Clinical and Microbiological Efficacy of Lactobacillus Reuteri in the Supportive Therapy of Periodontitis: a 6 Months Randomized Controlled Trial
Phase: NA
Conditions: Periodontal Diseases
Sponsor: Azienda Socio Sanitaria Territoriale degli Spedali Civili di Brescia

PROTOCOL #PERIOPRO 
Versione 1 del 13/01/2020 
 
  1 
 
 
CONFIDENTIAL AND PROPRIETARY 
Information provided herein is the property of the University-Hospital of Brescia. Nothing herein is to be reproduced, 
published, or disclosed to others in any way without the prior expressed written permission of the University-Hospital 
of Brescia. 
 
 
Title of Study: Clinical and microbiological efficacy of Lactobacillus reuteri in the supportive 
therapy of periodontitis: a 6 months Randomized Controlled Trial study.  
 
Type of Study: Clinical and microbiological 
 
Protocol No: PROTOCOL #PERIOPRO 
 
Expected study start date: APRIL, 2020 (study initiation) 
 
Study Location/Country: Brescia, Italy 
 
Study Originator(s)/ Contact (name, phone, fax): Magda Mensi, Piazzale Spedali Civili 1, 
25123 Brescia, phone: +390303995784, email: magda.mensi@unibs.it 
 
Test Products w/ PDM Numbers:  
CE marked Medical Device to be used as an adjunct therapy to standard in patients with 
moderate to severe periodontal disease (Confidential information). 
 
 
 

 
Versione 1 del 13/01/2020 
 
  2 
Study Title: 
Clinical and microbiological efficacy of Lactobacillus reuteri in the 
supportive therapy of periodontitis: a 6 months Randomized 
Controlled Trial study. 
 
Study sites: 
 
 
Dr. Magda Mensi (lead site) 
 
 
 
 
University-Hospital of Brescia 
 
 
 
 
Brescia, Italy. 
 
 
 
 
 
Study Phase: 
 
IV.  
 
Name of Medical Device: 
GUM® Periobalance® (SUNSTAR SUISSE S.A. Rte de Pallatex 11, 
CH-1163 Etoy SWIZERLAND) 
  
 
Objective: 
The objective of the clinical study is to assess the efficacy of the 
addition of Lactobacillus reuteri in the supportive therapy of 
periodontitis in terms of reduction of PD (Probing Depth). 
 
Patient Population: 
44 periodontal patients, systemically healthy, male and female 
adults (18 – 75 years), with history of periodontitis grade B or C and 
stage III or IV, with at least 2 residual sites/pockets with probing 
depth ≥6 mm or pockets of 5 mm with bleeding on probing in two 
different quadrants.  
 
Structure:                     
Parallel arms: Number of treatments: 1 (after a session of non-
surgical supportive therapy one group takes probiotic (Lactobacillus 
reuteri) lozenges 2 times a day for 21 days, another group takes 
placebo lozenges 2 times a day for 21 days).  
                                                                                              
Duration of study: 36 months.  
 
Number of Centers:    
One. 
 
Blinding: 
Patient, Examiner, Operator and Statistician -Blind. 
 
Method of Patient Selection: Periodontal pockets: at least 2 sites with probing depth ≥6 mm or 
pockets of 5 mm with bleeding on probing in two different 
quadrants. 
 
Total Sample Size: 
44 adult subjects will be recruited (randomized) to participate in 
this study, with 40 expected to complete the study. 
 
Primary Efficacy Variable: 
Primary outcome measure will be the changing of PD in 
millimeters. 
 
 
 
 

 
Versione 1 del 13/01/2020 
 
  3 
 
 
Clinical and microbiological efficacy of Lactobacillus reuteri in the supportive 
therapy of periodontitis: a 6 months Randomized Controlled Trial study. 
PROTOCOL #PERIOPRO 
____________________________________________________________ 
 
 
 
Protocol Date: JAN 13, 2020 
 
 
 
 
Protocol  Approval 
 
Signature Page 
 
 
 
 
 
  
 
 
_________________________ 
 ________________________ 
 
Dr. Magda Mensi 
 
 
 
 
 
 
Date 
University-Hospital of Brescia, Italy 
   
 
 
 
 
 
 
 
 
 
 

 
Versione 1 del 13/01/2020 
 
  4 
ADDRESSES AND RESPONSIBILITIES 
 
Addresses 
Study Centres: 
Section of Periodontics, School of Dentistry, Department of 
Surgical Specialities, Radiological Science and Public Health 
University of Brescia 
Piazzale Spedali Civili 1  
25123, Brescia, Italy 
 
 
 
 
 
 
 
 
 
 
 
 
Dr. Magda Mensi (Principal Investigator) 
 
 
 
 
 
Tel. +390303995784 
 
 
 
 
 
Email: magda.mensi@unibs.it 
 
Study coordinator: 
 
