Microbiota as Early Diagnostic and predictivE Factor for Osteoarthritic Degeneration and Microbial Contamination (MILESTONE)
Phase: NA
Conditions: Knee Prosthesis, Knee Prosthesis Infection, Disbiosis
Sponsor: Istituto Clinico Humanitas

 
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PROTOCOL 
 
MIcrobiota as earLy diagnostic and predictivE factor for oSTeOarthritic degeNEration and microbial 
contamination (MILESTONE) 
 
 
Protocol Code: MILESTONE 
 
 
Version No. (Date): V2.0 (13/01/2025) 
 
 
Sponsor (Institution): IRCCS Istituto Clinico Humanitas 
 
Coordinating Investigator: 
Tommaso Bonanzinga  
tommaso.bonanzinga@hunimed.eu  
0282245425 
 
 
 
  
Confidentiality Statement 
The information contained in this document, especially unpublished data, is provided to you in confidence 
as an investigator, potential investigator or consultant. It is understood that this information will not be 
disclosed to others than the applicable Competent Ethics Committee(s) and Regulatory Authority(ies) 
without written authorization from Istituto Clinico Humanitas except to the extent necessary to obtain 
informed consent from those who will participate in the study. 
 

 
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CONTACT ADDRESSES 
 
 
  
 
Coordinating Investigator:  
Name: Tommaso Bonanzinga  
Phone: 0282245425  
e-mail: tommaso.bonanzinga@hunimed.eu 
 
Study Research Coordinator: 
Name: Margherita Vacca 
Phone: 0282247770 
e-mail: margherita.vacca@humanitas.it 
 
Name: Martina di Martino 
Phone: 0282245206 
e-mail: martina.dimartino@humanitas.it 
 
Name: Elisa Pupino 
Phone: 0282245931 
e-mail: elisa.pupino@humanitas.it 
Statistician: 
Name: Emanuela Morenghi 
Phone: 0282244556 
e-mail: emanuela.morenghi@humanitas.it 
 
Central Laboratory 
 
Name: Istituto Clinico Humanitas 
Reference Person: Chiara Pozzi 
Phone: 0282245432 
e-mail: chiara.pozzi@humanitasresearch.it 

 
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COORDINATING INVESTIGATOR 
I have approved this Protocol entitled <MIcrobiota as earLy diagnostic and predictivE factor for oSTeOarthritic 
degeNEration and microbial contamination (MILESTONE)= and I agree to conduct the study as detailed herein 
and according to the current version of the World Medical Association Declaration of Helsinki, Good Clinical 
Practice guideline and applicable regulatory requirements. I will provide all study personnel under my 
supervision with all information needed to perform the study and I will inform them about their responsibilities 
and obligations 
 
 
Printed name 
Tommaso Bonanzinga 
Role 
Principal Investigator 
Department 
Center for the functional and biological reconstruction of the knee joint 
Signature 
 
Date 
13/01/2025 
 
STATISTICIAN 
I have approved this Protocol entitled <MIcrobiota as earLy diagnostic and predictivE factor for oSTeOarthritic 
degeNEration and microbial contamination (MILESTONE)= 
 
 
Printed name 
Emanuela Morenghi 
Signature 
 
Date 
13/01/2025 
 

 
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CENTRE SIGNATURE – PRINCIPAL INVESTIGATOR 
 
I have read this Protocol Amendment relevant to the study entitled <MIcrobiota as earLy diagnostic and 
predictivE factor for oSTeOarthritic degeNEration and microbial contamination (MILESTONE)= and I agree 
to conduct the study as detailed herein and in compliance with guidelines for Good Clinical Practice and 
applicable regulatory requirements. I will provide all study personnel under my supervision with all 
information provided by the Coordinating Investigator/Sponsor and I will inform them about their 
responsibilities and obligations. 
 
 
Printed name 
Tommaso Bonanzinga 
Role 
Principal Investigator 
Department 
Center for the functional and biological reconstruction of the knee joint 
Signature 
 
Date 
13/01/2025 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
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Abbreviations 
 
CA 
 
Competent Authority 
EC 
 
Ethics Committee 
eCRF  
electronic Case report form 
GCP 
 
Good Clinical Practice  
IMP 
 
Investigational Medicinal Product 
 
 
 
 
 
 
 
 
  
 

 
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INDEX 
 
1. SUMMARY .................................................................................................................................................. 9 
2. STUDY FLOW-CHART ........................................................................................................................... 14 
3. BACKGROUND ........................................................................................................................................ 16 
4. RATIONALE ............................................................................................................................................. 16 
5. STUDY OBJECTIVES ............................................................................................................................. 16 
6. STUDY POPULATION ............................................................................................................................ 16 
7. INCLUSION AND EXCLUSION CRITERIA ....................................................................................... 17 
7.1. INCLUSION CRITERIA....................................................................................................................... 17 
7.2 EXCLUSION CRITERIA ...................................................................................................................... 17 
7.3. RECRUITMENT .................................................................................................................................... 17 
8. STUDY ASSESSMENT ............................................................................................................................ 18 
9. STATISTICAL CONSIDERATIONS ..................................................................................................... 20 
9.1. Sample size .............................................................................................................................................. 20 
9.2. Analysis .................................................................................................................................................... 20 
10. QUALITY ASSURANCE AND CONTROL ........................................................................................ 21 
10.1. Data handling and record keeping / archiving................................................................................... 21 
10.2. Case Report Forms ............................................................................................................................... 21 
10.3. Source documents ................................................................................................................................. 21 
11. CONFIDENTIALITY OF PATIENT RECORDS ............................................................................... 21 
12. ETHICAL CONSIDERATIONS ........................................................................................................... 21 
13. INFORMED CONSENT ......................................................................................................................... 22 
14. DATA OWNERSHIP .............................................................................................................................. 22 

