Clinical Trial Details
— Status: Recruiting
Administrative data
| NCT number |
NCT06074861 |
| Other study ID # |
PER-ECL-2022-02 |
| Secondary ID |
|
| Status |
Recruiting |
| Phase |
N/A
|
| First received |
|
| Last updated |
|
| Start date |
December 4, 2023 |
| Est. completion date |
January 31, 2025 |
Study information
| Verified date |
September 2023 |
| Source |
Universitat Internacional de Catalunya |
| Contact |
Giuseppe Mainas, DDS, PhDc |
| Phone |
00393345921869 |
| Email |
gmainas[@]uic.es |
| Is FDA regulated |
No |
| Health authority |
|
| Study type |
Interventional
|
Clinical Trial Summary
Periodontitis (gum disease) leads to the formation of gum pockets. Its treatment involves
deep cleaning of the teeth, to remove soft and hard tooth deposits under the gum line.
Although in the long term this leads to improvement of the gum conditions and reduced
inflammation, in the hours and days post-treatment, inflammation may increase, sometimes also
associated with a high temperature. A method to reduce this response has not been found yet.
The goal of this multi-centre randomized controlled trial is to to test if a diet which acts
to mimic periodic fasting can influence responses in the mouth and throughout the body after
treatment of gum disease in patients with advanced gum disease but general health conditions.
Five Spanish centres (Universidad Complutense de Madrid, Universidad Internacional de
Catalunya, Universidad de Murcia, Universidad de Santiago de Compostela, Universidad de
Granada) will perform the clinical part of the study, whereas the King's College of London
(Guy's Hospital) will provide the analyses and processing of the data. Researchers will
include 24 patients in total.
The main question it aims to answer is:
- Is a mimic periodic fasting (together with the classical gum treatment) effective at
reducing the local and systemic inflammation provoked by the gum disease (and by the same
treatment) in the short- and medium term?
Although all participants will receive the necessary gum treatment (deep cleaning),
researchers will randomly assign them to one of two groups.
The test group will follow three cycles (the same day of the treatment, and 45- and 85 days
after treatment) of 5 days each of a fasting-mimicking diet (FMD). The FMD program is a
plant-based diet program designed to attain fasting-like effects while providing both macro-
and micronutrients to minimize the burden of fasting and adverse effects. The FMD consists of
100% ingredients that are generally regarded as safe (GRAS) and comprises proprietary
vegetable-based soups, energy bars, energy drinks, cracker snacks, olives, herbal teas, and
supplements. All items to be consumed per day are individually boxed.
In contrast, the control group will continue with their current diet. All patients will
complete dietary diaries to estimate calorie intake during this time period.
Researchers will collect blood, stool, plaque and gingival crevicular fluid samples from each
patient at different time points.
Besides, they will call the patients one or two times during each FMD cycle to check that
everything is proceeding properly and to solve any problems or concerns they may have. In
addition, a trained registered dietitian will be available during the whole study period in
case participants need some support or have doubts and/or questions.
If participants miss multiple visits (e.g. 2/3) will be considered as a drop-out and, if they
develop a serious medical condition, they might be excluded from the study. However,
independently from the participation into this study, each patient will have their gum
disease regularly treated.
People with periodontal disease usually have a bad dietary-habits. This investigation with
its holistic approach might lead people to modify their unbalanced diet due to the possible
related local and systemic benefits. In addition, cycles of 5 days might consistently raise
the adherence and willingness to follow such a fasting regime.
