Probiotics to Reduce Ventilator-Associated Pneumonia: Study Protocol for a Double-Blind Multicenter Randomized International Clinical Trial (PROACT)

Probiotics to Reduce Ventilator-Associated Pneumonia: Study Protocol for a Double-Blind Multicenter Randomized International Clinical Trial (PROACT) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Probiotics to Reduce Ventilator-Associated Pneumonia: Study Protocol for a Double-Blind Multicenter Randomized International Clinical Trial (PROACT) Alberto Corriero, Rossana Soloperto, Mariateresa Giglio, Michele Salvagno, and 16 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6813480/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Nov, 2025 Read the published version in Trials → Version 1 posted 5 You are reading this latest preprint version Abstract Background : Ventilator-associated pneumonia (VAP) remains a significant complication among critically ill patients, with associated mortality approaching 50%. Despite the implementation of established preventive strategies, additional interventions are urgently needed to further reduce the incidence of VAP. Probiotic therapy has emerged as a promising adjunctive approach; the benefits of probiotic therapy may be more pronounced in critically ill patients without pre-existing infections. The PROACT study will evaluate the prophylactic role of probiotics in a critically ill population with acute brain injury to reduce the VAP incidence, while also exploring microbiological endpoints and mortality to refine patient selection criteria. Methods : The PROACT study is a prospective, randomized, double-blind, placebo-controlled, multicenter trial designed to evaluate the efficacy of probiotic prophylaxis in adult critically ill patients requiring invasive mechanical ventilation following acute brain injury (e.g. head trauma, ischemic or hemorrhagic stroke). Patients with suspected pulmonary aspiration or pre-existing pulmonary infections at enrollment are excluded to reduce confounding. Participants are randomized in a 1:1 ratio to receive either a placebo (glucose polymer) or a probiotic formulation containing Lactobacillus acidophilus LA-5, Lactobacillus plantarum , Bifidobacterium lactis BB12, and Saccharomyces boulardii . The assigned intervention is administered twice daily via nasogastric tube and oropharyngeal application for up to 30 days or until Intensive Care Unit (ICU) discharge. The primary endpoint is the incidence of VAP, as defined by current international guidelines, in the intention-to-treat (ITT) population. Secondary endpoints include the incidence of VAP in the modified intention-to-treat (mITT) population, catheter-related bloodstream infections, occurrence of sepsis and septic shock, 30-day all-cause mortality, ICU length of stay and duration of mechanical ventilation. The study is powered at 80% (α=0.05) to detect a clinically meaningful difference based on effect size estimates based on the PROVAP study, requiring a total sample size of 186 patients. All participating centers implement standard VAP prevention bundles as part of routine care. Discussion : By evaluating the efficacy of probiotic therapy in preventing VAP and the impact on mortality among critically ill brain-injured patients, this trial has the potential to generate high-quality evidence supporting the incorporation of probiotics into standard VAP prevention protocols. The findings may have significant implications for clinical practice guidelines and public health policy related to infection control and microbiome-targeted interventions in the intensive care setting. Trial Registration Registered at ClinicalTrials.gov (identifier: [NCT06092554]). Registered on 2023-10-15. Ventilator-associated pneumonia probiotics randomized controlled trial mechanical ventilation gut-lung axis microbiota dysbiosis Figures Figure 1 Figure 2 Administrative information Title {1} PROACT - Probiotics in ICU to Reduce Ventilator-Associated Pneumonia: Study Protocol for a Double-Blind Multicentre Randomized International Clinical Trial Trial registration {2a and 2b}. ClinicalTrials.gov Registry ( NCT06092554). Registered on 2023-10-15. Protocol version {3} 28 th of February 2025 (3.0) Funding {4} This study is not funded. Author details {5a} ¹ Department of Interdisciplinary Medicine – ICU Section, University of Bari Aldo Moro, Piazza G. Cesare 11, 70124, Bari, Italy; [email protected] (AC); [email protected] (MTG); [email protected] (FP) 2 Department of Precision and Regenerative Medicine and Jonian Area – ICU Section, University of Bari Aldo Moro, Bari, Italy; [email protected] (VMR); [email protected] (SG); [email protected] (MR) 3 Department of Intensive Care, Hôpital Universitaire de Bruxelles (HUB), Université Libre de Bruxelles (ULB), Route de Lennik 808, 1070, Brussels, Belgium; [email protected] (FST); [email protected] (MS); [email protected] (RS) 4 Department of Interdisciplinary Medicine – Medical Statistics Section, University of Bari Aldo Moro, Piazza G. Cesare 11, 70124, Bari, Italy; [email protected] (PT) 5 Department of Interdisciplinary Medicine – Microbiology Section, University of Bari Aldo Moro, Piazza G. Cesare 11, 70124, Bari, Italy; [email protected] (AM) 6 UOC Anesthesia and Intensive Care, Ospedale "Di Venere", ASL Bari, Via Ospedale Di Venere 1, 70131 Bari, Italy; [email protected] (CP); [email protected] (NDT) 7 ICU Section, Ospedale Miulli, Acquaviva delle Fonti, Bari, Italy; [email protected] (GM) 8 Intensive Care Unit, Azienda Ospedaliera di Perugia, Piazzale G. Menghini 6/8, 06156 Perugia, Italy; [email protected] (AG); [email protected] (RA) 9 Department di Medicine and Surgery, Anaesthetics, Analgesia and Intensive Care Section, University of Perugia, P.le Lucio Severi, 1, 06132 Perugia, Italy 10 Department of Interdisciplinary Medicine, University of Bari Aldo Moro, Piazza G. Cesare 11, 70124, Bari, Italy; [email protected] (AMo); [email protected] (RMG); [email protected] (MF); [email protected] (MC) Name and contact information for the trial sponsor {5b} UNI-PHARMA S.A. Greece Tel: +30 210 8072512. Role of sponsor {5c} Generates center-specific randomization lists , prepares pre-randomized study product packages , and ensures identical appearance of placebo and probiotic . Introduction Background and Rationale {6a} Ventilator-associated pneumonia (VAP) remains a significant burden in the Intensive Care Unit (ICU), with reported mortality rates approaching 50% (1,2). To mitigate this risk, a VAP prevention care bundle, comprising a set of evidence-based, high-impact interventions, has been widely adopted. These strategies include (3–6): a) elevation of the head of the bed (to reduce the risk of tracheal micro-aspiration); b) daily sedation interruption and assessment of readiness to extubate (to shorten mechanical ventilation duration); c) subglottic secretion drainage; d) avoidance of scheduled ventilator circuit changes; e) peptic ulcer prophylaxis (to reduce stress-related mucosal damage and subsequent complications); and f) venous thromboembolism (VTE) prophylaxis (to prevent immobility-related complications). Despite growing interest, probiotic administration is not currently included among these standard recommendations. However, several studies have demonstrated promising results in reducing VAP incidence, particularly in high-risk populations, such as patients with traumatic brain injury. In a randomized controlled trial (7), the use of a four-strain probiotic preparation significantly reduced VAP incidence in patients with head trauma, from 28.3% in the placebo group to 11.9% in the intervention group. The same probiotic formulation was also evaluated in patients with multiple trauma, where it was associated with a reduction in surgical site infections, suggesting a broader prophylactic potential beyond respiratory infections (8). Additionally, a separate study assessing the use of a dual-strain probiotic preparation in patients with acute stroke demonstrated a reduction in VAP incidence in the probiotic group (9). Notably, patients with stroke and those with traumatic brain injury may share overlapping pathophysiological mechanisms and similar risk for VAP (10). Recent systematic reviews and meta-analyses (11,12) support the potential role of probiotics in reducing VAP incidence. Available data suggest that the prophylactic benefits of probiotics may be most pronounced in patients without active infections at baseline. However, the quality of evidence remains variable, and further high-quality randomized controlled trials with low risk of bias are warranted. The underlying mechanisms of probiotic efficacy in VAP prevention are not yet fully elucidated, but several hypotheses have been proposed. One involves modulation of the gut microbiota (GM), oral microbiota (OM), and lung microbiota via the gut–lung and gut–bone marrow–lung axes (13,14). Importantly, short-chain fatty acids (SCFAs), including butyrate, acetate, and propionate, produced by colonic bacterial fermentation of dietary fiber may exert anti-inflammatory effects and contribute to the maintenance of the respiratory epithelium's integrity (15). In critically ill patients, dysbiosis, defined as a disruption of the normal gut microbial balance, develops rapidly, often within six hours of ICU admission (16). This leads to a marked decrease in microbial diversity and the overgrowth of opportunistic, potentially pathogenic organisms (17,18). Interrupting this process by restoring eubiosis through early and targeted probiotic administration could help maintain the functionality of the gut-lung axis and reduce secondary infectious complications such as VAP. Nevertheless, a recent large-scale randomized controlled trial conducted in Canada failed to demonstrate a reduction in VAP incidence with probiotic therapy (19). However, a substantial proportion of patients in that study were already receiving antibiotics for non-specific infections at enrolment, and the protocol did not mandate microbiological confirmation for VAP diagnosis, potentially confounding the results. Objectives {7} This study aims to address key uncertainties that currently limit the incorporation of probiotic therapy into clinical guidelines and public health policies for VAP prevention in the intensive care setting. This randomized controlled trial will enroll critically ill patients with primary non-anoxic brain injury; patients with suspected pulmonary aspiration or pre-existing pulmonary infection at the time of enrolment will be excluded to minimize confounding. The primary objective is to evaluate whether a four-strain probiotic formulation ( Lactobacillus acidophilus LA-5, Lactobacillus plantarum , Bifidobacterium lactis BB12, and Saccharomyces boulardii ; Lactolevure®, Unipharma, Greece) can reduce the incidence of VAP and improve 30-day all-cause mortality. Trial design {8} The PROACT study is a randomized, double-blind, placebo-controlled, multicenter trial designed to evaluate the efficacy of probiotic therapy in preventing VAP in critically ill patients. The study design is illustrated in Figure 1. A total of 186 eligible ICU patients will be randomized in a 1:1 ratio to receive either a four-strain probiotic preparation (intervention group) or placebo (control group). The intervention (e.g. probiotics or placebo) will be administered twice daily for up to 30 days via nasogastric tube and oropharyngeal application. The study employs a double-blind methodology, ensuring that both participants and treating clinicians remain blinded to group allocation. The primary outcome analysis will be conducted on an intention-to-treat (ITT) basis. The trial is powered to detect a reduction in VAP incidence from 28.3% to 11.9%, based on effect size estimates observed in the PROVAP study (7). Methods: Participants, interventions and outcomes Study setting {9} This will be a prospective, randomized, double-blind, placebo-controlled clinical trial. The study protocol will be submitted for approval to the Ethics Committees (EC) and Institutional Review Boards (IRB) as per participating study site regulations. The study shall be governed by: the international standards for Good Clinical Practice (GCP) developed by the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use; the directive 2001/20/EC for Clinical trials; general Data Protection Regulation 679/2016 (EC). Eligibility Criteria {10} Patients eligible for enrolment are admitted to the hospital from the emergency department or transferred from another ICU. A legal representative will be asked for written informed consent once a patient is classified as having acute traumatic brain injury (TBI), ischemic stroke or hemorrhagic stroke (e.g. including intracerebral hemorrhage and subarachnoid hemorrhage). Therefore, eligibility screening