 
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15. PUBLICATION POLICY ...................................................................................................................... 22 
16. FUNDING AND SUPPORT ................................................................................................................... 22 
 
 

 
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1. SUMMARY   
 
 
Study Title 
MIcrobiota as earLy diagnostic and predictivE factor for 
oSTeOarthritic degeNEration and microbial. Contamination 
Study code  
 
Acronym: 
MILESTONE 
Version and Date 
V 2.0 13/01/2025 
Sponsor (Institution) 
IRCCS Istituto Clinico Humanitas 
Coordinating Investigator 
Tommaso Bonanzinga 
Supporter 
NA 
Product Name 
NA 
Study indication  
Osteoarthritis 
Background and rationale 
Recent studies have highlighted the role of gut microbiota in the 
pathogenesis of a variety of human metabolic, immunological, and 
neurological diseases such as inflammatory bowel disease (IBD) and 
irritable bowel syndrome (IBS), cardiovascular disease, and certain 
types of cancer [1]. In the field of orthopedics, the gut microbiota has 
gained attention as an environmental factor that can influence the 
development and progression of osteoarthritis (OA) [2]. It is now 
understood that OA is not solely a disease of mechanical wear and tear 
but involves complex interactions between genetic, environmental, 
and lifestyle factors [3]. The gut microbiota has gained attention as 
one of the environmental factors that can influence the development 
and progression of OA. It has been postulated that gut dysbiosis could 
exert its influence on the joint by establishing a chronic low-grade 
inflammation, altering the production of fatty acids which influences 
cartilage and modulation of pain perception [4]. Pre-clinical studies on 
animals have indeed confirmed this link but no study on humans has 
yet been published in the literature. [5] 
The goal of this study is to investigate how gut microbiota could 
influence the correct functioning of the body’s main joint and how it 
affects the development of early OA, Periprosthetic Joint Infection 
(PJI), and recovery after total joint replacement. 
We hypothesized that gut microbiota could influence the correct 
functioning of the main joint of the body, and therefore affect the 
development of early OA, of PJI and even the recovery after total joint 
replacement. If this hypothesis was confirmed by the results of the 
current study, it would change our understanding of osteoarthritis and 
periprosthetic joint infections providing a novel target for our 
preventive and possibly even therapeutic strategies. For example, 
probiotics, prebiotics, postbiotics and fecal microbiota transplantation 

 
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(FMT) could become new weapons in the orthopedic field. If gut 
dysbiosis will be found to be a predictive factor of early osteoarthritis, 
this will allow early recognition of the condition and therefore a 
precocious management of the condition [6]. In addition, the discovery 
that microbiota influences recovery after joint replacement could help 
us improving the post-operative care and ameliorating the recovery. 
This would translate in an improvement of the quality of life for the 
patient and a reduction of the costs for the national healthcare system.  
[1] Gebrayel P. et al. Microbiota medicine: towards clinical 
revolution. J Transl Med 2022 
[2] Hao X. et al. The gut microbiota in osteoarthritis: where do we 
stand and what can we do? Arthritis Res Ther 2021 
[3] Martel-Pelletier J. et al. NRDP 2016  
[4] Hao X. et al. AR Ther 2021  
[5] Tan TC et al. IJRD 2021  
[6] Chu, C.R., Williams, A.A., Coyle, C.H. et al. Early diagnosis to 
enable early treatment of pre-osteoarthritis. Arthritis Res Ther 14, 212 
(2012).  
 Study Objectives 
Objective #1: to estimate the prevalence of early OA among patients 
with gut dysbiosis. 
Objective #2: to investigate the intra-articular microbiota during 
surgery;  fecal and oral microbiota, before surgery and during recovery; 
permeability markers from blood and feces before surgery. 
Objective #3: to describe gut microbiota environment predisposing to 
gut dysbiosis, which negatively affects life expectancy in these 
patients, in periprosthetic joint infections (PJI) patients. 
Study design 
Multicentric observational 
Eligibility Criteria 
 
For objective #1 (to estimate the prevalence of early OA among 
patients with gut dysbiosis) 
Inclusion criteria: gut dysbiosis; age range 18-50 years old 
Exclusion criteria: musculoskeletal symptoms; previous shoulder or 
knee surgery; previous shoulder or knee known pathological 
conditions; rheumatological diseases 
For objective #2 (to investigate the intra-articular microbiota during 
surgery;  fecal and oral microbiota, before surgery and during 
recovery; permeability markers from blood and feces before surgery) 
Inclusion criteria: patients undergoing total knee replacement surgery 
Exclusion criteria: any concurrent or previous diseases or conditions 
which might negatively affect the surgery 
For objective #3 (to describe gut microbiota environment predisposing 
to gut dysbiosis, which negatively affects life expectancy in these 
patients, in periprosthetic joint infections (PJI) patients) 

