain or restore a natural microbiome by interference and/or inhibition of
other microorganisms, and especially pathogens. This might open up alternative ways of
fighting infectious diseases with less harmful side effects and may also help in the
treatment of disorders that seem to have nothing to do with bacteria, such as asthma,
obesity and diabetes. Probiotic bacteria, defined as "Live microorganisms which, when
administered in adequate amounts, confer a health benefit on the host" (WHO), are commonly
suggested candidates for bacteriotherapy. The potential mechanisms of action are still not
fully understood but it seems clear that there are local (direct) as well as systemic
(indirect) effects. According to Reid the potential avenues are: i) co-aggregation with
pathogens and growth inhibition, ii) bacteriocin and hydrogen peroxide production, iii)
competitive exclusion through antagonistic activities on adhesion sites and nutrition, iv)
systemic immunomodulation.
In vitro studies have shown effect on co-aggregation and growth inhibition and clinical
studies have shown effect on clinical parameters of gingival inflammation but still little
is known about the immunomodulatory effect and the host-microbe interaction.
Periodontal diseases are among the most widespread infectious diseases in man and can lead
to tooth loss. If left untreated gingivitis can lead to periodontitis.
The different surfaces of the oral cavity are all continuously bathed in saliva and several
pathogenic species in saliva have been associated with periodontal disease. Studies on the
microbial effect of probiotic bacteria on oral health have so far been based on conventional
cultivation methods and have been focused on one or a few strains. The new high-throughput
techniques provide a possibility for more comprehensive and precise picture of the bacterial
profiles, but to date only a few studies have used sequencing to determine the bacterial
composition of saliva after intake of probiotic bacteria. Most analyze the composition after
a very short term intake of the probiotic bacteria. Hence, there is only one study which
have analyzed the composition after longer intervention period and correlated this to
clinical outcomes.
Aim:
The aim of the study is to investigate the effect of tablets containing probiotic candidate
strains, on gingival inflammation and the levels of selected pro- and anti-inflammatory
cytokines in gingival crevicular fluid (GCF). A secondary aim is to describe the effect of
the tablets on the salivary microbiome. The null hypothesis is that the clinical
measurements, levels of cytokines and microbial composition of saliva will not differ from
baseline or between the study and control group.
Study design:
The study is planned as a double-blind; placebo controlled randomized trial with two
parallel arms. The intervention period is planned to be 4 weeks. The study plan will be
submitted for ethical approval.
Material:
The participants will be recruited among patients at the clinic at School of Dentistry and
through www.forsogsperson.dk. 80 participants are planned to be enrolled after informed
consent and then randomly allocated to either the probiotic group or the placebo group.
Sample size was determined by a power analysis (α= 0.05 and β=0.2) based on values of BOP
obtained previously. Seventy-four participants are needed and 80 are enrolled in order to
compensate for expected drop-outs.
Recruitment: If the participants after reading the notice about the study express interest
in the study they will be invited to an information appointment with assistant professor and
dentist Mette Kirstine Keller. Prior to the appointment, the participants will be informed
that they have the right to bring a companion to the information meeting. They will also
receive the written information about the study. The appointment will take place in a
meeting room without other people present than the investigator, the participant and
possibly a companion. The oral information will be based on the written information and will
be given in a plain language without any value-laden terms. The investigator is responsible
for ensuring that the participant has understood all given information before the project
begins. All comments from the participant are taken into relevant consideration both during
the information interview and the study. At any time the participant can withdraw from the
project. The informed consent for participation in the trial is given on the basis of oral
and written information, and such consent may be given after at least 24 hours of reflection
time. The project does not include subjects who, because of physical or mental handicap,
cannot give the informed consent.
Methods:
Intervention:
After informed consent, baseline examination and sampling, the participants are randomly
assigned to one of the study groups and given supply of either probiotic tablets or placebo
tablets.
The probiotic tablets contain Lactobacillus rhamnosus PB01 and Lactobacillus curvatus P2-2
at a dose of 1 * 109 CFU/tablet. The strains are chosen based on in vitro assays
investigating growth inhibition of pathogens and immunomodulation. The placebo tablets are
identical in size and composition but without the addition of the probiotic strains. Both
tablets are provided by Bifodan A/S. The participants will be instructed to take one tablet
in the morning and one in the evening thirty minutes after tooth brushing. The tablets are
packed in identical pots with color coding. The code will be kept from the investigators
until the analysis are finished.