can start, with exclusion criteria assessed first. If the patient does not meet any exclusion criteria, he will also be considered for inclusion criteria. If the patient meets all the inclusion criteria, he can be successfully enrolled in the study. Inclusion and exclusion criteria are shown in (Table 1). Table 1. Inclusion and exclusion criteria of the PROACT study. Inclusion Criteria Exclusion Criteria a) Adults aged 18–80 years b) Recent trauma (including TBI with ≥1 other organ injury) OR stroke/brain hemorrhage without aspiration/lung infection c) Intubation and mechanical ventilation initiated immediately after the event d) Expected mechanical ventilation ≥6 days (treating physician judgement) e) Written informed consent (patient or legal representative) a) Mechanical ventilation >72 hours before enrollment b) Pregnancy or lactation c) High infection risk (immunosuppression, HIV <200 CD4+, recent chemotherapy, neutropenia) d) Critical gastrointestinal conditions (severe pancreatitis, ischemic bowel) e) Inability to receive enteral medications f) Intent to withdraw advanced life support g) Endovascular infection risk * h) Sepsis and/or septic shock TBI: Traumatic Brain Injury; ED: Emergency Department; HIV: Human Immunodeficiency Virus; CD4+: Cluster of Differentiation 4 positive cells. * Includes: previously documented rheumatic heart disease, congenital valve disease, surgically repaired congenital heart disease, unrepaired cyanotic congenital heart disease, any intracardiac repair with prosthetic material, previous or current endocarditis, permanent endovascular devices such as inferior vena cava filters, dialysis vascular grafts, tunnelled (not short-term) hemodialysis catheters, pacemakers or defibrillators. Who will take informed consent? {26a} Before enrollment, a trained investigator or a designated research team member at each participating center will obtain written informed consent from patients or their legal representatives. Given the critical condition of eligible patients, consent will typically be sought from the patient’s legally authorized representative in accordance with local regulations and ethical guidelines. Additional consent provisions for collection and use of participant data and biological specimens {26b} The main informed consent document includes consent for collecting, storing and analyzing biological specimens (blood, urine, and stool samples). By signing the consent form, participants or their legal representatives agree to the use of these samples for microbiota analysis and related research on ventilator-associated pneumonia. All data and specimens will be anonymized and handled according to ethical and regulatory guidelines. Interventions Explanation for the choice of comparators {6b} A placebo was selected as the comparator to ensure blinding and to isolate the specific effects of probiotic administration on the incidence of VAP. The placebo consists of an inert glucose polymer powder, which has no known impact on gut microbiota composition or host immune function. This approach enables a rigorous assessment of probiotic efficacy while preserving methodological integrity within the double-blind, randomized study design. The choice of this placebo is consistent with prior clinical trials evaluating probiotics in critically ill populations. Intervention description {11a} Participants in both study arms will receive two capsules twice daily for up to 30 days: one capsule will be administered via nasogastric tube (NGT) and the other applied to the oropharyngeal mucosa. The NGT capsule will be dissolved in 100 mL of tap water, while the oropharyngeal capsule will be mixed with a sterile, water-based surgical lubricant for application. In cases where oropharyngeal mucosal injury is present, both capsules will be administered via the NGT. For patients with a percutaneous endoscopic gastrostomy (PEG), the NGT capsule will be delivered through the PEG tube. All administrations will occur at least 30 minutes after oral antiseptic care to minimize any potential interference with probiotic activity. Intervention group Participants in the intervention group will receive a four-strain probiotic preparation containing Lactobacillus acidophilus LA-5 [1.75 × 10⁹ Colony Forming Units (CFU)], Lactobacillus plantarum [0.5 × 10⁹ CFU], Bifidobacterium lactis BB12 [1.75 × 10⁹ CFU], and Saccharomyces boulardii [1.5 × 10⁹ CFU]. The probiotic capsules will be indistinguishable in appearance from the placebo capsules. Control group Participants in the control group will receive identical placebo capsules containing an inert glucose polymer with no probiotic activity. The placebo will be administered following the same procedure as the probiotic intervention to maintain blinding. Criteria for discontinuing or modifying allocated interventions {11b} Participants will discontinue the allocated intervention if they experience a serious treatment-emergent adverse event attributable to the study product, such as probiotic-associated infections, severe hypersensitivity reactions, or significant gastrointestinal complications. The intervention will also be terminated in cases where probiotic strains are isolated from a normally sterile site, suggesting potential microbial translocation or infection. Additional criteria for discontinuation include withdrawal of informed consent by the participant or their legally authorized representative, or the inability to continue enteral administration due to prolonged ileus, bowel obstruction, or exclusive reliance on parenteral nutrition. Importantly, participants who discontinue the intervention will remain in the study for outcome assessment, and their data will be included in the intention-to-treat (ITT) analysis to preserve the integrity of the trial’s primary endpoint evaluation. Strategies to improve adherence to interventions {11c} To ensure adherence to the allocated intervention, trained research staff will oversee the administration of probiotics or placebo at each study site, following standardized protocols. The administration schedule will be integrated into routine ICU workflows, with clear documentation in patient charts to ensure timely and consistent delivery. Regular training sessions will be conducted for ICU staff to reinforce protocol adherence, including proper handling and administration techniques. Study monitors will perform periodic