 
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Inclusion criteria: patients undergoing joint revision surgery for septic 
failure of a knee or hip prostheses or patients undergoing revision 
surgery for aseptic mobilization for PJI 
Exclusion criteria: major predisposing factor for gut dysbiosis 
(antibiotic therapy in the last 6 months, BMI > 40 and inflammatory 
bowel disease); chronic inflammatory joint diseases (e.g., rheumatoid 
arthritis, psoriatic arthritis); acute (< 90 days after the index procedure) 
and late hematogenous (symptoms of less than three weeks duration) 
infections; an inadequate amount of synovial fluid (< 10 mL) for 
culture, WBC, and PMN (neutrophil) percentage determinations 
Study Procedures   
The study is a multicentre observational study divided into 3 
objectives: 
Objective #1 is to enrol 40 patients with established intestinal dysbiosis 
and no musculoskeletal symptoms. Patients with potential dysbiosis 
will undergo an initial (V1) gastroenterological examination at the 
Operative Unit of Gastroenterology and Digestive Endoscopy in 
Humanitas, where they will be asked to join the clinical study. Each 
patient will be explained the study through the means of the informed 
consent process, and those who wish to join the study will sign the 
informed consent form. For each patient, the Bristol scale will be used 
as a tool to categorize the patients’ stool as reported by the patient; a 
stool sample collection kit will be provided to the patient together with 
its instructions for use. Patients will be instructed to provide a stool 
sample (V2) at the immunology and microbiota unit laboratory. On the 
same occasion, the clinician will perform a buccal swab to collect a 
saliva sample from the patient (see section 8 for specifics). Both 
samples will initially be stored at the Biobank - Cancer Center (Istituto 
Clinico Humanitas - Via Manzoni 56, 20089 Rozzano (MI)) under the 
responsibility of Dr. Daniela Pistillo and will then be processed at the 
laboratory of the immunology and microbiota unit and will be used for 
confirming the dysbiosis diagnosis. If the patient is not positive for 
dysbiosis markers, he/she will be classified as SCREENING 
FAILURE; if the dysbiosis is confirmed, the patient will be asked to 
come to another visit (V3) where he/she will undergo a MRI of the 
knee and a shoulder ultrasound scan in order to check for early 
osteoarthritis signs. Optional orthopaedic examination may be 
performed. Blood test will also be performed part of the samples of 
which will be used to evaluate biomarkers and will initially be stored 
at the Biobank - Cancer Center (Istituto Clinico Humanitas - Via 
Manzoni 56, 20089 Rozzano (MI)) with Dr. Daniela Pistillo in charge 
and will subsequently be processed. The other part of the blood 
samples on which insulin, glycated haemoglobin and glucose will be 
analysed will be sent to the Central Analysis Laboratory within ICH 
(see section 8 for details). 
Objective #2 is to enrol 50 patients undergoing total knee arthroplasty 
(TKA). Patients scheduled to undergo TKA will undergo a pre-
hospitalization orthopaedic examination (V1) where he/she will be 
informed about the clinical study. If he/she agrees to join the study, 

 
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he/she will sign the informed consent form and will follow study 
procedures: questionnaires will be administered and the patient will 
receive a stool sample collection kit together with its instructions for 
use. The stool sample will be used for DNA extraction at the 
immunology and microbiota unit laboratory and subsequent 
metagenomic analysis. Patient will be instructed to hand over the stool 
sample during hospital visit for TKA (V2), during which intra-articular 
tissue samples will be collected for metagenomics. In addition,  buccal 
swab and blood samples of the patient undergoing the surgery will be 
collected before surgery. Saliva, stool and blood samples for 
biomarkers will initially be stored at the Biobank - Cancer Center 
(Istituto Clinico Humanitas - Via Manzoni 56, 20089 Rozzano (MI)) 
under the responsibility of Dr. Daniela Pistillo and will subsequently 
be processed at the laboratory of the immunology and microbiota unit. 
The other part of the blood samples on which insulin, glycated 
haemoglobin and glucose will be analysed will be sent to the ICH's in-
house Central Analysis Laboratory. On discharge, patient will be 
provided another stool collection kit. One month after surgery (V3), a 
follow-up examination will be performed, where patient will bring the 
second stool sample and will undergo another buccal swab which will 
also be analysed by the same laboratory (see section 8). For stool and 
saliva samples of V3, the storage and analysis procedure is the same as 
for V2. In addition, the patient will fill in the KSS questionnaires. 
Objective #3 involves the enrolment of 80 patients undergoing joint 
revision surgery who are further divided into:  
Group 1: 40 patients for septic failure of a knee or hip replacement.  
Group 2: 40 patients for aseptic loosening for PJI 
Patients will undergo a pre-hospitalization visit (V1) with orthopaedic 
check-up, where they will be asked to join the clinical study. Each 
patient will be explained the study through the means of the informed 
consent process, and those who wish to join the study will sign the 
informed consent form. Patient will be provided a stool sample 
collection kit for DNA extraction at the immunology and microbiota 
unit laboratory and subsequent metagenomic analysis. The patient will 
be instructed to hand over the stool sample during hospital visit for 
revision surgery (V2) (see section 8). 
Number of patients (planned) 
Three objectives are set and a total of 170 patients are divided as 
following:  
Objective #1: 40 patients with proven gut dysbiosis and without 
musculoskeletal symptoms undergoing MRI of the knee and the 
ultrasound of the shoulder. 
Objective #2: 50 patients undergoing total knee replacement.  
Objective #3: 80 patients, which are further subdivided into:  
Group 1: 40 patients undergoing joint revision surgery for septic 
failure of a knee or hip prostheses.  
Group 2: 40 patients undergoing revision surgery for aseptic 
mobilization for PJI.  