Description:
Introduction
Periodontitis is a microbially-driven inflammatory disease of the periodontium. It has been
shown that the periodontitis-associated inflammation is not limited to the periodontium, but
may have far-reaching consequences in the rest of the body, and that this may be partly
mediated by systemic spread of oral microorganisms. Steps 1 and 2 of periodontal therapy is
also associated with a short-term acute phase inflammatory response, characterised by
elevation of circulating acute-phase inflammatory markers, which in turn is followed, if
successful, by a longer-term reduction in these molecules. In the intermediate to longer term
this is accompanied by changes in other markers of peripheral vascular health alongside
improvements in metabolic control lasting at least 12 months. Periodontitis is known to have
a bidirectional relationship with metabolic disruption and dysregulation. A very recent work
corroborated that periodontitis is associated with systemic inflammation as measured by serum
C-reactive protein (CRP) levels. Furthermore, it has been reported that intensive
non-surgical periodontitis treatment (steps 1 and 2) led to a progressive reduction of CRP
after 90 days with a moderate increase at 180 days. It is thought that improvements in
metabolic control may also improve periodontal outcomes, yet the exact mechanisms for this
are not fully clear. Several human and experimental investigations have also been published
concerning the possible effects of nutrition on management of periodontitis. A study on
monkeys found that long-term dietary caloric restriction (diet enriched with vitamins and
minerals) dampens clinical inflammation and reduces the degree of periodontal breakdown
secondary to acute microbial challenge. However, in another experimental study on monkeys,
long-term hypocaloric diet (30% less of daily calories intake plus vitamins and minerals)
appeared to differentially reduce the production of local inflammatory mediators and the risk
for inflammatory periodontal disease among males but not females. In addition, a recent
systematic review on animal models concluded that macronutrients that have an effect on
oxidative stress or immune system (i.e., n-3 fatty acid) seem to be important for the
prevention or treatment of periodontitis. In terms of human research, a Korean group, after a
four-week weight control intervention in periodontally healthy obese people, observed a
reduction of matrix metalloproteinase (MMP)-8, MMP-9, and interleukin-1β (IL-1β) in gingival
crevicular fluid (GCF). A German team reported that a diet consisting of low carbohydrate
intake, richness in Omega 3-fatty acids and fibre, higher vitamin C and D values, reduction
of animal proteins, and focused intake of nitrate-containing plants might lead to a
significant reduction in periodontal and gingival inflammation, even though serological
inflammatory parameters and the subgingival microbiome seem to be unaffected. More recently,
a study found that there was no overall association between Western dietary patterns
(characterized by a higher intake of processed meat, red meat, butter, high-fat dairy
products, eggs and refined grains) or prudent dietary patterns (characterized by a higher
intake of vegetables, fruit, legumes, whole grains, fish and poultry) and periodontitis;
however when solely obese individuals were considered, a Western diet was related to a higher
risk of periodontitis. Another very recent investigation, based on use of a dietary
inflammatory index, showed that consuming a pro-inflammatory diet was associated with
moderate-severe periodontitis in the U.S. general adult population. A novel mode of fasting
has been introduced in order to increase patient compliance, due to the fact that people are
usually not able to follow a strict diet for a long period. In particular, the fasting
mimicking diet (FMD), consisting of 30-50% of the normal caloric intake for 4-7 consecutive
days followed by a refeeding ad libitum period once a month, showed very promising
beneficious effects in terms of reduction of risk factors for aging, diabetes, autoimmunity,
cardiovascular disease, neurodegeneration and cancer. Overall, there is still uncertainty
about a potential role of periodic fasting on periodontal clinical and inflammatory
parameters. To our knowledge, no studies have investigated the potential effect of FMD on
systemic and periodontal response after periodontitis treatment.
Objectives
The main aim of this study is to test if a fasting mimicking diet can influence the systemic
and periodontal response following non-surgical periodontal therapy. The null hypothesis is
that a fasting mimicking diet does not modify the local and systemic inflammatory
post-treatment response.
Study Design
This is a double-blind (outcome-assessor and statistician), multi-centre, randomized clinical
study with parallel groups and a 1:1 allocation ration, with internal pilot. All recruited
patients will receive the same protocol for periodontal therapy (steps 1 & 2). One group
(test) will be submitted to three cycles of FMD. The FMD program is a plant-based diet
program designed to attain fasting-like effects while providing both macro- and
micronutrients to minimize the burden of fasting and adverse effects. The FMD consists of
100% ingredients that are generally regarded as safe (GRAS) and comprises proprietary
vegetable-based soups, energy bars, energy drinks, cracker snacks, olives, herbal teas, and
supplements. All items to be consumed per day are individually boxed to allow the subjects to
choose when to eat while avoiding accidentally consuming components of the following day. The
FMD consists of a 5-days regimen that provides approximately 1.100 kilocalories for the first
day; approximately 750 kilocalories per day for the second to the fifth day. On the contrary,
the other group (control) will continue with their normal diet.
