The Role of VSL#3® in the Treatment of Fatigue and Other Symptoms in Long Covid-19 Syndrome: a Randomized, Double-blind, Placebo-controlled Study (DELong#3)
Phase: NA
Conditions: Long COVID
Sponsor: Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico

 
 
 
 
 
Internal Medicine Department 
Gastroenterology and Endoscopy Unit   
 
 
 
 Head: Prof. Maurizio Vecchi 
Tel. 02 5503 3418 
mail: maurizio.vecchi@policlinico.mi.it 
 
 
 
The role of VSL#3® in the treatment of fatigue and 
other symptoms in Long COVID 19 syndrome: a 
randomized, double-blind, placebo-controlled study 
 
Acronym: DELong#3/2022 
Protocol version number: v.1.0 
Date: 14/05/2022 
 
Sponsor: Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico 
Address: Via Sforza 28, 20122 Milan, Italy 
 
Coordinating Center: Gastroenterology and Endoscopy Unit 
Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico 
Address: Via Francesco Sforza 35, 20122 Milan, Italy 
 
Principal Investigator: Prof. Flavio Andrea Caprioli 
 
 
 
 
 
 
        
 
 
<Firma>______________________________ 
 
 
PRIVACY STATEMENT All information contained in this document will be considered 
confidential and shall remain the exclusive property of Fondazione IRCCS Ca' Granda Ospedale 
Maggiore Policlinico. The use of such confidential information should be limited to the intended 
recipient for the agreed-upon purpose and shall not be disclosed, published, or otherwise 
communicated to unauthorized individuals, for any reason, in any form without the prior written 
consent of Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico. 
 

 
 
 
 
 
 
Dipartimento di Medicina Interna 
UOC Gastroenterologia ed Endoscopia  
 
 
 
 Direttore: Prof. Maurizio Vecchi 
Tel. 02 5503 3418 
mail: maurizio.vecchi@policlinico.mi.it 
                              
FLOWCHART 
 
 
 
 
Clinical Trial 
 
Screening 
Randomization 
Procedure 
Follow-up 
Period 
Visit 1  
(-2t) 
Visit 2 
(t0) 
Visit 3 
(t4) 
Visit 4 
(t8) 
Enrollment 
  
  
 
 
Informed consent 
 X 
 
   
 
Inclusion/exclusione criteria 
 X 
 
   
 
Procedures 
  
  
 
 
 VSL#3® 
  
X 
X 
 
Placebo 
  
X 
X 
 
Assessments 
  
  
 
 
[stool and blood sample] 
 X 
X 
  
 
[primary endpoint] 
  
X 
X 
X 
[secondary endopoint] 
  
X 
X 
X 
 
 
 
 
 
 
 
t=week 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
 
 
 
Dipartimento di Medicina Interna 
UOC Gastroenterologia ed Endoscopia  
 
 
 
 Direttore: Prof. Maurizio Vecchi 
Tel. 02 5503 3418 
mail: maurizio.vecchi@policlinico.mi.it 
                              
ABBREVIATION LISTS  
CCL: Chemokine Ligand  
EC: Ethical Committee  
IC: Informed Consent  
ChFS: Chalder Fatigue Scale  
CFS/ME: Chronic Fatigue Syndrome / Myalgic Encephalomyelitis  
CRF: Case Report Form  
CSF: Colony Stimulating Factor  
CXCL: C-X-C Motif Chemokine Ligand FGF:  
Fibroblast Growth Factor  
FLTL3: FMS-like Tyrosine Kinase 3 
Ligand FM: Fibromyalgia  
GCP: Good Clinical Practice  
GRZ: Granzyme  
IBS: Irritable Bowel Syndrome  
IEO: Institute Europeo of Oncology 
IFN: Interferon  
IL: Interleukin  
MERS: Middle-East Respiratory Syndrome  
PDGF: Platelet-Derived Growth Factor  
SARS: Severe Acute Respiratory Syndrome  
TGFA: Transforming Growth Factor Alpha  
TNF: Tumor Necrosis Factor  
TRAIL: Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand 
VEGF: Vascular Endothelial Growth Factor 
 

 
 
 
 
 
 
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UOC Gastroenterologia ed Endoscopia  
 
 
 
 Direttore: Prof. Maurizio Vecchi 
Tel. 02 5503 3418 
mail: maurizio.vecchi@policlinico.mi.it 
                              
1.RESPONSABILITIES (role of the sponsor and collaborators)  
The sponsor of the study is Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico. The 
coordinating center will be the UOC of Gastroenterology and Endoscopy at Fondazione IRCCS Ca' 
Granda Ospedale Maggiore Policlinico.  
The role of the Principal Investigator (PI) will be fulfilled by Prof. Flavio Andrea Caprioli, UOC of 
Gastroenterology and Endoscopy - Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico. 
The Principal Investigator will be responsible for patient recruitment, evaluation of inclusion and 
exclusion criteria, data acquisition, and interpretation of the data.  
Dr. Guido Basilisco - study concept and design  
Dr. Dario Consonni - statistical analyses 
 