 
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Investigational Sites (planned)  
Single-centre study   □     
Multi-centre study    x 
1: Centres: IRCCS Istituto Clinico Humanitas (Rozzano) 
2: AORN Antonio Cardarelli (Napoli) 
 
Sample size and statistical 
consideration 
Regarding the different objective of the study, Experimental design 
objective #1 and Experimental design objective #3, had the primary 
aim to describe the sample, so the study will enroll all the patients 
fulfilling inclusion and exclusion criteria, estimable in around 40 for 
Experimental design objective #1 and 80 for Experimental design 
objective #3. 
Regarding Experimental design objective #2, we can expect a 
difference in KSS between patient with or without microbiota of 9 
points. With an alpha error of 0.05 and a power of 0.8, considering a 
standard deviation of 11, the study will enroll 25 patients with 
microbiota and 25 without, for a total of 50 patients. 
Study timetable 
Provide the following study milestones: 
 
Planned date of the First Patient In (FPI - date of the Informed 
Consent signature of the first study patient): february 2025 
 
Planned date of the Last Patient In (LPI - date of the Informed 
Consent signature of the last study patient): february 2026 
 
Planned date of the Last Patient Out (LPO – date of the last visit of 
the last study patient): february 2026 
 
Planned study duration (from FPI to LPO): 24 months 
GCP Statement: 
 
This study will be conducted in compliance with the protocol, the 
current version of the Declaration of Helsinki and applicable 
guidelines as well as all national legal and regulatory requirements.  
 
 

 
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2. STUDY FLOW-CHART 
 
Objective #1 
 
Assessment / events  
V1 
V2 
V3  
ICF signed 
x 
 
 
Demographic data 
x 
 
 
Medical history 
x 
 
 
Gastroenterological examination 
x 
 
 
Kit for stool sample collection handover 
x 
 
 
Stool sample collection 
 
x 
 
Saliva sample collection 
 
x 
 
Blood analysis 
 
 
x 
Magnetic resonance imaging of the knee   
 
 
x  
Ultrasound of the shoulder 
 
 
x 
Orthopedic examination 
 
 
x* 
*: optional 
 
 
Objective #2 
 
Assessment / events 
V1 
V2 
V3 (1 M after 
surgery) 
ICF signed 
x 
 
 
Demographic data 
x 
 
 
Medical history 
x 
 
 
Kit for stool sample collection handover 
x 
x 
 
Stool sample collection 
 
x 
x 
Blood analysis  
 
x 
 
Intra-articular sampling 
 
x 
 
Total knee replacement 
 
x 
 
Saliva sample 
 
x 
x 
KSS score  
x 
 
x 
Orthopaedic visit 
x 
 
x 
 
 
Objective #3 
 
 
V1  
V2 
Assessment / events  
x 
 

 
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ICF signed 
x 
 
Demographic data 
x 
 
Medical history 
x 
 
Kit for stool sample collection 
handover  
x 
 
Stool sample collection 
 
x 
Reintervention 
 
x 
 
 

 
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3. BACKGROUND  
Recent studies have highlighted the role of gut microbiota in the pathogenesis of a variety of human metabolic, 
immunological, and neurological diseases such as inflammatory bowel disease (IBD) and irritable bowel 
syndrome (IBS), cardiovascular disease, and certain types of cancer[1]. In the field of orthopedics, the gut 
microbiota has gained attention as an environmental factor that can influence the development and progression 
of osteoarthritis (OA)[2]. It is now understood that OA is not solely a disease of mechanical wear and tear but 
involves complex interactions between genetic, environmental, and lifestyle factors [3]. The gut microbiota 
has gained attention as one of the environmental factors that can influence the development and progression 
of OA. It has been postulated that gut dysbiosis could exert its influence on the joint by establishing a chronic 
low-grade inflammation, altering the production of fatty acids which influences cartilage and modulation of 
pain perception [4]. Pre-clinical studies on animals have indeed confirmed this link but no study on humans 
has yet been published in the literature. [5] 
The goal of this study is to investigate how gut microbiota could influence the correct functioning of the body’s 
main joint and how it affects the development of early OA, Periprosthetic Joint Infection (PJI), and recovery 
after total joint replacement. 
[1] Gebrayel P. et al. Microbiota medicine: towards clinical revolution. J Transl Med 2022 
[2] Hao X. et al. The gut microbiota in osteoarthritis: where do we stand and what can we do? Arthritis Res 
Ther 2021 
[3] Martel-Pelletier J. et al. NRDP 2016  
[4] Hao X. et al. AR Ther 2021  
[5] Tan TC et al. IJRD 2021  
[6] Chu, C.R., Williams, A.A., Coyle, C.H. et al. Early diagnosis to enable early treatment of pre-osteoarthritis 
. Arthritis Res Ther 14, 212 (2012).  
 