 
Dr. Magda Mensi 
 
 
Section of Periodontics, School of Dentistry, Department of 
Surgical Specialities, Radiological Science and Public Health 
 
 
University-Hospital of Brescia 
 
 
 
 
 
Brescia, IT 
 
Data Management & Monitoring: 
Dr. Magda Mensi 
 
 
Section of Periodontics, School of Dentistry, Department of 
Surgical Specialities, Radiological Science and Public Health 
 
 
University-Hospital of Brescia 
 
 
 
 
 
Brescia, IT 
 
Statistician: 
 
 
 
Prof. Stefano Calza 
 
 
Unit of Biostatistics and Bioinformatics, 
 
 
 
 
 
Department of Molecular and Translational Medicine 
 
 
University of Brescia 
 
 
 
 
 
Brescia, IT 
 
Microbiologist:  
 
 
Prof. dr. Egija Zaura 
Head of the Department of Preventive Dentistry 
 
 
 
 
 
Professor in Oral Microbial Ecology 
 
 
 
 
 
Academic Centre for Dentistry Amsterdam 
 
 
 
 
 
Vrije Universiteit Amsterdam and University of Amsterdam 
 
 
 
 
 
Amsterdam, the Netherlands 
 
 
 
 
 
 
 

 
Versione 1 del 13/01/2020 
 
  5 
Responsibilities 
The principal investigator’s responsibilities are described in detail in ISO norm 14155:2011 for 
the clinical investigation of medical devices for human subjects. 
The role of the principal investigator is to implement and manage the day-to-day conduct of the 
clinical investigation as well as ensure data integrity and the rights, safety and well-being of the 
subjects involved in the clinical investigation. 
 
The principal investigator shall be qualified by education, training and experience to assume 
responsibilities for the proper conduct of the clinical investigation. In particular, the principal 
investigator is responsible for:  
- 
qualification of investigation site; 
- 
communication with Ethics Committee; 
- 
compliance with Informed Consent process; 
- 
compliance with Clinical Investigation Plan. 
Under the direct supervision of the Principal Investigator, certain duties may be delegated during 
the course of the study. These responsibilities will be documented on the transfer of responsibilities 
form maintained in the Investigator Site file. 
 
The Statistician will be responsible for: 
- 
sample size calculation; 
- 
statistical plan; 
- 
statistical section of the protocol; 
- 
statistical evaluation; 
- 
final statistical report. 
 
The Microbiologist will be responsible for: 
- 
sample microbiological processing and analysis; 
- 
final microbiological report. 
 
 
 
 
OBJECTIVE 
The study will be performed to assess the efficacy of the addition of Lactobacillus reuteri containing 
lozenges in the supportive therapy of periodontitis in terms of reduction of PD (Probing Depth). 
 

 
Versione 1 del 13/01/2020 
 
  6 
INTRODUCTION 
Probiotics are defined as ‘live microorganisms which, when administered in adequate 
amounts, confer a health benefit on the host’. The influence of probiotics on pathogens flora can 
derive from three principal modes of action: innate and acquired host defense modulation, 
production of antibacterial substances and competitive exclusion mechanism. In particular, 
Lactobacillus reuteri has been studied for its antibacterial and anti-inflammatory properties. 
It is a heterofermentative bacterium and the distinct strains have different characteristics. In 
particular it acts as an antibiotic, induces oxidative stress on pathogens, is resistant to proteolytic 
and lipolytic and present anti-inflammatory properties. 
Literature pointed out to potential effectiveness of L. reuteri as an adjunct to non-surgical 
periodontal therapy in initial treatment of periodontitis patients but underlined the limits of their 
conclusions due to the heterogeneity of the studies and to the few patients included. There is thus 
a need for more long-term randomized controlled studies. In particular, only one study addressed 
the use of this probiotic during the supportive therapy and in particular in patients with severe forms 
of periodontitis but few patients were included.  
Patients meeting the criteria of periodontitis stage III and IV, grade C are considered to be affected 
by severe and advanced forms of periodontitis with a rapid rate of progression. This group of 
patients could particularly benefit from supplements in the maintenance of periodontal health. 
 
STUDY OUTCOMES 
a. 
Primary outcome measure: change in PD  
b. 
Secondary outcomes:  
• 
changes in the following clinical parameters: Pocket closure, REC (Clinical Gingival 
Recession), CAL (Clinical Attachment Level), BOP (Bleeding on Probing), PI (Plaque 
Index). 
• 
Risk progression of the periodontitis and the need of periodontal surgery. 
c. 
Microbiological outcomes: changes in microbial composition and proportion of sequences 
identified as Lactobacillus reuteri in the deepest residual pockets. 
 
STUDY POPULATION 
Forty-four (44) adults, aged 18-75 years, will be entered into study (randomized). It is expected that 
forty (40) subjects will complete the study.  