 
Internal collaborations  
Operating Unit 
Participant Name 
Role and Functions in the Study 
Gastroenterology and 
Endoscopy Unit 
Dott. Guido Basilisco 
Conceptualization 
Occupational Medicine Unit 
Dott. Dario Consonni 
Statistical Analysis 
Infectious Diseases Unit 
Prof. Andrea Gori 
Identification of eligible patients 
to the PI 
Pneumology Unit 
Prof. Blasi 
Identification of eligible patients 
to the PI 
 
 
 
 
 

 
 
 
 
 
 
Dipartimento di Medicina Interna 
UOC Gastroenterologia ed Endoscopia  
 
 
 
 Direttore: Prof. Maurizio Vecchi 
Tel. 02 5503 3418 
mail: maurizio.vecchi@policlinico.mi.it 
                              
External collaborations (biological sample analysis, data analysis, diagnostic procedures, etc.) 
Istitution 
Operating Unit 
Paticipants name 
Role and Functions in 
the study 
Istituto Europeo di 
Oncologia 
Mucosal Immunology 
Laboratory 
Dott.ssa Federica 
Facciotti 
Immunological and 
microbiological 
analyses 
 
 
 
 
 
 
 
 
 

 
 
 
 
 
Internal Medicine Department 
Gastroenterology and Endoscopy Unit   
 
 
 
 Head: Prof. Maurizio Vecchi 
Tel. 02 5503 3418 
mail: maurizio.vecchi@policlinico.mi.it 
 
 
INDEX  
1. INTRODUCTION 
1.1 Background  
2. STUDY AIMS 
2.1 Primary objectives 
2.2 Secondary objectives 
3. STUDY DESIGN 
3.1 Study Design 
3.2 Inclusion Criteria 
3.3 Exclusion Criteria 
4. PROCEDURES  
4.1 Procedures 
4.2 Randomization 
4.3 Masking 
5. ENDPOINT 
5.1 Primary Endpoint 
5.2 Secondary Endpoint 
6. STUDY DURATION/TIMELINE 
7. STATISTICAL ANALYSIS 
7.1 Sample size 
7.2 Statistical Analysis 
8. AVERSE EVENTS 
9. RISK BALANCE EVALUTATION 
10. STUDY MANAGEMENT GESTIONE DELLO STUDIO  
10.1 Data Collection and Management 
10.2 Regulatory and Ethical Considerations 

 
 
 
 
 
 
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     10.2.1 Approval by Competent Authority 
     10.2.2 Approval by Ethics Committee 
     10.2.3 Informed Consent 
10.3 Duties of the Investigator 
10.4 Study Monitoring 
10.5 Assurance of Study Quality  
10.6 Study Closure 
10.7 Document Archiving 
10.8 Dissemination of Scientific Findings 
      10.8.1 Confidentiality 
      10.8.2 Publications 
10.9 Intellectual Property Rights on Study Results 
11. Compensation and Damages in Case of Harm 
12 Protocol Amendments 
13 Financial Agreements  
14 Disclosure of Conflicts of Interest  
15 References 
16 Appendix 

 
 
 
 
 
 
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1. INTRODUZIONE 
1.1 Background 
Viral, bacterial, and protozoal infections are associated with a diverse clinical spectrum of 
symptoms, 
including 
fatigue, 
difficulty 
concentrating, 
depression, 
sleep 
disturbances, 
gastrointestinal disorders, and musculoskeletal pain [1-3]. In previous coronavirus epidemics such 
as Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS), 
studies have linked the infection to the possible development of Chronic Fatigue Syndrome/Myalgic 
Encephalomyelitis (CFS/ME) [4-6]. In particular, a study by Lam et al. conducted on 233 SARS 
survivors revealed that after 4 years, 27.1% of patients met the criteria for a CFS/ME diagnosis [7]. 
In the field of gastroenterology, several studies have demonstrated that previous infections of the 
gastrointestinal tract can lead to Irritable Bowel Syndrome (IBS) and CFS/ME. A study conducted 
on a Norwegian population of 576 individuals followed for 10 years after a Giardia Lamblia 
epidemic showed that the relative risk of developing IBS was 4.74 compared to an uninfected 
control group, while the relative risk of CFS/ME was 3.01 [8]. Another significant German study 
involving a large number of patients (508,278) found an increased relative risk of IBS and CFS/ME 
(1.73 and 2.08, respectively) in patients diagnosed with infectious gastroenteritis [9]. Several 
mechanisms may be involved in the pathogenesis of CFS and IBS associated with previous 
infection. Among these, it is possible that viral, bacterial, or protozoal infections lead to a 
modification of the intestinal microbiota in qualitative and quantitative terms. This alteration could 
result in a pro-inflammatory phenotype, leading to the release of bacterial toxins and inflammatory 
cytokines into the systemic circulation through areas of altered intestinal permeability (leaky gut) 
[10-17]. The same alteration of intestinal permeability and the microbiota may also be involved in 
various neurological and psychiatric disorders reflecting an alteration of the so-called "brain-gut 
axis" [11,18-20]. Additionally, cognitive impairment may play a role in the perception of fatigue. 
Persistent cognitive impairments have been documented following SARS-CoV-2 infection [21-23], 