4. RATIONALE 
We hypothesized that gut microbiota could influence the correct functioning of the main joint of the body, and 
therefore affect the development of early OA, of PJI and even the recovery after total joint replacement. If this 
hypothesis was confirmed by the results of the current study, it would change our understanding of 
osteoarthritis and periprosthetic joint infections providing a novel target for our preventive and possibly even 
therapeutic strategies. For example, probiotics, prebiotics, and fecal microbiota transplantation (FMT) could 
become new weapons in the orthopedic field. If gut dysbiosis will be found to be a predictive factor of early 
osteoarthritis, this will allow early recognition of the condition and therefore a precocious management of the 
condition [1]. In addition, the discovery that microbiota influences recovery after joint replacement could help 
us improving the post-operative care and ameliorating the recovery. This would translate in an improvement 
of the quality of life for the patient and a reduction of the costs for the national healthcare system. 
 
[1] Chu, C.R., Williams, A.A., Coyle, C.H. et al. Early diagnosis to enable early treatment of pre-osteoarthritis. 
Arthritis Res Ther 14, 212 (2012). 
 
 
5. STUDY OBJECTIVES 
 
Experimental design Objective #1: to estimate the prevalence of early OA among patients with gut dysbiosis. 
Experimental design Objective #2: to investigate the intra-articular microbiota during surgery; fecal and oral 
microbiota, before surgery and during recovery; permeability markers from blood and feces before surgery. 
Experimental design Objective #3: to describe gut microbiota environment predisposing to gut dysbiosis, 
which negatively affects life expectancy in these patients, in periprosthetic joint infections (PJI) patients 
 
6. STUDY POPULATION 
A total of 170 patients are divided as following across the three study objectives:  
 

 
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Experimental design objective #1: 40 patients with proven gut dysbiosis and without musculoskeletal 
symptoms undergoing MRI of the knee and the ultrasound of the shoulder.  
 
Experimental design objective #2: 50 patients undergoing total knee replacement.  
 
Experimental design objective #3: 80 patients are further subdivided into:  
Group 1: 40 patients undergoing joint revision surgery for septic failure of a knee or hip prostheses.  
Group 2: 40 patients undergoing revision surgery for aseptic mobilization for PJI. 
 
7. INCLUSION AND EXCLUSION CRITERIA 
7.1. INCLUSION CRITERIA  
For objective #1 (to estimate the prevalence of early OA among patients with gut dysbiosis) 
Inclusion criteria: proven gut dysbiosis (increased Firmicutes/Bacteroidetes (F/B) phyla ratio and increased 
permeability); age range 18-50 years old. 
For objective #2 to investigate the intra-articular microbiota during surgery;  fecal and oral microbiota, before 
surgery and during recovery; permeability markers from blood and feces before surgery.  
Inclusion criteria: patients undergoing total knee replacement surgery 
For objective #3 (to describe gut microbiota environment predisposing to gut dysbiosis, which negatively 
affects life expectancy in these patients, in periprosthetic joint infections (PJI) patients) 
Inclusion criteria: patients undergoing joint revision surgery for septic failure of a knee or hip prostheses; 
patients undergoing revision surgery for aseptic mobilization for PJI 
 
7.2 EXCLUSION CRITERIA 
For objective #1 (to estimate the prevalence of early OA among patients with gut dysbiosis) 
Exclusion criteria: musculoskeletal symptoms; previous shoulder or knee surgery; previous shoulder or knee 
known pathological conditions; rheumatological diseases 
 
For objective #2  to investigate the intra-articular microbiota during surgery;  fecal and oral microbiota, before 
surgery and during recovery; permeability markers from blood and feces before surgery. 
Exclusion criteria: any concurrent or previous diseases or conditions which might negatively affect the surgery 
 
For objective #3 (to describe gut microbiota environment predisposing to gut dysbiosis, which negatively 
affects life expectancy in these patients, in periprosthetic joint infections (PJI) patients) 
Exclusion criteria: major predisposing factor for gut dysbiosis (antibiotic therapy in the last 6 months, BMI > 
40 and inflammatory bowel disease); chronic inflammatory joint diseases (e.g., rheumatoid arthritis, psoriatic 
arthritis); acute (< 90 days after the index procedure) and late hematogenous (symptoms of less than three 
weeks duration) infections; an inadequate amount of synovial fluid (< 10 mL) for culture, WBC, and PMN 
(neutrophil) percentage determinations 
 