 
Versione 1 del 13/01/2020 
 
  7 
Randomized subjects who deviate from the protocol (major protocol deviation) and, for this reason, 
are excluded from the analysis, will be replaced to guarantee that the sample required for the 
analysis (40) is reached. 
Patients will be treated at the Department of Surgical Specialities Radiological Science and Public 
Health, School of Dentistry, Section of Periodontics, Brescia, Italy from 04/2020. 
                
Inclusion Characteristics 
- Signed Informed Consent Form. 
- Male and female subjects, aged 18-75 years, inclusive. 
- Good general health (free of systemic diseases such as diabetes, HIV infection or genetic 
disorder, ongoing malignant disease of any type that could influence the outcome of the 
treatment and might interfere with the evaluation of the study objectives). 
- History of periodontitis staging III or IV grading B or C  
- At least 2 sites with probing depth ≥6 mm or pockets of 5 mm with bleeding on probing in two 
different quadrants. 
- Previous periodontal non-surgical treatment at least 3 months maximum 6 months. 
- Availability for the 6-month duration of the study for an assigned subject. 
 
Exclusion Characteristics  
- Not willing to follow the agreed protocol. 
- Presence of orthodontic appliances.  
- Smokers more than 10 cigarettes per day 
- Chronic obstructive pulmonary disease and asthma. 
- Tumors or significant pathology of the soft or hard tissues of the oral cavity. 
- Current radiotherapy or chemotherapy. 
- Pregnant or lactating women. 
- Current or past (within 3 months prior to enrollment) assumption of medications that may 
influence periodontal conditions and/or interfere with healing following periodontal treatment (i.e., 
corticosteroids, calcium channel blockers, systemic antibiotics, ...). 
- History of allergy to Erythritol or chlorexidine. 
- Restorations on the teeth to be treated which may interfere with treatment administration and/or 
scoring procedures, at the discretion of the examiner. 
- Use of systemically administered antibacterial agents or probiotics 3 months prior to enrollment. 

 
Versione 1 del 13/01/2020 
 
  8 
 
A written informed consent will be obtained from each included patient after explanation of the risks 
and benefits of participating to this study. No change in the trial design will be made after approval 
of the Ethical Committee. 
 
 
1. 
STUDY DESIGN 
The study design was chosen according to the current standards required by ISO, GCP and 
according to national requirements. 
a. 
A Parallel-Arm, Statistician, Operator and Examiner are blinded, RCT will be established; 
b. 
The RCT will be run as mono-center; 
c. 
A total of 44 subjects will be recruited to participate in the study. It is expected that all of 40 
subjects will complete the study in total; 
d. 
Admission into the study will be via rolling admission (estimated recruitment time: 12 months); 
e. 
Each subject must have at least 2 residual sites/pockets with probing depth ≥6 mm or pockets 
of 5 mm with bleeding on probing in two different quadrants; 
f. 
Each subject will follow the following treatment protocol: 
i. – Visit 1 (Screening): (within 29 days prior to baseline) – consent secured; screening and 
entry into study; baseline data collection; full periodontal charting (full-mouth PD, REC, 
BOP, PI); identification the sites for microbiological sample. 
ii. – Visit 2 (T0 – Baseline Visit+Treatment): collection of microbiological samples from the 
two deepest sites in two different quadrants. All patients receive a full mouth periodontal 
treatment (GBT) and OHI (oral hygiene instruction). According to the list of randomization 
the patient will be allocated into one of the two arm of probiotic treatment: study lozenges 
are given to all patients to consume at home. The patients are instructed to dissolve 
these on their tongue twice a day, preferably after brushing, for 3 weeks. The patients of 
the probiotic group will receive probiotic lozenges containing Lactobacillus reuteri DSM 
17938 and Lactobacillus reuteri ATCC PTA 5289 (a minimum of 2 × 108 colony-forming 
units L. reuteri Prodentis/lozenge, BioGaia AB). The patients of the control group will 
receive control lozenges without live bacteria. Furthermore, the probiotic and control 
lozenges will be identical in taste, texture and appearance. 
iii. – Visit 3 (T1) – 3 weeks: the participants will be asked to return the empty packages of 
the study medication to examine the adherence. At that time, side effects will also be 
questioned by the examiner by means of an open question. Collection of the 