compliance checks, reviewing records and addressing any deviations. In cases where enteral administration becomes temporarily challenging, alternative administration routes, such as PEG, will be used whenever feasible to maintain adherence. Relevant concomitant care permitted or prohibited during the trial {11d} Per institutional guidelines, all participants will receive standard ICU care, including VAP prevention measures. Routine oral care with antiseptics (e.g., chlorhexidine) is permitted but must be administered at least 30 minutes before probiotic or placebo administration to prevent potential neutralization. The use of other probiotics, prebiotics, or symbiotics outside the study protocol is strictly prohibited. Patients receiving systemic antifungal or antibiotic therapy will remain in the study, as these treatments are part of routine ICU management, but their potential impact on study outcomes will be recorded and analyzed. The study investigators will document and review any deviation from the protocol regarding concomitant care. Provisions for post-trial care {30} Regardless of the assigned intervention, participants will continue to receive standard ICU care after the study's conclusion. All adverse events will be monitored and handled in the routine clinical practice, and if needed, appropriate medical treatment will be provided. Any serious adverse reaction that a participant may have towards the study intervention will get the proper follow-up care at no extra charge. The study team will ensure that all the relevant findings are conveyed to the treating physicians and that participants or their legal representatives are informed of any clinically significant results that may have implications for further care. Outcomes {12} The study will evaluate the effect of probiotic administration on VAP incidence and other clinical outcomes in critically ill patients. Outcomes will be assessed in both the ITT and modified ITT (mITT) populations, with detailed microbiological and clinical evaluations performed throughout the study period. Primary Outcome The primary outcome is VAP incidence in the ITT population, defined as the first episode of lower respiratory tract infection occurring more than 48 hours after initiation of mechanical ventilation, meeting predefined clinical, radiological and microbiological criteria. VAP is defined as the first episode of a lower respiratory tract infection that meets all the following criteria: (a) onset >48 hours after initiation of mechanical ventilation; (b) increase in Sequential Organ Failure Assessment (SOFA) score by ≥1 point; (c) new infiltrate in chest X-ray or chest computed tomography; (d) core temperature >38°C; (e) purulent tracheobronchial secretions; (f) clinical pulmonary infection score (CPIS) >6; (g) a sustained increase in the daily minimum fraction of inspired oxygen (FiO₂) of ≥0.20 and/or in the daily minimum positive end-expiratory pressure (PEEP) of ≥3 cmH₂O, both lasting for at least 2 consecutive days; (h) and isolation of a pathogen from bronchoalveolar lavage (BAL) fluid (≥1 × 10 4 cfu/mL) or tracheal aspirate (≥1 × 10 5 cfu/mL) (20,21).VAP incidence will be calculated as follows: (Number of cases with VAP/Total number of patients in each group) = VAP rate per 100 patients. Secondary Outcomes Secondary outcomes include VAP incidence in the mITT population , defined as patients without pre-existing lung colonization at baseline, as well as other relevant ICU outcomes: catheter-related bloodstream infections (CRBSI), sepsis, septic shock, 30-day mortality, ICU length of stay, and duration of mechanical ventilation . Sepsis is defined as a new infection with at least a 2-point increase in the total SOFA score (22). Septic shock is defined as sepsis aggravated by hypotension and serum lactate level >2 mmol/L (18 mg/dL) that requires vasopressor administration despite adequate volume resuscitation (22). CRBSI, according to the Infectious Diseases Society of America, is defined when one of the following criteria is met: 1) isolation of the same pathogen from a quantitative blood culture drawn through the central line and from a peripheral vein with the single bacterial colony count at least threefold higher in the sample from the central line as compared to that obtained from a peripheral vein; 2) same organism recovered from percutaneous blood culture and from quantitative (>15 colony-forming units) culture of the catheter tip; 3) a shorter time to positive culture (>2 hours earlier) in the central line sample than the peripheral sample (differential time to positivity [DTP])(23).Mechanical ventilation days are the total days during which the patient underwent mechanical ventilation. All secondary endpoints will be analyzed for both the ITT and mITT populations. Tertiary Outcome The agreement rate between investigator-assigned and adjudicated VAP diagnoses will be analyzed as a tertiary outcome, to assess the consistency and robustness of clinical endpoint adjudication across participating centers. Participant timeline {13} Each participant will follow a structured timeline from enrollment to final follow-up, ensuring consistent data collection and adherence to the study protocol (Figure 2). Screening, Randomization, and Enrollment (Day 0) Eligible ICU patients will be identified based on the inclusion and exclusion criteria. Informed consent will be obtained from the participant or their legally authorized representative. Participants will then be assigned to a pre-randomized list, ensuring allocation concealment. Baseline data will be collected before intervention initiation, including demographic information, clinical scores, and microbiological samples. Intervention Period (Day 1-30) Participants will receive the allocated intervention twice daily for up to 30 days or until extubation. If a tracheostomy is present, administration will continue until permanent disconnection from the ventilator. Daily assessments will include the following: vital signs, presence of sepsis or septic shock, CRBSI, catecholamine support, enteral feeding, parenteral feeding, combined enteral and parenteral feeding, use of prokinetics, antibiotics (including beta-lactamase inhibitors, second- and third-generation cephalosporins, piperacillin/tazobactam, metronidazole, clindamycin, amikacin, and glycopeptides), microbiological results and antibiogram (if available), Glasgow