 
7.3. RECRUITMENT 
Objective #1: young (18-50 years old) patients with potential dysbiosis who are scheduled to undergo a 
gastroenterological consultation will be explained the purposes of the clinical study during the consultation 

 
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through the means of the informed consent process. Patients will be asked to provide a stool sample and a 
saliva sample (buccal swab), which will be assessed in order to confirm the dysbiosis. Upon diagnosis 
confirmation, patients will join the clinical study and will undergo follow up examinations as scheduled in the 
protocol. 
Objective #2: during the course of a routine pre-surgery assessment visit, patients who comply with the 
inclusion/exclusion criteria who are already scheduled to undergo total knee replacement will be explained the 
clinical study; if the patients agree to join the study, they will undergo follow up examinations as scheduled in 
the protocol. 
Objective #3: patients who comply with the inclusion/exclusion criteria who present with failed or painful total 
hip or knee arthroplasty will undergo standardized diagnostic protocol to identify those with a PJI. The 
diagnosis of chronic infection (> 90 days after the index procedure) will be made based on the International 
Consensus Group (ICM) definition of PJI. Site staff will explain the clinical study and patients will then be 
asked to join the clinical study during the pre-surgery assessment visit. If the patients agree to join the study, 
they will undergo follow up examinations as scheduled in the protocol. 
 
8. STUDY ASSESSMENT  
Prior to conducting any of the pre-entry test not performed routinely in the treatment of the patient, the 
investigator will: 
 
explain the study to the patient by means of the informed consent process  
 
collect a signed copy of written informed consent. 
 
The study is a multicentre observational study divided into 3 objectives: 
Objective #1 is to enrol 40 patients with established intestinal dysbiosis and no musculoskeletal symptoms. 
Patients with potential dysbiosis will undergo an initial (V0) gastroenterological examination at the Operative 
Unit of Gastroenterology and Digestive Endoscopy in Humanitas, where they will be asked to join the clinical 
study. Each patient will be explained the study through the means of the informed consent process, and those 
who wish to join the study will sign the informed consent form. For each patient, the Bristol scale will be used 
as a tool to categorize the patients’ stool as reported by the patient; a stool sample collection kit will be provided 
to the patient together with its instructions for use. Patients will be instructed to provide a stool sample (V1) 
at the immunology and microbiota unit laboratory. On the same occasion, the clinician will perform a buccal 
swab to collect a saliva sample from the patient. (see section 8 for specifics). Both samples will initially be 
stored at the Biobank - Cancer Center (Istituto Clinico Humanitas - Via Manzoni 56, 20089 Rozzano (MI)) 
under the responsibility of Dr. Daniela Pistillo and will then be processed at the laboratory of the immunology 
and microbiota unit and will be used for confirming the dysbiosis diagnosis. be used for confirming the 
dysbiosis diagnosis. If the patient is not positive for dysbiosis markers, he/she will be classified as 
SCREENING FAILURE; if the dysbiosis is confirmed, the patient will be asked to come to another visit (V3) 
where he/she will undergo a MRI of the knee and a shoulder ultrasound scan in order to check for early 
osteoarthritis signs. Optional orthopaedic examination may be performed. Blood test will also be performed 
part of the samples of which will be used to evaluate biomarkers and will initially be stored at the Biobank - 
Cancer Center (Istituto Clinico Humanitas - Via Manzoni 56, 20089 Rozzano (MI)) with Dr. Daniela Pistillo 
in charge and will subsequently be processed. The other part of the blood samples on which insulin, glycated 
haemoglobin and glucose will be analysed will be sent to the Central Analysis Laboratory within ICH (see 
section 8 for details).  
Objective #2 is to enrol 50 patients undergoing total knee arthroplasty (TKA). Patients scheduled to undergo 
TKA will undergo a pre-hospitalization orthopaedic examination (V1) where he/she will be informed about 
the clinical study. If he/she agrees to join the study, he/she will sign the informed consent form and will follow 
study procedures: questionnaires will be administered and the patient will receive a stool sample collection kit 

 
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together with its instructions for use. The stool sample will be used for DNA extraction at the immunology 
and microbiota unit laboratory and subsequent metagenomic analysis. Patient will be instructed to hand over 
the stool sample during hospital visit for TKA (V2), during which intra-articular tissue samples will be 
collected for metagenomics. In addition, buccal swab and blood samples of the patient undergoing the surgery 
will be collected before surgery. Saliva, stool and blood samples for biomarkers will initially be stored at the 
Biobank - Cancer Center (Istituto Clinico Humanitas - Via Manzoni 56, 20089 Rozzano (MI)) under the 
responsibility of Dr. Daniela Pistillo and will subsequently be processed at the laboratory of the immunology 
and microbiota unit. The other part of the blood samples on which insulin, glycated haemoglobin and glucose 
will be analysed will be sent to the ICH's in-house Central Analysis Laboratory. On discharge, patient will be 
provided another stool collection kit. One month after surgery (V3), a follow-up examination will be 
performed, where patient will bring the second stool sample and will undergo another buccal swab which will 
also be analysed by the same laboratory (see section 8). For stool and saliva samples of V3, the storage and 
analysis procedure is the same as for V2. In addition, the patient will fill in the KSS questionnaires. 
Objective 3 involves the enrolment of 80 patients undergoing joint revision surgery who are further divided 
into:  
Group 1: 40 patients for septic failure of a knee or hip replacement.  
Group 2: 40 patients for aseptic loosening for PJI 
Patients will undergo a pre-hospitalization (V1) with orthopaedic check-up, where they will be asked to join 
the clinical study. Each patient will be explained the study through the means of the informed consent process, 
and those who wish to join the study will sign the informed consent form. Patient will be provided a stool 
sample collection kit for DNA extraction at the immunology and microbiota unit laboratory and subsequent 
metagenomic analysis. The patient will be instructed to hand over the stool sample during hospital visit for 
revision surgery (V2) (see section 8). 
 