 
Versione 1 del 13/01/2020 
 
  9 
microbiological sample will be performed from the same subgingival sites as during 
Baseline (T0) and reinforcement of OHI. 
iv. – Visit 4 (T2) - 3 months: Collection of the microbiological sample from the same 
subgingival sites as during Baseline (T0), collection of full periodontal charting (PD, REC, 
PI, BOP) and reinforcement of OHI. 
v. – Visit 5 (T3) - 6 months: Collection of the microbiological sample from the same 
subgingival sites as during Baseline (T0), collection of full periodontal charting (PD, REC, 
PI, BOP) and full mouth GBT will be done in both groups. Subjects will either be entered 
into a routine recall system for treatment or scheduled for any further periodontal 
treatment as clinically indicated, and exit the study. 
g. 
All treatments are made by the same clinician; 
h. 
Calibration: the examiner is calibrated showing an intra-examiner reproducibility of 96% for 
duplicate measurement of probing pocket depth (PPD) with a maximum difference of 1 mm in 
5 patients; 
i. 
Data collection: A PCP-UNC 15 periodontal probe is used on six sites per tooth (mesio-buccal, 
buccal, disto-buccal, mesio-lingual, lingual and disto-lingual) with a gentle probing force by the 
same clinician. Measurements are rounded to the nearest millimeter; 
j. 
Clinical assessment data will be recorded via hard copy CRFs. CRFs will be submitted to a 
data entry staff member for entry into the statistical database. At the conclusion of the study, 
such statistical database will be submitted to the statistician for the preparation of the statistical 
analysis and the final study report.  
 
2. 
TREATMENTS 
Study lozenges (both: test and placebo) will be delivered to the study center prior study start.  
Specification of test treatment – Microbiological analysis 
Microbiological analysis will assess changes in microbial composition and the proportion of 
sequences assigned as Lactobacillus reuteri in subgingival plaque samples. 
Sample for microbiological analysis: 
 
1. Subgingival plaque collection 
Subgingival plaque from 2 sites with probing depth ≥6 mm or pockets of 5 mm with bleeding on 
probing in two different quadrants (the two deepest sites) will be collected using sterile paper point. 

 
Versione 1 del 13/01/2020 
 
  10 
After isolating the site with cotton rolls, supragingival plaque was removed using a Gracey curette 
and the site was dried with airflow. Subsequently, two paper points were inserted in the pocket 
for 10 s. The paper points with subgingival plaque will be placed in empty sterile Eppendorf vial 
and stored on ice until transport to the laboratory (max 2 h), and stored at -80°C.   
 
1. Sample processing 
Samples will be shipped on dry ice to the Department of Preventive Dentistry, at the Academic 
Centre for Dentistry Amsterdam (ACTA). Bacterial DNA will be extracted, purified and quantified 
and prepared for 16S rRNA gene amplicon sequencing using Illumina MiSeq technology. The 
obtained sequencing reads will be quality filtered and processed into zero radius operational 
taxonomic units (zOTUs) and assigned taxonomy using HOMD database. 
The zOTU-table will be randomly subsampled to even depth. The Shannon diversity index per 
sample will be calculated using dedicated software (PAST). This diversity index will be used to 
characterize the diversity of the microbial community and accounts for both the abundance and 
evenness of the OTUs present in the sample. Data ordination plots will be obtained using the 
Principal Component Analysis (PCA) on the log2 transformed zOTU-data. The statistical difference 
in microbial profiles between the study groups at each time point will be assessed using 
permutational analysis of variance (PERMANOVA) with Bray-Curtis similarity distance. 
Metagenomic biomarker discovery tool LEFSE will be used to identify microbial taxa that 
significantly discriminate the groups. The Bray-Curtis similarity index will be calculated between the 
dependent samples collected at different time points and the similarity distances between the study 
groups will be compared statistically.  
zOTUs that taxonomically will be assigned to genus Lactobacillus, will be analyzed separately, in 
order to assess if the probiotic strains have established in the oral cavity. For this, first, a 
phylogenetic tree will be created based on the representative sequence of each zOTU. Sequences 
that will be phylogenetically related to L. reuteri 16S rRNA gene fragment amplified, will be marked 
as potential probiotic sequences. Relative abundances of all genus lactobacillus sequences and L. 
reuteri sequences between groups per timepoint will be compared. 
 
 
Concomitant Therapy 
A necessary concomitant medication or therapy is permitted as long as it is not excluded in the 
exclusion criteria. Every medication has to be reported in the CRF (Appendix 1) and in the 

 
Versione 1 del 13/01/2020 
 
  11 
Concomitant Medication form (Appendix 2). All changes during the study are to be reported in the 
CRF. Usage of prohibited therapy will lead to the exclusion of the subject. 
 
Blinding and Randomization  
 
Blinding: The statistician will be blinded to the group identification. The patient and the operator 
will not know the treatment. Knowledge of the randomization list will be limited to the persons 
responsible for creation of the randomization list, provider of randomization numbers to study site, 
and delivery of the test/control treatment until the last subject (i.e. final examination of the last 
subject), quality control and verification of the CRFs is completed.  
Randomization and allocation concealment: The randomization of the study protocols will be 
performed by a staff member who is not further involved in this study. This will be done based on 
a computer-generated table (www.randomization.com) that links each patient to one of the 
treatment groups. The same staff member will be responsible for blinding the study products. This 
will be done by labelling the packaging of these products with a letter indicating the treatment 
group. Additionally, the jars containing the lozenges will be identical in appearance and non-
transparent. When a patient will be included in the study, the study medication will be handed out 
to the researcher according to group to which that patient will be assigned based on the patient 
number and the randomization list. The similarity of the packaging, and the identical appearance, 
texture and taste of the study products make the double-blinding of the researcher and patient 
possible. 
Implementation: The Principal Investigator will be informed immediately of Serious Adverse 
Events (SAEs), regardless of the causality relationship between the administration of the 
investigational treatment and the SAE.  
 