The participants will take two tablets a day for four weeks. Follow up registrations are
conducted after two weeks, four weeks and six weeks.
Clinical procedures:
At each visit, saliva and GCF (Gingival Crevicular Fluid) will be collected and then plaque
index (PI)) and bleeding on probing (BOP) will be registered. For the collection of GCF, two
contralateral buccal sites from the upper incisors, canines or premolars will be selected
from each participant. Visual supragingival plaque on the selected teeth will be removed and
the sites will be dried with cotton pellets. GCF will be collected using periopaper strips
(ProFlow, Amityville, NY, USA) gently inserted in the gingival sulcus for 20 s. The position
of the strip is marked on a chart to secure the identical sample site at the follow ups. In
case of blood contamination, the strip is discharged and the sampling is repeated after 5
min. The volume of GCF is recorded using a Peritron 8000 (Proflow) and expressed in μL. One
strip holds approximately 0.1-0.4 μL GCF. The strips are then transferred to plastic tubes
and stored at -80ºC until further analysis.
For the saliva samples, paraffin-stimulated whole saliva is collected during a 3-minute
period at least two hours after any food intake. The approximate amount of saliva at normal
rate will be 3-6 ml after 3 minutes. The collected saliva is immediately frozen in 1.5 ml
aliquots and stored at -80ºC until further analysis. The remainder of the sample will be
destroyed after completion of the study.
Laboratory procedures:
Analysis of cytokines on GCF: The absorbed GCF is eluated in 120 μL PBS (phosphate buffered
saline) containing 0.05 % Tween 20. The levels of INFγ, TNF-α, IL-8, IL-10 and IL-12 is
determined using luminex and multiplex immunoassay panels (LINCOplex, LINCO Research, Inc,
USA) in accordance with the manufacturer's manual. The concentrations of the cytokines are
expressed as pg/ml.
Analysis of the salivary microbiome: Total bacterial DNA will be extracted from 2 ml of
thawed saliva sample for identification and comparison of the composition and stability of
the total microbial communities in the two trial groups through amplicon sequencing of the
phylogenetic marker gene 16S rRNA. The hypervariable region V3-V4 of bacterial and archaeal
16S rRNA genes will be PCR amplified using specific primers 341F and 806R and analyzed by an
established high-throughput sequencing pipeline developed by the University of Copenhagen
Microbiology partner based on state-of-the-art sequencing technologies (Illumina Mi-Seq) and
custom made bioinformatic tools specifically designed for analysis of large microbiome
datasets.
Statistical methods:
All data will be processed with SPSS software (v 22.0; Chicago, Ill, USA). The follow-up
values are compared to baseline within each group by Wilcoxon paired two-sided test and
differences between groups are analyzed by Wilcoxon unpaired test. A p-value < 0.05 is
considered statistical significant.
Data management:
All collected data will be treated in compliance with the Act on Processing of Personal Data
and "The Health Law". At inclusion the participants will be assigned a unique number and a
separate protocol without personal identification number.
Scientific importance:
The study may provide evidence that the probiotic tablets can influence clinical markers of
gingival inflammation and/or pro- and anti-inflammatory cytokines, which can lead to new
strategies in managing gingivitis and maintaining oral health. The use of probiotic bacteria
in treatment of dental diseases could be one option the use of antibiotics. Furthermore it
will provide new insights to the effect of introducing new strains to the salivary
microbiome which is supposed to be a significant event in bacteriotherapy.
Risks and Adverse Effects:
The participants are fully covered by the patient insurance of The School of Dentistry which
cover any damage to the trial participant during the study .There is no expected side
effects to the treatment with probiotics. Probiotics are generally considered safe (GRAS:
Generally Recognized As Safe) by The European Food Safety Authority (EFSA).
The participants will be asked to report any unexpected adverse effect.
Ethical considerations:
The participants will not benefit directly from the treatment, but the knowledge gained from
the study might contribute to the prevention of microbial derived illnesses in the mouth,
e.g. caries and periodontitis, in the future. The sample collection does not cause any
discomfort to the participants and the tablets have no known side effects. Therefore, the
personal benefit of the participants corresponds to the minimal risk and discomfort to the
participants. The participants who complete the trial will be compensated with a gift
voucher of 500 DKK to Magasin. It should be emphasized that positive, negative and
inconclusive results will be published.
Time Schedule:
Preparation of the study will be carried out in the winter 2014/2015. After ethical
approval, patients can be recruited to the study during winter/spring 2015.