Coma Scale (GCS) score, SOFA score, presence of diarrhea (Bristol stool chart type 6/7), mechanical ventilation status, peripheral capillary oxygen saturation (SpO₂), PEEP, culture results and adverse events. VAP screening will be ensured every day by the attending physician and the research team. The screening protocol begins with clinical suspicion. The presence of new or progressive radiographic infiltrates and at least two of three clinical criteria (24) (e.g. fever/hypothermia, leukocytosis/leukopenia and/or purulent secretions) represent the protocol trigger. Leukocytosis is defined as an elevation in the absolute White Blood Cell (WBC) count (>10,000 cells/μL), while leukopenia is defined as a reduction in the WBC count below 3500 cells/μL (25). Hypothermia is defined as a core body temperature of <35.0 °C (95.0 °F) (26), while fever is defined as a core body temperature of ≥38.3°C (101° F) (27). Once the clinical suspicion is present, samples of respiratory secretions, e.g. tracheal aspirate and BAL, are obtained. Samples will be quantitatively cultured at the local hospital laboratory. Part of the samples will be stored at -80 °C and shipped by air transport to the Laboratory of Immunology of Infectious Diseases of the 4 th Department of Internal Medicine of ATTIKON University General Hospital (124 62 Athens, Greece) for molecular testing. A VAP diagnosis will be established if a quantitative culture yields ≥10 5 CFU/mL of one pathogen in a tracheal aspirate, or if ≥10 4 copies of a pathogen are detected in BAL through molecular testing (in addition to meeting all the criteria for VAP diagnosis listed in the study endpoints section). The volume to be taken for tracheal aspirates and BAL is 3 mL. For patients with low secretions, perform tracheobronchial tree lavage with 5 ml of 0.9 % saline, specifying in the notes that this is a diluted sample. In all cases, the total quantity must not exceed the indicated volume of 3mL. Follow-up and Outcome Assessment (Day 30 or 72h post-extubation) The data collection will continue for at least 30 days unless the patient is successfully extubated or disconnected from the ventilator if a tracheostomy is present, interrupting mechanical ventilation. In case of extubation, the data collection and the probiotic administration will continue for 72 hours after successful weaning to pass the time window for a late VAP diagnosis. In case of tracheostomy and disconnection from the ventilator, the data collection and the probiotic administration will continue for 72 hours: during this time frame, if the patient is reconnected to the ventilator and the tracheostomy cuff has never been deflated, then the protocol will resume until the completion of the total 30 days. The data collection will continue for 3 additional days if a patient is still in the ICU after the thirtieth day, although the intervention stops. If VAP is diagnosed, the patient will continue the probiotic administration for up to 30 days as per the study protocol since there may still be a beneficial effect on the secondary outcomes. Sample size {14} The study is powered to detect a statistically significant reduction in the incidence of VAP between the probiotic and placebo groups. The sample size was calculated using MedCalc® Statistical Software version 19.6.3 (MedCalc Software Ltd, Ostend, Belgium) to compare two independent proportions (28). Sample size calculations were based on findings from the PROVAP trial, which reported a VAP incidence of 28.3% in the placebo group and 11.9% in the probiotic group (7). Using a two-sided alpha level of 0.05 and a beta of 0.20 (corresponding to 80% power), the required sample size was determined to be 186 participants, with 93 patients per group. To account for potential losses to follow-up and unusable data, we considered a 10% oversampling rate , resulting in a final sample size of 206.7 patients, rounded to 208 to ensure equal sample sizes. Recruitment {15} For the current study, participants will be recruited from various ICU centers using consecutive enrollment, where every potentially eligible patient is considered for the study. Recruitment will be consecutive until the required number of 186 participants is obtained. The research team at each site will be responsible for identifying the newly admitted ICU patients, obtaining informed consent, and for verifying eligibility before assigning the patients to the pre-randomized list. The enrollment rate will be monitored regularly. If it falls below expectations, additional centers may be included to ensure timely study completion. Assignment of interventions: allocation Sequence generation {16a} Participants will be randomized 1:1 to the probiotic or placebo group using a computer-generated randomization list stratified by study center. Each participating center will receive its own pre-randomized list, ensuring a balance between groups within each site. Randomization will follow a blocked design to prevent significant imbalances and will be concealed until allocation. Each participant will be assigned to the next available allocation on the site-specific pre-randomized list upon enrollment. Concealment mechanism {16b} Each participating center will receive pre-randomized study product packages based on a centrally generated randomization list specific to that center, created by the study sponsor. The probiotic and placebo products will be identical in appearance to ensure blinding. Investigators, clinicians, participants, and outcome assessors will remain fully blinded to treatment allocation, as the study products will be pre-assigned before arrival at each center. The allocation sequence will not be disclosed to study personnel at the sites, and strict allocation concealment will be maintained throughout the trial. Implementation {16c} The study sponsor's designated personnel will prepare and label the packages before shipment, ensuring that the randomization sequence remains concealed from site investigators. Upon enrollment, participants will receive the following available pre-assigned study products at their center. The Principal Investigator at each site will oversee the trial implementation and ensure adherence to the protocol. Investigators, clinicians, and participants will remain blinded, as the probiotic and placebo products appear identical. Assignment of interventions: Blinding Who will be blinded {17a} The trial will be double-blind, ensuring that participants, investigators, clinicians, and outcome assessors remain unaware of treatment allocation. To maintain blinding, the probiotic and placebo products will be identical in appearance, packaging, and administration. Only the designated personnel at the sponsor site responsible for generating the randomization list and preparing the pre-randomized study product packages will be unblinded. These personnel will not be involved in patient care, data collection, or outcome assessment. Procedure for unblinding if needed {17b} Unblinding will only occur to ensure patient safety, for instance, in the case of a serious adverse event (SAE) that the study intervention may have caused. When unblinding is needed, the Principal Investigator must submit a formal request to the trial’s unblinded safety monitor, who will then consult the randomization list kept by the sponsor. Data collection and management Plans for assessment and collection of outcomes {18a} All outcomes will be systematically monitored and documented by trained research personnel at each participating center. The following will be assessed clinically on a daily basis: VAP, sepsis, septic shock, CRBSI, length of stay in the ICU, and duration of mechanical ventilation. The diagnosis of VAP will be made according to pre-defined clinical, radiological and microbiological criteria, including CPIS, quantitative cultures of tracheal aspirates or BALn and sustained changes in ventilatory parameters. All collected data will be entered into a secure electronic Case Report Form (eCRF) (https://data.castoredc.com/) and regularly monitored for accuracy and completeness. Investigators will ensure adherence to standardized sample collection and storage protocols to maintain consistency across all study sites. Plans to promote participant retention and complete follow-up {18b} To guarantee high retention and complete follow-up, research staff will perform daily patient assessments during the ICU stay and strictly monitor patient compliance with the study protocol. In participants who are extubated before day 30, the follow-up will extend to 72 hours after extubation to include any late-onset VAP. The research team will contact the receiving facility if a participant is transferred to another unit or hospital to get the outcome data. Data quality control and site visits will be performed regularly to prevent missing data and to enforce the protocol. Data management {19} All study data will be documented in a secure web based eCRF to enhance the quality and comprehensiveness of the data collected per the regulatory requirements. The participants’ data will be recorded in the study daily by the trained research personnel at each site, and the information will be entered into the eCRF regularly. Data will be collected daily by trained research staff at each participating center and entered into the eCRF in real-time. To maintain data integrity, the study monitors will conduct periodic data audits. At the end of the trial, the final dataset will be locked, and a copy will be securely archived for regulatory compliance. Data handling will follow GCP guidelines, and no personally identifiable information will be included in the dataset. Confidentiality {27} All participant data will be handled in strict compliance with confidentiality regulations, including GCP guidelines and the General Data Protection Regulation (GDPR 2016/679). All data will be de-identified before being entered into the eCRF after assigning a unique study ID to each participant. The final dataset will be stored on a password-protected server, with access restricted to authorized personnel only. The final dataset will contain no personally identifiable information and will be stored on a password-protected server that only authorized personnel can access. Any data shared for publication or external analysis will be fully anonymized. During the study, confidentiality will be maintained, and no personal data will be disclosed to anyone outside the research team unless required by law or regulatory authorities. Plans for collection, laboratory evaluation and storage of biological specimens for genetic or molecular analysis in this trial/future use {33} Biological specimens will be collected for two distinct purposes: (1) microbiota composition analysis using blood, urine, and stool samples and (2) initial assessment of pathogens in the airways at enrollment/VAP diagnosis using tracheal aspirates and BAL samples. No samples will be retained for future use beyond this trial, and all will be destroyed after analysis is completed. Airways initial assessment and VAP diagnosis Tracheal aspirates and BAL will be performed before enrollment and at each suspected VAP episode. All samples will undergo initial microbiological analysis at the local microbiology laboratory, and a portion will be stored at −80°C. At the end of the study, frozen samples will be shipped by air transport to the Laboratory of Immunology of Infectious Diseases of the 4th Department of Internal Medicine of ATTIKON University General Hospital (124 62 Athens, Greece) for molecular testing. For each collection, 3 mL of tracheobronchial secretions will be obtained; if secretions are scarce, 5 mL of saline will be flushed into the airways, and 3 mL will be aspirated for analysis. Biobanking procedures Biological samples, including feces, blood, and urine, will be collected pre- and post-treatment (after 30 days or 72 hours after successful extubation) to study how probiotics administration affects GM’s composition (11). Fecal samples will be taken pre- and post-treatment at admission and at the end of the probiotic treatment (after 30 days or after successful extubation). They should be placed immediately in the containers provided and refrigerated at +4°C for a maximum of 24 hours before being stored at -80°C until tested. Collection of faces at the patient’s bedside. Transfer at least 5/10 teaspoons of material with the help of the scoop placed on the inner face of the stool collection container cap. Use the scoop to break up the fecal material. Cap the container tightly. Freeze samples at -80°C. Blood samples will be taken before and after treatment at admission and at the end of the probiotic treatment (after 30 days or 72 hours after successful extubation). For each blood collection performed: The normal aliquot will be sent to the central laboratory for the routinely scheduled laboratory tests. One aliquot for serum (tubes normally used for serology). The aliquots obtained will be immediately frozen at -80°C. Two aliquots of 10 ml each (total of 20 ml) will be used to isolate peripheral blood mononuclear cells (PBMCs) (blood collected in tubes containing Lithium Heparin). Isolated PBMCs will be stored at -80°C. One aliquot of whole blood (tube containing EDTA, normally used for blood counts) will be stored at -80°C. Urine samples will be taken pre- and post-treatment at admission and at the end of the probiotic treatment (after 30 days or 72h after successful extubation). The samples will be collected in urine containers. Once delivered, a portion will be used for routine screening, and the remaining will be aliquoted into 15ml falcons. The urine aliquots will be stored at -80°C. Statistical methods Statistical methods for primary and secondary outcomes {20a} Descriptive statistical analyses will be conducted. An initial univariate analysis will compare the placebo and probiotic groups across all main variables. Subsequently, a multivariate logistic regression analysis will be performed to identify variables significantly associated with VAP incidence. For the primary outcome (VAP incidence in the ITT population), a multivariate logistic regression model adjusted for relevant baseline covariates - including age, sex, SOFA score at admission, Charlson Comorbidity Index, baseline GCS, baseline antibiotic use, catecholamine support, nutrition type (enteral, parenteral, or mixed), baseline PEEP levels, smoking history, and hospital center) - will be used to estimate odds ratios (OR) with 95% confidence intervals (CI) comparing the probiotic and placebo groups. The analysis will be performed through a random effect model to account for the center effect. A The analysis will be performed on the ITT population, with a sensitivity analysis on the mITT population. The mITT population comprises only patients with less than 10² cfu/mL of bacterial growth in tracheobronchial secretions before starting the study drug. Commensal bacteria will not be accounted for. For secondary outcomes, statistical methods will include: VAP incidence in the mITT population: Multivariate logistic regression adjusted for baseline covariates (age, sex, SOFA score at admission, Charlson Comorbidity Index, baseline GCS, baseline antibiotic use, catecholamine support, nutrition type, baseline PEEP, smoking history, and hospital center). 30-day mortality: Kaplan-Meier survival analysis with log-rank test; hazard ratios (HR) with 95% CI will be estimated using multivariate Cox regression adjusted for baseline covariates. Sepsis, septic shock, and CRBSI incidence: Multivariate logistic regression adjusted for baseline covariates. ICU length of stay and mechanical ventilation duration: Independent samples t-test (if normally distributed) or Mann-Whitney U test (if non-normally distributed); multivariable linear regression models adjusted for baseline covariates may also be applied if appropriate. All statistical tests will be two-sided, with a significance level of α = 0.05. Missing data will be handled using multiple imputation techniques where applicable. Interim analyses {21b} No interim analyses are pre-planned. Methods for additional analyses (e.g. subgroup analyses) {20b} Stratification Analysis for Antibiotic Use Given the possible variability in antibiotic administration practices among participating centers, despite the full adherence to VAP bundles, a stratification analysis will be conducted to assess the impact of empirical antibiotic use on VAP incidence. Patients will be stratified based on: Early Empirical Antibiotic Use: Defined as antibiotic administration within the first 48 hours of mechanical ventilation. Late or No Empirical Antibiotic Use: Defined as antibiotic administration after 48 hours or no antibiotic exposure prior to VAP diagnosis. Subgroup Analysis: Trauma vs. Stroke/Brain Hemorrhage As the study includes both TBI and stroke patients, a subgroup analysis will be performed to evaluate whether probiotics have a differential effect on VAP incidence between these two groups. The primary and secondary outcomes will be analyzed separately within each subgroup using logistic regression models adjusted for baseline characteristics to determine if the probiotic intervention has varying efficacy based on the underlying condition. Sensitivity Analysis Adjusting for ICU Site Effect Since this is a multicenter trial, a mixed-effects logistic regression model will be used to adjust for potential variability across participating ICUs. ICU site will be included as a random effect, allowing for the evaluation of potential center-to-center variations in patient management, baseline VAP incidence, and intervention response. This will help ensure that observed effects are not confounded by site-specific differences in ICU practices. Methods in analysis to handle protocol non-adherence and any statistical methods to handle missing data {20c} All analyses will follow the ITT principle, meaning all randomized participants will be analyzed in their initially assigned group, regardless of protocol deviations. As per predefined criteria, a mITT analysis will also be performed, excluding patients with bacterial growth ≥10² CFU/mL in tracheal aspirates or BAL at baseline. Missing data will be minimized through real-time data entry monitoring and automated validation checks in the eCRF. They are expected to be primarily related to observed patient characteristics such as age, baseline severity scores - including SOFA and Acute Physiology and Chronic Health Evaluation II (APACHE II) - comorbidities, and ICU type, rather than unobserved outcomes or treatment effects. Furthermore, when identified, missing data will be handled using multiple imputation techniques under the assumption of missing at random (MAR). Analyses will be conducted on the imputed datasets, and results will be pooled according to Rubin’s rules. In addition, sensitivity analyses will be performed using complete case analyses to evaluate the robustness of the findings. Plans to give access to the full protocol, participant level-data and statistical code {31c} Due to the Italian data protection laws and GDPR regulations , access to participant-level data is restricted. The