3. 
PROCEDURE 
 
Screening and Selection of Subjects 
 
 
Patients visiting the Department of Dentistry at Brescia University will be asked to participate 
in this study. Subjects will report to the clinical facility and be screened by the dental examiner 
to identify those subjects who meet the inclusion/exclusion characteristics. The findings of 
this initial screening procedure will be recorded on the CRF. Subjects who meet the 

 
Versione 1 del 13/01/2020 
 
  12 
inclusion/exclusion characteristics and sign an Informed Consent Form will be entered into 
the study.  
 
 
 
 Baseline Oral Soft and Hard Tissue Assessment 
 
All subjects will receive an evaluation of their oral soft and hard tissues. This examination will 
include an evaluation of the soft and hard palate, gingival mucosa, buccal mucosa, muco 
gingival fold areas, tongue, sublingual and submandibular areas and the tonsillar and 
pharyngeal areas. The results of this evaluation will be recorded on the CRF. 
 
 
 Clinical Periodontal Evaluation 
 
All patients receiving treatment will be evaluated by the dental examiner for the following 
assessments (see Study Flow Chart): 
● Collection of subgingival plaque samples; 
● Full Mouth Plaque Score (PI); 
● Periodontal Inflammation (BOP);  
● Probing Pocket Depth (PD);  
● Recession (REC); 
● Clinical Attachment Level (CAL). 
 
 
Clinical attachment level (CAL) is the measurement of the position of the soft tissue in relation 
to the cemento-enamel junction (CEJ) that is a fixed point that does not change throughout 
life. All parameters will be recorded in the CRF. 
 
  
 
 Treatment 
 
Patients who have been identified for treatment receive a GBT session and after OHI will be 
randomized according to a randomization table generated. 
 
  Follow up 
 
Following completion of GBT and OHI the subject will be given the appropriate number of 
interproximal cleaning devices and discuss with the operator about the appropriated 
toothbrush and toothpaste for home use. Subjects will be asked to refrain from using any 
other adjunctive oral home care products during the study period. Every necessary study 
lozenges will be given to all patients to consume at home. The patients will be instructed to 
dissolve these on their tongue twice a day, preferably after brushing, for 3 weeks. 

 
Versione 1 del 13/01/2020 
 
  13 
After 3 weeks the compliance will be recorded and microbiological sample will be collected 
again. At 3 and 6 months the following assessments (see Study Flow Chart) will be recorded: 
● Collection of subgingival plaque samples; 
● Full Mouth Plaque Score (PI); 
● Periodontal Inflammation (BOP);  
● Probing Pocket Depth (PD);  
● Recession (REC); 
● Clinical Attachment Level (CAL). 
 
 
Study Flow Chart 
 
 
Study phase 
Enrolment 
Treatment 
Follow-up 
Follow-up 
Follow-up 
Study visit 
Visit 1 
Visit 2 (T0) 
Visit 3 (T1) 
Visit 4 (T2) 
Visit 5 (T3) 
Study Week 
-29 days 
0 
3 weeks 
3 months 
6 months 
Selection criteria 
+ 
 
 
 
 
Informed consent 
+ 
 
 
 
 
Demographics, ethnics 
+ 
 
 
 
 
General health status 
+ 
+ 
+ 
+ 
+ 
Medical & dental history 
+ 
 
 
 
 
Concomitant therapy/treatment 
+ 
+ 
+ 
+ 
+ 
Safety (adverse events and serious adverse events) 
 
+ 
+ 
+ 
+ 
Data collection (PD,REC, BOP, PI) 
+ 
 
 
+ 
+ 
Microbiological Sample 
 
+ 
+ 
+ 
+ 
Randomisation  
 
+ 
 
 
 
GBT 
 
+ 
 
 
+ 
OHI 
 
+ 
+ 
+ 
+ 
Dispensing standard interdental floss 
 
+ 
 
 
 
Dispensing study lozenges 
 
+ 
 
 
 

 
Versione 1 del 13/01/2020 
 
  14 
 
 
4. 
STATISTICAL ANALYSIS  
 
Sample size determination 
The sample size was calculated via simulation assuming an expected difference of 1mm pocket 
probing depth and using a linear mixed effect model considering patients as random effects. We 
simulated 1000 realizations of pocket probing depth applying the data correlation structure from 
the AgPro study (fixed effect correlation = -0.664; random effects intercept SD = 0.974; random 
effects residual SD = 1.65) and assuming an average of 7 experimental sites per patient. A sample 
size of 20 patients per group allowed for a power of at least 80% at the chosen α = 0.05. Taking 
into account a dropout rate of 10%, we estimated a total sample size of 44 patients. The simulation 
was performed using R (version 3.6.1). 
 