 
 
 
 
 
 
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Tel. 02 5503 3418 
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and a recent Italian study on a group of 38 hospitalized COVID-19 patients demonstrated deficits in 
processing speed and verbal memory [23]. Several studies have attempted to intervene in the 
dysbiotic intestinal pathogenic mechanism of CFS/ME. A systematic review with meta-analysis 
from 2018 analyzed the use of probiotics in the context of CFS/ME and IBS in 25 studies. The 
possible therapeutic role of probiotics is recognized, but the low quality of the trials, overall lack of 
evidence, and the different composition of these products do not allow for the affirmation of their 
actual effectiveness [24]. Similar conclusions are reported in recent American guidelines on IBS, 
which confirm the role of infections and the microbiota in the pathogenesis of this disease but also 
lament the poor quality of evidence (mostly based on monocentric studies with small sample sizes) 
to recommend their systematic use [25]. The same guidelines cite a meta-analysis by Ford et al. 
from 2014, which summarizes the results of 37 randomized clinical trials in IBS and concludes that 
probiotics have been associated with improvements in overall symptomatology, abdominal pain, 
bloating sensation, and flatulence [26]. However, the authors themselves declare that the evidence 
of probiotic effects is influenced by the different composition of commercially available products 
and the poor quality of the conducted studies [26]. 
During the current COVID-19 epidemic, a series of scientific studies have demonstrated an 
alteration in the composition of the intestinal microbiota during SARS-CoV-2 infection [27-29]. 
This alteration, compared to the gut flora of healthy controls, appears to persist beyond the 
hospitalization period, be independent of the administered therapy, be associated with the presence 
of inflammatory taxa, and be more extensive and radical than that induced by H1N1 influenza 
infection [27-29]. 
Furthermore, starting from the first wave of COVID-19, a new post-infectious syndrome 
called "Long-Covid19 Syndrome" has been described in several countries, characterized by the 
persistence of clinical symptoms after recovery from COVID-19 according to WHO criteria [30,31]. 
Carfì et al. analyzed 143 hospitalized patients who were discharged after an average of 60 days 
from the onset of the disease and found that up to 87.4% of patients had at least one persistent 

 
 
 
 
 
 
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 Direttore: Prof. Maurizio Vecchi 
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symptom, with fatigue being the main one (53.1%) [32]. In a study conducted using an online 
platform, Goërtz et al. demonstrated that in a cohort of 2113 patients (including patients with a 
confirmed or suspected diagnosis of COVID-19), the number of symptoms experienced 79 days 
from the onset of the illness had significantly reduced, but at the same time, only 0.7% of patients 
were symptom-free. Once again, fatigue was the most reported symptom (87%) [33]. A study by 
Townsend et al. from Ireland, involving 128 patients, approximately half of whom were 
hospitalized for COVID-19, evaluated after a median time of 72 days, showed that only 42% of 
these patients had a health status comparable to that before the infection. 52% of the subjects met 
the diagnostic criteria for fatigue, with Chalder Fatigue Scale (ChFS) scores similar to those of 
patients with CFS/ME. Surprisingly, the state of fatigue does not seem to be correlated with the 
clinical severity of SARS-CoV-2 infection or the presence of inflammatory changes (e.g., white 
blood cell count, neutrophil count, neutrophil-to-lymphocyte ratio, and levels of PCR, LDH, IL-6), 
suggesting a more subtle etiopathogenic mechanism in line with other observations in CFS/ME [34]. 
Another Norwegian study observed the prevalence of fatigue in a cohort of non-hospitalized SARS-
CoV-2 positive patients, using the ChFS via postal survey conducted at a distance from the 
diagnosis of COVID-19. The 458 enrolled patients responded after a median time of 117 days from 
SARS-CoV-2 infection, and 46% showed a ChFS score (in dichotomous mode) compatible with 
clinically significant fatigue, while the Likert Scale score of the same scale differed significantly 
from that obtained from a cohort of healthy subjects [35]. Finally, in our own case series of 164 
patients studied five months after SARS-CoV-2 infection, compared to a control group of 183 
subjects, the relative risk of CFS was increased by 2.24. 
VSL#3® is a patented high-concentration blend (450 billion bacterial units) of live and 
lyophilized lactic acid bacteria and bifidobacteria, belonging to eight different strains (one strain of 
Streptococcus thermophilus BT01; three strains of Bifidobacteria: B. breve BB02, B. animalis 
subsp. lactis BL03 (formerly known as B. longum BL03), and B. animalis subsp. lactis BI04 
(formerly known as B. infantis BI04); four strains of Lactobacilli: L. acidophilus BA05, L. 