Details for sampling 
The following samples will be collected: 
1. Feces (microbiota and biomarker of intestinal permeability) 
2. Saliva (buccal swab) (microbiota) 
3. Intra-articular tissue (microbiota) 
4. Peripheral blood (serum collected in EDTA) for the evaluation of biomarkers associated with 
intestinal permeability biomarkers, fasting glucose and insulin, glycated haemoglobin (HbA1c). 
Fecal sampling 
The feces will be collected by the patient within 24 hours preceding the hospital visit. Detailed instructions 
and the container for the collection will be provided to the patient. The container will be transferred to the 
hospital and then to the laboratories at +4°C. Once in the laboratories the sample will be stored at -80°C. 
Oral sampling: Saliva samples will be collected using a buccal swab performed by a healthcare professional, 
and frozen as soon as possible at – 80°C for subsequent microbiome analysis.  It is recommended to perform 
the test when the mouth is clean, as external substances can skew the analysis results. It is advised to collect 
the sample before taking any oral medications and to avoid smoking, eating, chewing gum, drinking coffee, or 
any beverages other than water for 30 minutes prior to the collection. It is recommended to drink water 15 
minutes before the collection to facilitate salivation. To increase saliva production, the patients should massage 
the inside of their cheeks with their tongue and to push the tip of their tongue against their teeth. Yawning or 
making chewing motions can also help increase salivation. 

 
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Intra-articular tissue sampling for microbiome analysis:  For each patient, 5 intra-articular samples will be 
taken intraoperatively: two SF (sinovial fluid) samples, one from ST (sinovial tissue), one from cartilage and 
one from Hoffa's fat pad. They are then analyzed as follows: bacterial DNA will be extracted using DNeasy 
PowerSoil Pro Kits (Qiagen) according to the manufacturer’s procedures. DNA concentration will be measured 
using the NanoDrop spectrophotometer (Thermo Fisher scientific) and stored at −80 °C. Libraries will be 
prepared using Illumina® DNA prep according to the manufacturer’s procedures. 16S rRNA gene sequencing 
will be carried out. Standard microbiota diversity metrics (e.g., alpha diversity, including species richness and 
evenness, beta-diversity, rarefaction curves) and taxonomy will be calculated. 
Blood sampling: Prior to surgery, peripheral blood samples will be collected from each patient enrolled in the 
observational study into 2 BD Vacutainer SST II Advanced (REF 367953) and will be incubated 30' at room 
T°. Tubes will then be centrifuged 2000 g with maximum acceleration and slowing down (9/9) at Room T° 
21°C 15'. In sterile conditions, serum will be drawn with P1000 pipette tip with sterile filter. The serum will 
be employed for assessing intestinal permeability-associated biomarkers, fasting glucose and insulin, glycated 
haemoglobin (HbA1c). 
Oral, and fecal microbiome analysis: Bacterial DNA will be extracted using DNeasy PowerSoil Pro Kits 
(Qiagen) according to the manufacturer’s procedures. DNA concentration will be measured using the 
NanoDrop spectrophotometer (Thermo Fisher scientific) and stored at −80 °C. Libraries will be prepared using 
Illumina® DNA prep according to the manufacturer’s procedures. Shotgun metagenomic sequencing will be 
performed for buccal (~ 15 Gb/sample) and fecal (~ 5-7.5 Gb/sample) samples. Standard microbiota diversity 
metrics (e.g., alpha diversity, including species richness and evenness, beta-diversity, rarefaction curves) will 
be calculated.Taxonomic profiling will be performed by using the latest version available of MetaPhlAn and 
the latest taxonomic database available. Output: MetaPhlAn 4 taxonomy tables with relative abundances of 
species/SGBs. Functional profiling will be performed by using the latest version available of HUMAnN. 
Output: HUMAnN tables with gene families UniRef90, RPKMs, BioCyc path RPKMs and path coverage in 
.tsv files. 
Analysis of biomarkers associated with intestinal permeability: Fecal and blood samples will be used to 
measure, by specific ELISA kits, intestinal permeability biomarkers: e.g Zonulin will be measured both in the 
serum and in the feces; bacterial DNA, I-FABP and LBP will be measured in the serum. 
 