Statistical Analysis  
Data will be described using standard summary statistics such as mean and standard deviations 
for quantitative variables and proportions for categorical variables. The primary outcome is pocket 
probing depth (PPD). Only the sites with PPD greater or equal to 6mm at baseline were considered 
or PPD equal to 5mm and BoP+. For the primary outcome a linear mixed effect model will be 
used considering patients as random effects. 
Secondary continuous outcomes will be modelled using a linear mixed model while generalized 
mixed models will be applied to categorical outcomes. Data will be modelled both using a 1-
level hierarchical model (clustering level: patient) after averaging the outcome variables within 
subject, 
and 
using 
a 
3-level 
(patient, 
tooth 
and 
site) 
hierarchical 
model. 
Results will be expressed as estimates and relative 95% confidence intervals. A significant level 
of 5% will be used for all the comparisons and all analysis will be performed using R (version 
3.6.1 or higher). 
 
Evaluation of Safety 
The following safety variables will be used for the evaluation of safety: 
• 
Adverse events (AE); 
• 
Adverse reactions (AR). 
The total number of AEs and the total number of AEs at least possibly related to the study treatment, 
as well as the total number of patients affected by at least one adverse event will be calculated per 
treatment group.  

 
Versione 1 del 13/01/2020 
 
  15 
The type of AE classified by organ system (according to MedDRA terminology) will be tabulated. 
Serious and/or unanticipated adverse events and AEs resulting in discontinuation or 
reduction/withdrawal of the study treatment will be presented separately. 
 
Safety analysis set 
Safety summaries will be based on the safety analysis set which will consist of all patients who 
received the test/control treatment. 
 
5. 
MONITORING AND AUDIT 
Study Monitoring 
The study will be monitored by the Principal Investigator at periodic intervals during the course of 
the study to ensure that the study is being conducted according to Good Clinical Practice.  
The Investigators will be contacted by the PI on a regular basis. On this occasion, the progress of 
the study will be discussed with the Investigator and the CRFs will be checked for completeness 
and consistency and to verify compliance with the study protocol. 
 
6. 
SUBJECT TERMINATION/WITHDRAWAL PROCEDURES 
All efforts will be made to determine the reason(s) why a patient is withdrawn from the study. 
Subjects could be withdrawn from the study if any of the following occur: 
 
 
 
1. 
Subject fails to substantially comply with the protocol requirements; 
2. 
Subject develops a serious adverse reaction. The Study Investigator will immediately notify 
the PI and information will be recorded on a Serious Adverse Event Form (Appendix 3); 
3. 
Subject elects to terminate participation in the study. Participation in the study is voluntary. 
A subject has the right to withdraw from the study at any time for any reason. 
 
 
The Investigator may terminate the study at any time if the risk-benefit ratio is no longer favourable. 
Before discontinuation, the Investigator should inform the PI and ask for advice. If the Investigator 
is concerned about continuation of the study, his/her concerns should be transmitted immediately 
to the PI. 
 
7. 
DOCUMENTATION AND DATA MANAGEMENT 
Data collection in the case report form (CRF) 

 
Versione 1 del 13/01/2020 
 
  16 
All study data will be recorded in the case report forms identified by the subject number. Only the 
principal investigator, co-investigators, or designated study personnel may make entries in the 
case report forms.  
The Investigator has to identify all data that were directly recorded into the CRF and to be 
considered to be source data. 
The CRFs will be checked for completeness and plausibility by the PI. The Investigator will resolve 
any queries. 
 
Investigator Site File (ISF) 
The ISF includes all documents that are required for the clinical study. During monitoring, the ISF 
will be checked regularly for completeness and actuality. After the clinical trial is finished or stopped, 
the ISF has to be stored 15 years in the study center. 
 
Data Management 
Data extraction from CRFs into a single, electronic database is performed by examiners. 
Discrepancies are to be clarified and corrected by authorized persons by means of documented 
data queries between Statistician and Investigator(s). 
After the study is finished and before data are analyzed, a blind data review meeting will be held 
between the investigator and the statistician. When the database has been declared to be 
complete and accurate, it will be locked. Any changes to the database after this procedure can 
only be made by joint written agreement among the clinical trial leader, the trial statistician, and 
the co-investigators. 
 
8. 
ADVERSE EXPERIENCES/EVENTS 
Subjects will be informed of any possible adverse reactions which they could experience and will 
be instructed to immediately report any event to the investigator. The investigators will record any 
and all adverse reactions and report this documentation to the Principal Investigator. In the event 
of an adverse experience, emergency or other problems or questions regarding participation in this 
study, the subject can contact the following investigators: 
 
 
for Brescia Site:  Dr. Magda Mensi (Principal Investigator) – tel. +39 030 3995784. 
 