 
 
 
 
 
 
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plantarum BP06, L. paracasei BP07, and L. helveticus BD08 (formerly identified as L. delbrueckii 
subsp. bulgaricus BD08)). 
Animal studies support the biological plausibility that modulation of the microbiome can 
affect perception and cognitive-behavioral mechanisms [36]. In an exploratory study in patients 
with IBS, Bifidobacterium Longum reduced depression scores and improved the patients' quality of 
life. These improvements were associated with changes in brain activation patterns indicating that 
the probiotic reduces limbic reactivity [37]. Recently, a comparative study of VSL#3 versus a 
LOW-FODMAP diet was published, showing that both treatments had fundamentally the same 
efficacy [38]. 
 
2. STUDY OBJECTIVE/HYPOTHESIS  
2.1 Primary Objectives 
The primary objective of this study is to test whether a high-concentration probiotic formulation 
(e.g., VSL#3®) can reduce the degree of clinically significant fatigue in ambulatory patients 
suffering from Long-Covid-19 Syndrome, i.e., the persistence of systemic symptoms 4 weeks after 
molecular detection of SARS-CoV-2 
 
2.2 Secondary Objective(s) 
1.To determine if this high-concentration probiotic formulation can improve scores of anxiety, 
depression, quality of life, gastrointestinal symptoms, cognitive deficits, performance, 
psychological distress, and clinical evaluation of the study patients. 
2.To assess the immunological profile of inflammation markers in blood and bacterial species in 
feces in patients treated with VSL#3® compared to patients treated with a placebo. Any differences 
before and after treatment will be identified. 
 
3.STUDY DESIGN  
3.1 Study Design 

 
 
 
 
 
 
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Our study is a double-blind, randomized, placebo-controlled trial. Anticipating a higher 
representation of female subjects in our sample, we hypothesized a stratified block randomization 
by sex. This is a single-center study conducted in an outpatient setting, and currently hospitalized 
patients will not be recruited. 
3.2 Inclusion Criteria 
Patients who meet all of the following criteria will be included: 
• 
Ambulatory patients who have signed informed consent. 
• 
Age above 18 years and below 65 years. 
• 
Previous diagnosis of SARS-CoV-2 infection, documented by molecular nasopharyngeal or 
antigen swab. 
• 
Not currently undergoing isolation or quarantine. 
• 
Not having used antibiotic products within the 30 days preceding the trial. 
• 
Chalder Fatigue Scale (in dichotomous form) >=4, possibly associated with signs and 
symptoms of Long-Covid-19 Syndrome [31,32]: signs and symptoms that develop during or 
after SARS-CoV-2 infection, persist for more than 4 weeks, and are not reasonably 
explained otherwise; signs and symptoms include fatigue, sleep disturbances, cognitive 
deficits (i.e., brain fogging, loss of concentration and memory, anxiety, depression), muscle 
weakness, joint pain, gastrointestinal alterations (reduced appetite, nausea, changes in bowel 
habits such as constipation and/or diarrhea, abdominal pain) 
3.3 Exclusion Criteria  
Patients with at least one of the following criteria will be excluded: 
• 
Moderate to severe cardiovascular and pulmonary diseases with organ dysfunction 
(NYHA>2, Borg scale >=2). 
• 
Decompensated endocrine and metabolic diseases (Child-Pugh cirrhosis >= B, 
decompensated hypo/hyperthyroidism, decompensated adrenal insufficiency). 
• 
Pre-existing diagnosis of FM, CFS/ME, and/or IBS prior to SARS-CoV-2 infection. 

 
 
 
 
 
 
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• 
Confirmed diagnoses of neurological disorders, psychiatric illnesses, and cognitive 
impairments prior to SARS-CoV-2 infection. 
• 
Previously confirmed diagnoses of chronic musculoskeletal disorders prior to SARS-
CoV-2 infection. 
• 
Refusal to participate in the study / refusal to allow the processing of personal data. 
• 
Pregnancy or breastfeeding. 
• 
Alcohol or drug dependency in the previous years. 
• 
Use of other probiotics during the trial. 
• 
Use of antibiotics during the trial and in the 30 days preceding it. 
• 
Substantial change in diet during the trial. 
• 
Participation in another clinical study in the 30 days preceding or previous participation 
in the same trial. 
• 
Known intolerance/hypersensitivity to the investigational product or the excipients of the 
placebo formulation. 
 