Subject selection
This is a multi-centre study. Participants will be screened by International University of
Catalunya (UIC), Complutense University of Madrid (UCM), University of Santiago de
Compostela, University of Murcia and University of Granada clinical staff/students among
periodontitis patients attending the respective hospitals. The investigators expect 24
eligible participants available per year and assume that 70% of them will agree to take part
in the trial after expressing initial interest.
Study procedures
Subject recruitment
International University of Catalunya's, Complutense University, Santiago de Compostela and
Granada Universities' patients, will be individually approached for recruitment on campus and
within the Dental faculty. After expressing an initial interest, potential participants will
be provided with an information sheet and the opportunity to ask questions to members of the
study team via email or telephone. If they then express a desire to participate, they will be
invited to a screening visit. Each individual will be given at least 24 hours and up to a
week to decide if they would wish to take part. Individuals will be reimbursed for their time
with a payment at completion of the study.
Screening Procedures
At the screening visit, consent will be sought by the trained applicant or study dentist,
which will be recorded through a written participant consent form detailing the complete
trial procedure and an agreement checklist for each data collection method. Once consent is
given, each enrolled participant will be recorded on a Participant Log form. Initial
screening will be to ensure that each individual conforms to the outlined inclusion and
exclusion criteria. This will be assessed by taking an oral history and will be recorded on a
hardcopy Screening Log form. Participants will need to undergo a periodontal examination from
the study dentist to ensure suitability.
Randomisation Procedures and allocation concealment
Initial central randomization will be done using the "Sealed Envelope" method by personnel
not directly involved in the study, at Guy's Hospital. For important influencing factors
('centre') stratified randomisation will be carried out as recommended. No stratification by
age will be carried out, because for small sample sizes, stratification should be limited to
one or very few factors and because the influence of age is limited by the study design and
will be adjusted for in statistical analyses. For each centre patients will be allocated to
one of the two treatment groups using a randomization list that was previously prepared by
the study statistician. The list will be gerenated by blocked randomization. Sealed envelope
will be used in each centre to ensure allocation concealment. Participant will be assigned
for test or control treatment out between days -7/-10 and day 0. The therapist will be
informed about treatment allocation by opening the envelope at the beginning of the treatment
appointment.
Schedule for each visit
Participants who have expressed a degree of interest in participating from face-to-face
discussions or from responses to advertisements will be invited to a Screening visit. This
will take place, as all study visits will, at the Hospital General de Catalunya (Barcelona)
and at the Odontostomatology Units (Clinica de Odontologia) of Madrid, Santiago de
Compostela, Murcia and Granada, and this should take approximately 45 minutes. This will take
place at a mutually convenient time and date for the potential participant and the study
dentist, within the confines of clinical space availability. Here they will be initially
asked about their personal information/medical history and undergo a dental examination. If
they are eligible, they will be informed when the next stage of the study will commence and
following an appointment is given in order to begin with the investigation. Treatment will be
provided by the post-graduate students of the Master of Periodontology of each centre.
One-two weeks later on Day -7/-10 (baseline appointment or visit 1), informed consent will be
given and signed. Blood, GCF, plaque and stool samples will be collected. Medical history,
adverse events and concomitant medications will be checked and an oral examination will be
carried out including recording number of teeth and at 6 sites per tooth probing depth,
recession (mm) and bleeding after probing. A very superficial prophylaxis, motivation and
oral hygiene instructions will be provided.
On Day 0 (morning) (visit 2), patients assigned to the test group will start the FMD for 5
consecutive days. All patients will undergo full-mouth subgingival instrumentation (PMPR)
procedures and more detailed hygiene instruction will be reinforced. Medical history, adverse
events and concomitant medications will be checked, and an oral examination will be carried
out in order to screen for complications. A trained registered dietitian (from one of the
centres) will discuss FMD with patients and also distribute survey to report items not eaten
and extra food consumed.
On day 1 (v3), samples of blood and GCF will be taken. Medical history, adverse events and
concomitant medications will be checked, an oral examination will be carried out in order to
screen for complications.