Adverse Events (AEs) and Serious Adverse Events (SAEs) are defined by the ICH Guideline 
Medical Device Directive 93/43/EEC and Guidelines on Medical Devices MEDDEVG 2.7/3rev. 3, 

 
Versione 1 del 13/01/2020 
 
  17 
May 2015 Clinical Investigations: Serious Adverse Event Reporting under Directives 90/385/EEC 
and 93/42/EEC for Good Clinical Practice (ICH GCP) as follows: 
 
An adverse event (AE) is any untoward medical occurrence, unintended disease or injury or any 
untoward clinical signs (including an abnormal laboratory finding) in subjects, users or other 
persons whether or not related to the investigational medical device.  
 
With respect to intensity, adverse events are classified as follows: 
- Mild 
 
Some awareness of symptoms, but easily tolerated; 
- Moderate Symptoms causing enough discomfort to interfere with usual activity; 
- Severe 
Incapacitating event causing inability to work or to perform usual activity. 
 
Adverse events are classified as either non-serious or serious. 
 
Serious Adverse Event (SAE) is an adverse event that: 
a) led to a death; 
b) led to a serious deterioration in health of the subject, that either resulted in: 
- a life-threatening illness or injury, or 
- a permanent impairment of a body structure or a body function, or 
- in-patient hospitalization or prolongation of existing hospitalization, or 
- in medical or surgical intervention to prevent life threatening illness or 
- injury or permanent impairment to a body structure or a body function. 
c) led to fetal distress, fetal death or a congenital abnormality or birth defect. 
 
Device deficiency: inadequacy of an investigational medical device related to its identity, quality, 
durability, reliability, safety or performance. This may include malfunctions, use error, or 
inadequacy in the information supplied by the manufacturer. 
 
AEs include any clinically significant deterioration of a subject’s medical status, after being enrolled 
and signing an informed consent form. The AE may involve any organs or systems and can be 
represented by the new onset or the deterioration of a disease, a syndrome, a symptom, a physical 
sign, as well as by findings and results of instrumental examinations and laboratory tests. Any 
medically relevant and untoward change from baseline, including frequency or pattern changes for 
a fluctuating condition (e.g., migraine), occurring after the administration of investigated treatments 

 
Versione 1 del 13/01/2020 
 
  18 
is an adverse event. All such occurrences must be recorded and reported accordingly, whether 
they appear causally related to the study medication, or not. 
 
Serious Adverse Device Effect (SADE) is an adverse device effect that has resulted in any of the 
consequences characteristic of a serious adverse event. 
 
Unanticipated Serious Adverse Device Effect (USADE) is a serious adverse device effect which 
by its nature, incidence, severity or outcome has not been identified in the current version of the 
risk analysis report or investigator’s brochure (IB). 
 
NOTE: Anticipated SADE (ASADE): an effect which by its nature, incidence, severity or outcome 
has been previously identified in the risk analysis report or IB. 
 
The following events are considered reportable: 
- any SAE; 
- any Device Deficiency that might have led to a SAE if: 
a) suitable action had not been taken or 
b) intervention had not been made or 
c) if circumstances had been less fortunate; 
- new findings/updates in relation to already reported events. 
 
Reportable events have to be reported by clinical investigators.  
 
Assessment of Causality 
The following criteria are to be used for the assessment of the causal relationship to the test/control 
treatment. For classification, all criteria of one of the following categories must be met: 
related, if  
• a timely correlation exists and 
• dechallenge and/or rechallenge and 
• a biological plausibility exists and 
• other factors are clearly excluded 
• a medical report or clinical proof exists; 
 
possibly related, if  

 
Versione 1 del 13/01/2020 
 
  19 
• a timely correlation exists and 
• dechallenge and/or rechallenge and 
• a biological plausibility exists and 
• a medical report or clinical proof does not exist; 
 
unrelated, if  
• a timely correlation does not exist or is uncertain and 
• dechallenge and/or rechallenge does not exist and 
• a biological plausibility exists and 
• a medical report or a clinical proof does not exist; 
 
unknown, if 
• not enough information exists for evaluation 
• no further contact with reporter possible. 
 
Sound medical/clinical judgment will be applied when assessing the causality and seriousness of 
the adverse events. 
 
Adverse Event Reporting 
The study center will provide the study participants with emergency telephone numbers for study 
related support and feedback. The emergency telephone number is operated by either an 
investigator of the study or by a qualified person designated by the principal investigator. The 
schedule of reachability of the emergency telephone number will be defined prior to start of the 
study. 
 
Serious Adverse Events 
The Investigator shall immediately after awareness (and in any event, not later than within 24 hours 
after awareness) inform the PI. The Investigator will send the SAE report to the following email 
address: magda.mensi@unibs.it. Appendix 3 has to be used for SAE reporting. Form has to be 
completed electronically and in English.  
 