3.4 Study Withdrawal Criteria 
• 
Onset of hypersensitivity to the study product or the placebo 
• 
Clinical necessity for the use of antibiotics, immunosuppressants or immunomodulators, 
glucocorticoids, opioids, antidepressants, or anxiolytics at regular and continuous 
dosages. 
 
4. PROCEDURES RELATED TO THE STUDY 
4.1 Intervention Patients  
Patients will be referred by the Pneumology and Infectious Diseases outpatient clinics for a 
preliminary evaluation of inclusion and exclusion criteria, within the context of scheduled visits for 
sequelae of SARS-CoV-2 infection. The recruitment phase is divided into an initial screening visit 
(Visit 1, t-2) to determine if the patient meets the inclusion/exclusion criteria. Following this visit, a 

 
 
 
 
 
 
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14-day run-in period will be conducted to screen for non-adherent patients, i.e., those who do not 
show up for the subsequent visit. The patient will return for a visit at 14 days (Visit 2, t0) when 
randomization will occur, and the patient will be assigned to the treatment or placebo group. The 
patient will be instructed to take the treatment (supplement or placebo) at the prescribed dosage of 
two sachets per day for a consecutive and continuous period of four weeks. During this visit, all 
immunological and microbiological analyses will be performed, including the collection of 20 mL 
of blood and 1 g of fecal material. The patient will also complete clinical questionnaires to assess 
the primary and secondary endpoints. The clinical questionnaires will be stored in a deidentified 
form at the Gastroenterology and Endoscopy Unit, while the biological samples will be shipped and 
analyzed at the Mucosal Immunology Laboratory of the IEO. Considering the short duration of the 
study and the lack of literature data demonstrating a clear benefit of VSL#3 on this syndrome, no 
crossover between study arms is planned under any circumstances. During the study, the use of 
immunosuppressants or immunomodulators, glucocorticoids, opioids, antidepressants, or 
anxiolytics at regular and continuous dosages is not allowed. If there is a clinical necessity for 
antibiotic therapy, it will result in discontinuation of the study. After completing the 28-day 
treatment period, the patient will return for a visit (Visit 3, t4) where immunological and 
microbiological analyses will be repeated, and questionnaires will be completed again to evaluate 
the primary and secondary endpoints. An additional visit with completion of clinical questionnaires 
will be scheduled 28 days after the end of the treatment (Visit 4, t8) to assess whether any response 
is maintained at the end of the treatment period. 
4.2 Randomization  
Randomization will be managed using the REDCap (Research Electronic Data Capture) software. 
Considering the relatively small number of anticipated patients (n = 96, see dedicated section), we 
have planned a permuted block randomization with a 1:1 ratio between the placebo and treatment 
groups. Given the higher expression of the Long-Covid-19 Syndrome in females, randomization 
will be stratified by sex. We do not anticipate any socio-health or ethical factors that could interfere 

 
 
 
 
 
 
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with the randomization process to bias patients into one arm over another. 
4.3 Blinding  
The study participants, healthcare professionals delivering the intervention, and outcome assessors 
will be blinded to the treatment being administered. Actial Farmaceutica will provide drug and 
placebo packages that are indistinguishable from each other and will disclose the nature of the two 
treatments at the end of the study analyses. In the event of serious adverse effects that require 
hospitalization and the patient's admission, an external collaborator not involved in the study will be 
informed of the treatment nature through direct contact with Actial Farmaceutica to intervene if 
necessary. 
 
5. ENDPOINTS  
 
5.1 Primary Endpoint  
Demonstration of a statistically significant difference between the placebo group and the treatment 
group in the scores of the Chalder Fatigue Scale at the end of the treatment period (t4). The Chalder 
Fatigue Scale is a validated scale used to assess fatigue in the context of CFS/ME [39-41]. 
 
5.2 Secondary Endpoints  
Demonstration of a statistically significant difference between the placebo group and the treatment 
group after 4 weeks of treatment (t4) and after an additional 4-week follow-up period (t8; 1 month 
after the end of treatment), unless otherwise specified, for the following variables: 
• 
Scores of the Chalder Fatigue Scale (t8) 
• 
Hospital Anxiety and Depression Scale (HADS). HADS is a validated scale that quantifies 
anxiety and depression experienced by the patient [42,43]. 
• 
Short Form Health Survey (SF-36). SF-36 is a validated scale that assesses health-related 
quality of life [44]. 
• 
Structured Assessment of Gastrointestinal Symptoms Scale (SAGIS). SAGIS is a validated 
scale that measures the intensity and impact of gastrointestinal symptoms [45]. 