On day 7 (v4), samples of blood and GCF will be taken. Medical history, adverse events and
concomitant medications will be checked, an oral examination will be carried out in order to
screen for complications.
On day 45±5 (v5), samples of blood and GCF will be taken. Oral hygiene instructions will be
reinforced and a session of supragingival scaling with polishing will be provided. The second
cycle of FMD will be started in patients of the test group. Medical history, adverse events
and concomitant medications will be checked, an oral examination will be carried out in order
to screen for complications.
On day 85 ±5 (v6), the third cycle of FMD will be started in patients of the test group.
Medical history, adverse events and concomitant medications will be checked, oral hygiene
instructions will be reinforced and a session of supragingival scaling with polishing will be
provided.
On day 90 ±5 (v7), full-mouth periodontal charting measurements will be recorded. Samples of
blood and GCF samples will be taken. Oral hygiene instructions will be reinforced and, if
necessary, a session of supportive periodontal therapy will be provided. Medical history,
adverse events and concomitant medications will be checked, an oral examination will be
carried out in order to screen for complications.
On day 180 ±10 (v8), full-mouth periodontal charting measurements will be recorded at 6 sites
per tooth: probing depth, recession (mm) and bleeding after probing. Blood, GCF, plaque and
stool samples will be taken. Oral hygiene instructions will be reinforced. Medical history,
adverse events and concomitant medications will be checked, an oral examination will be
carried out in order to screen for complications.
*Patients will be called one or two times during each FMD cycle to check that everything is
proceeding properly and to solve any problems or concerns they may have.
Follow up Procedures
If a participant misses multiple (2/3) visits they could be considered as a drop-out.
However, they will be included in the intention-to treat analysis.
End of Study Definition
The study will be complete once all the participants have finished all the phases of the
clinical period, and the processing/analysis on the biological material and clinical scores
has been performed.
Sample Collection/Labelling/Logging
The samples will be collected during the study visit from the patient by qualified dentists
of the International University of Catalunya, Complutense University of Madrid, University of
Santiago de Compostela, University of Murcia and University of Granada who will participate
in this study. During the collection of all sample types, proper hand hygiene, fitted gloves
and sterile single-use examination items will be used to ensure the integrity of the samples
collected and the safety of the examiner and participant. Pseudo-anonymised labelled tubes
will be prepared in advance of each visit, with the label consisting of the participant
number prefixed with the sample type designation e.g. G for GCF, B for blood, S for saliva,
SubP for sub-gingival plaque etc. Collection of samples will take place at each visit before
clinical examination takes place, to prevent the physical manipulation from disturbing the
sampling site or from causing slight gingival bleeding which may contaminate the sample being
collected. GCF Periopaper® Strips (Pro-flow Inc., Amityville, New York, USA) will be placed
in the gingival crevice of the teeth with the deepest pocket site (buccal aspects) in each
quadrant. They will then be immediately measured on a calibrated Periotron 8000 (Pro-Flow
Inc., Amityville, New York, USA) electronic transducer, which will quantify the amount of GCF
collected. Once measured, the GCF strips for that visit will all be stored at -80ºC in a
single 2ml tube and pooled for further analysis. 4ml of venous blood will be collected from
the participant's arm into Hemogard safety closure capped evacuated sterile vacutainers,
containing the anticoagulant K2 Potassium salt of EDTA (Ethylene Diamine Tetra Acetic acid)
that has been spray coated onto the interior surface of the tube. Samples will be taken by
experienced and trained phlebotomists. To reduce this risk of fainting, participants will be
on a semi- reclined plinth throughout the duration of the sampling and for five minutes
following sampling. A researcher will be present with the participant at all times to ensure
no falls or injuries are sustained in the event of fainting. Used needles will be discarded
into a secure sharps bin. The samples for serum will be centrifuged for 10mins at 11,000 RPM
then immediately divided into 0.2ml - 0.5ml aliquots and stored in a freezer at -80°C, until
required for analysis. Sub-gingival plaque samples will be collected from the buccal surface
of the tooth by the study dentist using a sterile curette, from below the gingival margin
respectively. The plaque will be collected from teeth with the deepest pocket site (buccal
surface) at each study visit. These teeth were selected to have a good mixture of molars and
incisors. The collected plaque from these four teeth will be pooled into 1ml of sterile 0.1x
Tris-EDTA and stored at -80ºC. Participants will be provided with home collection kits for
sampling of stool in preserving solution ([http://www.microbiome-standards.org)]
http://www.microbiome- standards.org) and this sample will be posted to the local laboratory
within 7 days of collection. On receipt of stool samples in the laboratory, samples will be
stored at -80 C. All biologically contaminated disposable items will be discarded into the
correct biological hazard bin for processing and removal. All samples taken will be logged
onto the participant's hardcopy CRF for that study visit, with the date and time of
collection recorded and the initials of the collecting study dentist. This will also be
logged onto the study Excel spreadsheet. If a particular sample is not able to be taken at
that visit, the reason for this will be stated in the appropriate 'Additional notes' section
of the CRF.