Adverse Events 
Adverse events will be assessed by the investigator or designee within 24 hours for severity, 
relationship to the study product, possible etiologies, and whether the event meets the criteria as a 

 
Versione 1 del 13/01/2020 
 
  20 
serious adverse event and therefore requires immediate notification to the PI. For data collection 
purposes, the outcome of all adverse events recorded on the Adverse Reaction section of the CRF 
will be designated as of the completion of the final evaluation or examination. However, the 
investigator is responsible for following all adverse events until resolution or until no longer of 
clinical concern, and providing these data to the PI. At the end of the study, the investigator will 
report all adverse events (serious and non-serious) to the PI on CRFs. Forms have to be completed 
electronically and in English. 
 
Pregnancy 
No pregnant women (according to medical history) will intentionally be enrolled in this study. In the 
event a woman enrolled in this clinical research study becomes pregnant during the course of the 
study, participation in this study will be terminated upon the clinical staff’s notification of the event. 
The subject’s medical records used in this study will be updated to reflect the pregnancy and there 
will be follow- up contact until the end of the pregnancy to record the outcome in the clinical file.  
 
9. 
ADHERENCE TO PROTOCOL/AMENDMENT(S) 
The Investigator will be required to adhere to the final protocol. Any changes to the protocol, except 
those necessary to eliminate apparent hazards, will require prior approval by the local reviewers 
through the submission of a protocol amendment.  In the event of emergency, the Investigator shall 
engage any medical procedures that he/she deems appropriate. However, all such procedures 
must be promptly reported to the PI and Ethical Committee.  
The Ethical Committee which granted approval for the study must be notified of all changes in the 
protocol and must provide written approval if changes are substantial (e.g. increase the risk to the 
subject, and/or affect the rights of the subject or validity of the investigation, change of/within study 
population, number of participants or changes of patients’ age group). 
Departures from eligibility requirements may be allowed on a case-by-case basis by the medical 
monitor or other authorized sponsor representative. Such departures must be medically and 
scientifically justified, must be pre-authorized, and must be documented in the CRF and tracked as 
official eligibility waivers. 
 
10. 
ADMINISTRATIVE ASPECTS 
Final Report 
Following the completion of the study, the PI shall prepare a final study report. The final report will 
include a general description of the conduct of the study including protocol deviations, subject 

 
Versione 1 del 13/01/2020 
 
  21 
withdrawals, discussion of any adverse events, safety and efficacy data, and statistical analysis of 
the data. This report will be shared with the Co-investigator at the Participating Centre by the 
Principal Investigator, and agreed upon parts before being sent to the Sponsor. 
 
Data Retention 
The information in this and any further document contains trade secrets and commercial 
information that are privileged or confidential and may not be disclosed unless such disclosure is 
required by law or regulations.  
In any event, persons to whom the information is disclosed must be informed that the information 
is privileged or confidential and may not be further disclosed by them. 
The files pertaining to this study will be kept by the University-Hospital of Brescia for a period of 15 
years from the day of delivery of the final report and will be available for consultation by competent 
authorities at any time. 
The Investigators will retain originals of the approved study protocol, copies of completed CRFs, 
subjects' participation agreements, relevant source documents and all other supporting 
documentation related to the study for a period of 5 years. These files must be made available for 
inspection upon reasonable request by an authorized representative of EMS or the competent 
authorities. 
 
11. 
ETHICAL ASPECTS AND REGULATIONS 
Ethical Conduct of the Study 
This study is to be conducted in accordance with the ethical principles of the Declaration of Helsinki 
and according to local laws and regulations. 
 
Independent Ethics Committee (IEC) and Relevant Authorities 
Before starting the study, the study protocol will be subject to review by the Ethical Committee of 
the University-Hospital of Brescia. As required by law, the study will be notified to authorities if 
applicable. No subject should be admitted to the study before the Ethical Committees issue their 
written favourable opinion of the study. Periodic status reports must be reported to the ethics 
committee at least annually as well as notification of the completion of the study. The investigator 
must maintain an accurate and complete record of all reports and documents submitted to and 
received from the ethics committee according to the ISO 14155:2011 Clinical Investigation of 
Medical devices for human subjects. 
 

 
Versione 1 del 13/01/2020 
 
  22 
Subject information and Informed consent form 
The purpose and description of the study in lay language, possible adverse reactions, risks and 
benefits of participation and the subject’s right to withdraw without prejudice at any time must be 
explained to each subject. Each subject must read, understand and sign the informed consent form 
provided before any study-related procedure. 
 
Subject data protection and Confidentiality 
The name of the subject as well as all other personal data will be kept strictly confidential by the 
Investigator. If due to medical reasons, it is necessary to identify the subject during the study 
course, this will be done under medical secrecy.  
The subject has given his/her consent before the beginning of the study. In case of withdrawal of 
this consent subject has to leave the study. 
All subjects will agree to verify, by letter, that they participated in this study, if called upon to do so. 
 
New Findings 
Subjects will be informed of any significant new findings related to study products or procedures 
when they become known during the course of this clinical research study. Such information may 
affect the subject’s decision to continue participation in the study.