 
 
 
 
 
 
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• 
Somatic symptoms of the Symptom Checklist-90 (SCL-90) assessed by the SCL 12. SCL-90 
is a validated questionnaire that assesses psychological distress in individuals [46-52,54]. 
• 
Karnofsky Performance Status (KPS) Scale. KPS is a validated scale consisting of 11 points 
that describes the overall functional status of a patient on a range from 100% (no evidence 
of disease, no symptoms) to 0% (death) [49,50,51,52]. 
• 
Visual Analog Scale (VAS) rating of the patient's overall health status by the examining 
physician. 
• 
Serum expression of cytokines (IL-2, IL-7, IL-15, IL-10, IL-33, IL-4, IL-5, IL-13, IL-1a, IL-
1b, IL-1Ra, IL-12p70, IFNa, IFNb, IFNg, TNFa, TRAIL, GRZb, IL-6, IL-17A, IL-17F, IL-
22, and IL-23), chemokines (CXCL1 (GROa), CXCL2 (GROb), CXCL8 (IL-8), CXCL10 
(IP-10), CX3CL1 (Fraktalkine), CCL2 (MCP-1), CCL3 (MIP1a), CCL4 (MIP-1b), CCL5 
(Rantes), CCL11 (Eotaxin), CCL19 (MIP3b), and CCL20 (MIP3a)), growth factors (FLT3L, 
IL-3, G-CSF, GM-CSF, TGFa, EGF, PDGFaa, PDGFab/bb, VEGF, and FGF basic), and 
immune activation markers (CD40L and PDL1). 
• 
Variation in the bacterial component of the fecal microbiota in terms of alpha and beta 
diversity and correlation of these parameters with clinical response on fatigue. 
 
6. STUDY DURATION / TIMELINE  
The study will have a total duration of 70 days per enrolled patient. Please refer to the attached 
Gantt chart. 
Study Start: 09/2022 Recruitment Closure: 12/2022 Study End: 07/2023 
 
7.STATISTICAL ANALYSIS 
7.1 Sample Size  
Assuming a placebo effect of 30% reduction in the primary outcome (fatigue assessed by Chalder 
Fatigue Scale - Likert 0-33), a statistical power of 80% with a significance level of 0.05 (two-tailed), 

 
 
 
 
 
 
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we estimate that 42 patients per arm are needed to detect a 60% efficacy of the probiotic in reducing 
the same parameter. This reduction, if confirmed, would have significant clinical relevance, 
indicating a substantial efficacy compared to placebo. Considering a dropout rate of 15%, we 
believe that recruiting 48 actual patients per arm is necessary to demonstrate this efficacy. 
7.2 Data Analysis  
Both intention-to-treat and per-protocol analyses will be conducted. Depending on the distribution 
of the data, Student's t-test or Wilcoxon Mann-Whitney test will be used for continuous variables, 
and Chi-square test or Fisher's exact test for categorical variables. Relationships between multiple 
variables will be assessed using linear or logistic regression tests based on the nature of the 
available data. Statistical analysis will be performed using the RStudio software (R Core Team, 
2020) and Stata software (StataCorp, 2013). No interim analyses are planned due to the short 
duration of the study and the relatively small number of enrolled patients. 
 
8.ADVERSE EVENTS  
The project does not involve the administration of drugs or other substances or invasive clinical 
procedures. Therefore, no significant adverse events are expected, except those related to venous 
blood collection. Serious adverse effects associated with the ingestion of the studied probiotic are 
not reported in the literature, except for a temporary increase in the sensation of abdominal bloating 
following the initial doses of the probiotic. 
 
9. RISK / BENEFIT ASSESSMENT  
Currently, there is no established cure for Long-Covid-19 Syndrome. The use of a probiotic 
formulation offers the advantage of an excellent safety profile compared to pharmacological 
formulations. However, since the pathogenic mechanism of this syndrome is unknown, the benefit 
of the probiotic formulation cannot be established a priori. A follow-up of the patients will be 
conducted one month after the end of the study, primarily to assess the persistence of any potential 

 
 
 
 
 
 
Dipartimento di Medicina Interna 
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 Direttore: Prof. Maurizio Vecchi 
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mail: maurizio.vecchi@policlinico.mi.it 
                              
benefits rather than the occurrence of adverse events. 
 