Sample Analysis Procedures
All experiments will be conducted in the Microbiome and Salivary laboratories, Guy's
Hospital. GCF samples will be thawed and eluted in PBS (pH 7.2±0.2) supplemented with
Easypack Protease Inhibitor Cocktail (Roche) as described in Curtis et al. 1988. Briefly, the
periopaper strips will be placed in a perforated 0.5ml microcentrifuge tube which will be
held inside a 1.5ml tube. The elution buffer (50μL) will be added on to the strips and placed
in a vortex shaker for 2 minutes before centrifugation for 15 minutes at 11,000 rpm and 4 ̊C.
The centrifugation will be repeated with a further 50μL of the elution buffer added on to the
strips, to yield ~90μL of total eluted GCF. Protein concentration will be quantified using an
LVis plate (BMG Labtech CLARIOstar) calibrated with Bovine Serum Albumin (Sigma). GCF
analysis for selected analytes will be performed by Luminex. Samples will be processed at the
KCL Centre for Host-Microbiome Interactions, fl.17 Guys Tower Wing. Plaque and stool samples
will undergo DNA extraction following standard procedures in our lab and will be analyzed
using next generation DNA sequencing to characterize the subgingival microbiota in order to
identify and determine the levels of key periodontal bacterial pathogens and microbial
community-wide changes in sites treated with both test and control protocols. Blood samples
will be tested for cytokines, acute phase proteins and inflammatory markers using various
techniques such as Western blots or enzyme-linked immunosorbent assays (ELISA), which will
determine the presence and amount of these in the blood. Gingival Crevicular Fluid will be
analysed for inflammatory mediators, antibodies, peptides and cytokines, again using
techniques such as ELISA. Circulating levels of IGF-1, fasting blood glucose level, blood
β-Hydroxybutyrate level will be assessed to monitor the compliance to FMD.
ELISA (high-sensitivity two-site enzyme-linked immunosorbent assay): IL-6 detection.
Cobas Integra 700 (automated immuno-turbidimetric high-sensitivity assay): HsCRP detection.
The data generated from these experiments will be stored on secure, password- protected
King's computers, including those in the Salivary and Microbiome laboratories and the study
laptop provided by King's.
Sample Storage Procedures
Blood, saliva, plaque and GCF samples will be stored at -80ºC in secure, locked HTA freezers
in the laboratories of International University of Catalunya, Complutense University of
Madrid, University of Santiago de Compostela and University of Granada. This will ensure long
term stability in preparedness for sample processing. Following collection, stool samples
will be sent to each participating centre by patients in sealed pre-stamped envelopes. As
they are stored, all samples will have their physical integrity checked and samples to be
frozen will then be logged onto a password-protected, secure computer database, which records
their location in the freezer and the time/date they were stored. A physical hardcopy
recording the samples being stored will also be featured on the CRF. The storage, transport,
use and ultimate disposal of the sample provided will be conducted in compliance with the UK
Human Tissue Act 2004 and with the EU Tissues and Cells Directives. If any samples are not
immediately processed, they will be pseudo-anonymously stored for further analysis at a later
date. Samples will be transferred to Salivary laboratory, Guy's Hospital and will be archived
for 10 years after completion. After ethics approval for the study has expired, the blood,
saliva and GCF samples will be disposed of in accordance with the Human Tissue Act 2004, and
any amendments thereto, or transferred to a tissue bank.