 
9. STATISTICAL CONSIDERATIONS  
9.1. Sample size 
Regarding the different objective of the study, Experimental design objective #1 and Experimental design 
objective #3, had the primary aim to describe the sample, so the study will enroll all the patients fulfilling 
inclusion and exclusion criteria, estimable in around 40 for Experimental design objective #1 and 80 for 
Experimental design objective #3. 
Regarding Experimental design objective #2, we can expect a difference in KSS between patient with or 
without microbiota of 9 points. With an alpha error of 0.05 and a power of 0.8, considering a standard deviation 
of 11, the study will enroll 25 patients with microbiota and 25 without, for a total of 50 patients. 
 
9.2. Analysis 
Data will be described as number and percentage, if categorical, or mean and standard deviation, if continuous, 
approximately Gaussian, or median and range, otherwise. Adherence to Gaussian distribution will be checked 
with Shapiro Wilks test. 
For Experimental design objective #1 and Experimental design objective #3 only descriptive statistics will be 
performed. For Experimental design objective #2, differences between the two groups will be explored with 

 
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chi square test, for continuous variables, or with t student test or with Mann Whitney test for numerical 
variables, according to their distribution. Association with arthroplasty recovery will be evaluated with logistic 
regression analysis, and the results will be expressed as odds ratio (OR), with 95% confidence interval 
(95%CI). 
Significance threshold will be set to 0.05. All analyses will be made with Stata version 18 or superior. 
 
10. QUALITY ASSURANCE AND CONTROL  
 
Quality Assurance and Quality Control systems based on written SOPs are in place at the Sponsor site.  
 
10.1. Data handling and record keeping / archiving  
The investigator must keep the documents on file for at least 7 years after completion or discontinuation of the 
study. After that period, the documents may be destroyed, subject to local regulations. Before proceeding to 
documents’ destruction, sites must inform the Coordinating Investigator/delegate in writing.  
Should the investigator wish to assign the study records to another party or move them to another location, the 
Coordinating Investigator/delegate must be notified in advance.  
 
10.2. Case Report Forms  
After having signed the informed consent form (ICF) and privacy form, patients’ clinical, demographic and 
radiological data will be recorded during each visit in the visit report. Any data relevant to the study protocol 
will be recorded in Excel files which are protected by a password and who will be accessed only by study 
staff. 
The investigator will ensure the accuracy, completeness and timeliness of the data reported in the files. 
Participants will not be identified in the Excel files by name or initials and birth date. Appropriate coded 
identification will be used. 
 
10.3. Source documents  
Source data must be available at the site to document the existence of the study participants. Source data 
include the original documents relating to the study, as well as the medical treatment and medical history of 
the participant. 
 
11. CONFIDENTIALITY OF PATIENT RECORDS 
The investigator assures that patients’ anonymity should be maintained and that their identities are protected 
from unauthorized parties. Particular attention should be paid whenever patient data are supplied to third 
parties and may be autonomously processed.  
The investigator should keep in a confidential way a patient identification log recording both patient code and 
name. The investigator should also maintain patients’ written consent forms, in strict confidence (i.e. not for 
submission to the Coordinating Investigator).  
Any investigator and/or research staff member who has a conflict of interest with this study (such as patent 
ownership, royalties, or financial gain greater than the minimum allowable by their institution) must fully 
disclose the nature of the conflict of interest.   
   
12. ETHICAL CONSIDERATIONS 
The responsible investigator ensures that this study is conducted in agreement with this protocol, the Good 
Clinical Practice, the current version of Declaration of Helsinki and the applicable regulations. 

 
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The protocol and any amendments are subject to review and approval by the competent Independent Ethics 
Committee(s) (<IEC=). 
 
13. INFORMED CONSENT 
All patients should be informed of the aims of the study. They should be informed as to the strict confidentiality 
of their patient data, but that their medical records may be reviewed for study purposes by authorized 
individuals other than their treating physician.  
It should be emphasized that the participation is voluntary and that the patient is allowed to refuse further 
participation in the protocol whenever he/she wants. This does not prejudice the patient’s subsequent care. 
Documented informed consent must be obtained for all patients included in the study before any study related 
procedure is performed. The written informed consent form should be signed and personally dated by the 
patient or by the patient’s legally acceptable representative, and by the Investigator who has provided the study 
information. 
 
14. DATA OWNERSHIP 
Istituto Clinico Humanitas is the owner of the data resulting from the study. All centers and investigators 
participating in the study should be made aware of such circumstance and not to disseminate information or 
data without the prior written consent by Istituto Clinico Humanitas. 
 
15. PUBLICATION POLICY 
After completion of the study, the Coordinating Investigator prepares a draft manuscript containing final results 
of the study on the basis of the statistical analysis. The manuscript is delivered to the co-authors for comments 
and then sent to a scientific journal for publication. 
All publications, abstracts, presentations, manuscripts and slides - issued by the Investigators of the 
collaborative sites and including data from the present study- should be submitted to and reviewed by the 
Coordinator Investigator at least 3 (three) weeks in advance the planned date for the submission to the scientific 
journal. 
 
16. FUNDING AND SUPPORT 
The present research project has been approved in the Piano Nazionale di Ripresa e Resilienza (PNRR-
MCNT2-2023-12377976) and will be funded by the Italian Ministry of Health, Direzione generale della 
Ricerca e dell’innovazione in sanità.