10. STUDY MANAGEMENT  
10.1 Data Collection and Management  
The study data will be collected using an electronic Case Report Form (CRF) managed through 
REDCap (Research Electronic Data Capture) software. A supplementary module will be available 
for the investigator to enter patient demographic data, SARS-CoV-2 infection information, and the 
results of the study questionnaires. The questionnaires to be used in the study, along with their 
respective references, are listed in the primary and secondary outcome sections. Immunological and 
microbiological investigation data will be uploaded in electronic format. Patient information will be 
entered in a pseudonymized manner into a database managed exclusively by the investigator or 
designated collaborators, with each patient identified by a unique code. The file linking the 
participant code with their identifying data will be kept separately on a password-protected 
computer. The study database will be password-protected and stored on a computer accessible only 
to authorized study personnel designated by the principal investigator. Deidentification of data will 
be done in a way that those accessing the database cannot link the data to the participants' identities. 
Only local investigators will have access to the identities of enrolled participants. 
10.2 Regulatory and Ethical Considerations  
10.2.1 Approval from Competent Authority  
In accordance with applicable regulations, the principal investigator must obtain approval 
from the appropriate Competent Authority before commencing the clinical study. This study will be 
conducted in compliance with the rules of ICH/GCP (International Conference of 
Harmonization/Good Clinical Practice) and all applicable laws, including the Declaration of 
Helsinki of June 1964, modified by the latest World Medical Association General Assembly in 
Seoul, 2008. 
10.2.2 Ethical Committee Approval  

 
 
 
 
 
 
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 Direttore: Prof. Maurizio Vecchi 
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mail: maurizio.vecchi@policlinico.mi.it 
                              
The investigator must ensure that the protocol has been reviewed and approved by the local 
independent Ethics Committee (EC) before commencing the study. The EC must also review and 
approve the Informed Consent (IC) form and all written information received by the patient prior to 
enrollment in the study. If it becomes necessary to modify the protocol and/or IC during the study, 
the investigator will be responsible for ensuring the review and approval of the modified document 
by the EC. The content of such modifications will be implemented only after approval by the EC. 
Until then, the previously approved version of the document must be referred to. 
10.2.3 Informed Consent (IC)  
The investigator or authorized personnel must inform individuals about all aspects and 
procedures of the study. The investigator or authorized personnel will obtain informed consent or 
assent from potential trial participants or their legal representatives. The process of obtaining 
informed consent must comply with current regulatory procedures. The investigator (or a 
designated collaborator) and the subject must date and sign the IC form before the patient 
undergoes any study-related procedures. The subject will receive a dated and signed copy of the IC 
form from both parties; the original copy will be retained in designated study archives. Neither the 
investigator nor the designated personnel should in any way coerce or unduly influence a subject to 
participate or continue participating in the study. The subject's decision to participate in the study 
must be completely voluntary. The investigator and designated personnel must emphasize to the 
subject that they may withdraw their consent at any time without penalty or loss of any entitled 
benefits. Written or oral information regarding the study, including the written IC form, must not 
contain any language that would require the subject to waive (even apparently) their legal rights or 
that would release the investigator, institution, or sponsor from liability for negligence. 
10.3 Investigator's Responsibilities  
In accordance with applicable local regulations, the investigator must provide periodic reports 
regarding the progress of the study at their center to the EC and notify the EC of the study's closure. 
Periodic reports and closure notification are part of the investigator's responsibilities. 

 
 
 
 
 
 
Dipartimento di Medicina Interna 
UOC Gastroenterologia ed Endoscopia  
 
 
 
 Direttore: Prof. Maurizio Vecchi 
Tel. 02 5503 3418 
mail: maurizio.vecchi@policlinico.mi.it 
                              
10.4 Study Monitoring  
In accordance with applicable regulations and Good Clinical Practice (GCP), the monitor must 
periodically visit or contact the study site. The duration, nature, and frequency of these 
visits/contacts depend on the recruitment rate, the quality of the documents held by the site, and 
their adherence to the protocol. 
Through these contacts, the monitor must: 
• 
Monitor and evaluate the progress of the study 
• 
Review the collected data 
• 
Perform source data verification 
• 
Identify any issues and propose solutions 
The purposes of the monitoring activities are to ensure that: 
• 
The rights and well-being of the subjects are protected 
• 
The study data is accurate, complete, and verifiable from the original documents 
• 
The study is conducted in compliance with the protocol, approved amendments, GCP, and 
applicable regulations 
The investigator must: 
• 
Provide the monitor with direct access to all relevant documentation 
• 
Dedicate time and personnel to meet with the monitor to discuss the monitoring results and 
address any other relevant aspects. 
The monitor should also contact the site prior to the study initiation to discuss the protocol and data 
collection procedures with the staff. 
10.5 Study Quality Assurance  
As the sponsor, Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico may, at its 
discretion, perform quality control of the study. In this case, the investigator must allow the monitor 
direct access to all relevant documentation and dedicate time and personnel to discuss the 
monitoring results and any other aspects of the study. Additionally, Regulatory Authorities may 