Shipping of sample from Spain to UK
Samples will be placed in an igloo that can contains sufficient ice/dry ice to ensure samples
are kept at the correct temperature for longer than the estimated journey time. Then, the
polystyrene igloo is partially filled with ice/dry ice allowing room for samples and extra
ice. Samples may be placed in a sealed plastic bag to aid cataloguing. The igloo is then
filled with dry ice to cover the samples and a dispatch form and, additionally, a copy of the
address label in a sealed plastic specimen bag is included. A lid will be placed on the igloo
and secured with packing tape. Following that, the igloo is wrapped in strong brown wrapping
paper and both an address label and very low temperature hazard labels are attached before
the delivery. It will be selected the fastest mean of transport (e.g., plane) in order to
complete the shipping as fast as possible.
Assessment of Safety
The investigators do not foresee any significant risks to the participant involved with the
sample collection, including blood samples, as these are well-established procedures. Based
on previous literature, a possible risk might concern some mild/moderate systemic adverse
events caused by the FMD diet regimen, including fatigue, weakness and headaches. There is
also a slight risk of fainting during or following blood sampling. To reduce this risk,
participants will be on a semi-reclined plinth throughout the duration of the sampling and
for five minutes following sampling. A researcher will be present with the participant at all
times to ensure no falls or injuries are sustained in the event of fainting. If a needle
stick injury occurs to the examiner during the collection of the blood sample, or a scratch
injury occurs, local procedures will be implemented to respond to such an incident. No
serious adverse events are expected to occur during the course of the study.
Ethics Reporting
An Adverse Event is any unfavourable and unintended signs, symptoms or disease associated
with the study. Should an Adverse Event occur, such as fainting during the taking of blood,
the investigator will inform the Chief Investigator, for inclusion in the study file CRF. A
Serious Adverse Event (SAE) is an event that causes death, requires the hospitalisation of
the participant, causes persistent disability, or causes a birth defect. Should this occur, a
report will be logged within 24 hours of learning of the event and a report to the ethics
committee within 15 days of learning of the event. The form should be completed in typescript
and signed by the chief investigator. The main REC will acknowledge receipt of safety reports
within 30 days.
Compliance and withdrawal
Subject compliance
Participant compliance will be assessed at each study visit. A self-reported medical history
will ensure that the participant has not taken any action (e.g., started to participate in
another clinical trial) or developed a condition or started taking medication (e.g. taking
systemic antibiotics) which is prohibited by the exclusion criteria. In addition, compliance
will be evaluated based on blood test taking into account the reductions in circulating
levels of IGF-1. This will be recorded on a checklist on the CRF. Each patient should fill a
diet diary to throughout the study including FMD cycles for test patients. Failure to
complete the dietary diary would also be recorded as drop-out, due to the crucial importance
of diet for the validity of the study. Lastly, if a participant unexpectedly fails to attend
a study visit, they will be requested to attend clinic at the very next possible opportunity.
Withdrawal / dropout of subjects
Participants may withdraw from the study at any time. This would be through a verbal
discussion between the participant and any of the study team, and their decision logged on a
written Withdrawal form. Reasons for withdrawal could be due to physical discomfort or too
great a time commitment for example. Upon completion of the data analysis, where all
anonymised data would be collated, participants would no longer be able to withdraw their
data from the study, which would be made clear at the initial recruitment stage. The latest
date for this is at the end of the study. Certain circumstances may require the study team to
decide to withdraw participants from the trial, such as significant non-compliance to the
protocol e.g., missing multiple study visits, or other events which would fulfil the
exclusion criteria. This would involve speaking with the participant to inform and explain
the decision to them, and the decision logged on a written Withdrawal form. If non-compliance
is revealed at a study visit, it would also be logged in the 'Additional Notes' section of
the CRF. Subjects will not be replaced when individuals withdraw.
Statistical considerations
Sample size calculation
No studies have been published using FMD in periodontitis patients. Therefore, no data about
feasibility and required sample size is available in the literature. it is very difficult to
estimate what the correct study sample size would be and, as a consequence, this study is
designed as an internal pilot. While Browne and co-workers (1995) suggested 30 patients would
be required as rule of thumb for sample size, Kieser & Wassmer (1996) found that a pilot
sample size between 20 and 40 would minimise the overall sample size of the larger study.
Following the suggestion by Julious 2009, 12 patients per study arm will be recruited for
this study.
Interim analysis and feasibility/progression criteria
The decision to progress with the larger trial (and the relative sample size) will depend on
the feasibility/progression criteria below.
FEASIBILITY ASPECTS PROGRESSION CRITERIA TO BE SATISFIED
Willingness to be randomized ≥ 50% of approached patients agree Self-reported compliance with
diet ≥ 66% of test group patients self-report compliance (defined as not more than 1 day in
which the diet was not followed) Tested compliance with diet ≥ 66% of test group patients
show compliance based on blood test (defined as 20 to 30% reductions in circulating levels of
IGF-1) (Wei 2017) Acceptance of blood samples ≥ 66% of recruited patients have all blood
samples taken as per protocol Absence of serious adverse events No patients report serious
adverse events which might be related to the test diet
If all criteria above are met, analysis of existing data up to 3 months will be carried out,
using data from the 24 'internal pilot' patients. Means and standard deviations of CRP values
at 3-months will be used to calculate the required sample size for the larger trial.
Follow-up of the study patients will continue up to 6 months post-treatment. Should the
difference between groups for the primary outcome (CRP at 3 months) be minimal, resulting in
a very large sample size, a decision may be made not to progress to recruiting a larger group
of patients and recruitment will be stopped. However, the patients still in the study will be
followed up until the last study follow up (180 days).
Statistical analysis
The primary outcome of the study is circulating CRP levels at 3 months post- treatment (test
vs. control). However, the internal pilot/feasibility part of the study will need to
completed first before considering whether to proceed with a fully-powered study sample size.
If feasibility criteria are met, the difference in CRP levels at 3 months post-treatment
between groups will be used to the sample size, and a formal statistical plan will be
formulated. Different statistical packages will be used for different purposes, Stata 14
(StataCorp LLC, College Station, Texas, USA) will be main package and R (with R-Studio
plugin), will be used for additional analyses, both of them with specific routines created
for this study. The Shapiro-Wilk Test will be applied to verify if the quantitative variables
analysed follow a normal distribution and logarithmic transformation will be performed in
those that do not follow it. The profile of the population and of the study groups will be
described. The results of the qualitative variables will be expressed in proportions and the
quantitative variables will be expressed as means and standard deviation, including the 95%
confidence intervals. The distribution of the population for each of the clinical,
biochemical and microbiological parameters will also be obtained. For the bivariate analysis,
chi-square test will be used to determine if there is a statistically significant association
between two qualitative variables, the data will be presented as contingency tables, when the
Chi- square test does not meet the validity conditions Fisher's exact test will be applied.
To compare the means of two independent variables for intergroup analyses, the Student's
T-test will be used, considering Student's T-test for paired samples in within-group
analyses. If the normal assumptions are not fulfilled, the No Parametric "Mann-Whitney U"
test will be applied. For intragroups analyses, ANOVA for repetitive measures or Friedman
test will be used. Multivariate analysis will be performed by means of a multiple linear
regression model and ordinal logistic regression introducing in it the independent variables
with a lower significance level of 0.20. The conditions will be checked by residual analysis
of heteroscedasticity and linearity. The level of statistical significance for this study
will be p<0.05.
Stopping / discontinuation rules and breaking of randomisation code
The investigators do not foresee any risks involved with the sample collection and FMD, as
these are well-established procedures. Therefore, the investigators foresee no part of the
study should need to be discontinued and the trial shouldn't need to be stopped due to
participant safety concerns. However, since FMD has not been tested in periodontitis
patients, in the unlikely event that there is a Serious Adverse Event, a report to the
Sponsor will be logged within 24 hours of learning of the event and a report to the ethics
committee within 15 days of learning of the event. A decision will then be made as to whether
or not the study should stop in conjunction with these bodies.