 
 
 
 
 
 
Dipartimento di Medicina Interna 
UOC Gastroenterologia ed Endoscopia  
 
 
 
 Direttore: Prof. Maurizio Vecchi 
Tel. 02 5503 3418 
mail: maurizio.vecchi@policlinico.mi.it 
                              
conduct inspections. In such cases, the investigator must authorize the inspector's direct access to all 
relevant documentation and dedicate time and personnel to discuss the monitoring results and any 
other aspects of the study. 
10.6 Study Closure  
At the time of study closure, the monitor and investigator must initiate a series of procedures, 
including: 
• 
Reviewing all study documentation 
• 
Reconciling study data 
• 
Resolving any outstanding queries 
10.7 Document Archiving  
In accordance with applicable national regulations, the investigator must retain a copy of all 
documentation in a dry and secure location after study closure. 
10.8 Disclosure of Scientific Findings 10.8.1 Confidentiality The investigator and other personnel 
involved in the study must treat all study-related information (including the protocol, obtained data, 
and all study-related documentation) confidentially and must not use such information, data, or 
findings for purposes other than those described in the protocol. These restrictions do not apply to: 
1. Information that becomes publicly available without negligence on the part of the 
investigator or their personnel 
2. Information that requires confidential disclosure to the EC solely for the purpose of 
evaluating the study 
3. Information that needs to be disclosed to obtain appropriate medical care for a study subject. 
10.8.2 Publications  
Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico is the sole owner of the data. 
10.9 Intellectual Property Rights on Study Results 
If, during the project, know-how, technical materials, and/or goods protected by industrial and/or 
intellectual property rights or susceptible to protection are used and shared to the extent strictly 

 
 
 
 
 
 
Dipartimento di Medicina Interna 
UOC Gastroenterologia ed Endoscopia  
 
 
 
 Direttore: Prof. Maurizio Vecchi 
Tel. 02 5503 3418 
mail: maurizio.vecchi@policlinico.mi.it 
                              
necessary for the conduct of the project, developed by the Promoter or the collaborating center 
("Background"), it is understood that such background will remain the property of the party that 
developed it, and the use or sharing of it by the owning party will not result in the acquisition of any 
rights by the other party. The results and data generated during the study will remain the exclusive 
property of the Promoter, who agrees to grant the Collaborating Center the right to use them solely 
for internal research activities after publication by the Promoter. 
 
11.COMPENSATION AND DAMAGES IN CASE OF HARM  
In the event of adverse events or potential damages resulting from participation in the research, the 
Insurance Policy of our Institute extends coverage to the participating subjects in research projects. 
 
12. PROTOCOL AMENDMENTS  
Not applicable. 
 
13. FINANCIAL AGREEMENTS  
This study is funded by Actial Farmaceutica Srl - Viale Shakespeare 47 00144 Rome (Italy), as 
specified in the attached funding agreement. 
 
14. DISCLOSURE OF CONFLICTS OF INTEREST 
The authors of this protocol declare no conflicts of interest. 
 
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Dipartimento di Medicina Interna 
UOC Gastroenterologia ed Endoscopia  
 
 
 
 Direttore: Prof. Maurizio Vecchi 
Tel. 02 5503 3418 
mail: maurizio.vecchi@policlinico.mi.it 
                              
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Dipartimento di Medicina Interna 
UOC Gastroenterologia ed Endoscopia  
 
 
 
 Direttore: Prof. Maurizio Vecchi 
Tel. 02 5503 3418 
mail: maurizio.vecchi@policlinico.mi.it 
                              
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Dipartimento di Medicina Interna 
UOC Gastroenterologia ed Endoscopia  
 
 
 
 Direttore: Prof. Maurizio Vecchi 
Tel. 02 5503 3418 
mail: maurizio.vecchi@policlinico.mi.it 
                              
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Dipartimento di Medicina Interna 
UOC Gastroenterologia ed Endoscopia  
 
 
 
 Direttore: Prof. Maurizio Vecchi 
Tel. 02 5503 3418 
mail: maurizio.vecchi@policlinico.mi.it 
                              
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Dipartimento di Medicina Interna 
UOC Gastroenterologia ed Endoscopia  
 
 
 
 Direttore: Prof. Maurizio Vecchi 
Tel. 02 5503 3418 
mail: maurizio.vecchi@policlinico.mi.it 
                              
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UOC Gastroenterologia ed Endoscopia  
 
 
 
 Direttore: Prof. Maurizio Vecchi 
Tel. 02 5503 3418 
mail: maurizio.vecchi@policlinico.mi.it 
                              
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