full study protocol and statistical analysis plan will be made available upon reasonable request to authorized regulatory authorities, ethics committees, and academic institutions in compliance with legal and ethical requirements. Oversight and monitoring Composition of the coordinating center and trial steering committee {5d} The coordinating center will oversee the day-to-day management of the trial, including site coordination, regulatory compliance, and data monitoring. Regular meetings will be held to review trial progress and address operational challenges. The Trial Steering Committee will provide scientific oversight, ensuring protocol adherence and evaluating study milestones. A Data Management Team will ensure accurate and timely data collection, with regular quality control checks. An Endpoint Adjudication Committee will independently review VAP diagnoses and key clinical outcomes for consistency. The committee will independently confirm or refute the diagnosis of VAP according to pre-specified clinical and microbiological criteria, remaining blinded to the treatment allocation. This adjudication process will serve to validate the primary outcome (VAP incidence) and ensure consistency and objectivity across participating centers. Additionally, the agreement rate between investigator-assigned and adjudicated VAP diagnoses will be analyzed as a tertiary outcome. Composition of the data monitoring committee, its role and reporting structure{21a} The data monitoring function of the study is performed by an independent pharmacologist responsible for ongoing safety monitoring, compliance verification, and data integrity assessments. The role of the monitor is independent of that of the trial steering committee and principal investigators. The data monitor has the responsibility of reviewing adverse events and serious adverse events related to the intervention at regular intervals, reviewing VAP incidence and other safety endpoints to detect safety issues and reporting any protocol deviations or significant findings to the trial steering committee. The data monitor makes recommendations for continuation or possible changes to the study if necessary. The steering committee will receive all reports from the data monitor and recommend any needed study changes. Regular monitoring will occur throughout the trial, with additional safety reviews conducted if specific concerns arise. Adverse event reporting and harms {22} Investigators will closely monitor the patient daily for any possible treatment-emergent adverse events (TEAEs) and Serious TEAEs (STEAEs). Any unfavorable and unanticipated medical occurrence in a patient receiving a pharmaceutical product that is not necessarily related to the therapy is referred to as a TEAE. The TEAEs might be a symptom, a sign or a laboratory result. If a TAEs meets any of the following criteria, it is defined STEAEs: Results in death Life-threatening situation i.e. the patient is at risk of death Requires In-patient hospitalization or prolongation of existing hospitalization Persistent or significant disability/incapacity Congenital anomaly/ birth defect an important medical event that may not result in death, be life-threatening, or require hospitalization may be considered an SAE when, based upon appropriate medical judgment, the event may jeopardize the subject’s health and may require medical or surgical intervention to prevent one of the outcomes listed above, e.g. death. Grading of severity The severity of the TEAE shall be graded as follows: Mild: the TEAE is transient and well tolerated by the patient Moderate: the TEAE causes discomfort and affects the patient’s usual activities. Severe: the TEAE affects the patient’s usual activities to an important degree and may cause disability or be life-threatening. Relationship to the supplement The investigator will use the following definitions to assess the relationship between the adverse event and the probiotic administration: Probably related: strong relationship; another etiology is improbable or clearly less probable. Possibly related: strong relationship; an alternative etiology is as probable or less probable. Probably not related: slight or no time associated with the probiotic administration. Unrelated: no relationship with the probiotic administration, TEAEs due to an underlying or concomitant disease, or another pharmaceutical product. Investigators will actively look for any TEAEs that are probably related or possibly related to the administration of the probiotics. Specifically, any culture obtained by the ICU and processed by the microbiology laboratory positive for either Lactobacillus plantarum, Lactobacillus acidophilus , Bifidobacterium lactis and Saccharomyces boulardii will be recorded. If the microorganism is isolated from a sterile site or is the predominant strain in a non-sterile setting, the patient will be withdrawn from the trial, and the intervention will be discontinued. Other TAEs include allergy, abdominal distension, diarrhea, pleural effusion, deep venous thrombosis and Clostridioides Difficile infection. Abdominal distension is defined as the objective physical manifestation of an increase in abdominal girth (29). The percussion note over the abdomen will be tympanic, and the abdominal X-ray will reveal dilated bowel loops with multiple air-fluid levels (30). Diarrhea is defined as three or more bowel movements per day with loose or watery stools (Bristol chart of 6 or 7) (31). Pleural effusion is defined as the excessive accumulation of fluid in the pleural space, identified by chest X-ray or chest ultrasonography (32). Deep venous thrombosis (DVT) is defined as the formation of a blood clot in the body’s deep veins, most commonly the leg, but it can also occur in the arms and the mesenteric and cerebral veins. Once clinical (Wells criteria) and laboratory (D-Dimer) suspicions arise, these are confirmed by point-of-care ultrasound (33). DVT will be recorded only if clinically suspected and subsequently confirmed by diagnostic imaging (e.g., point-of-care ultrasound or formal vascular Doppler studies). No routine screening for asymptomatic DVT will be performed. Clostridium difficile infection is defined as the presence of BOTH the following criteria: 1) presence of diarrhea (see previous definition), 2) positive stool test for toxigenic C. difficile or its toxins, or colonoscopic/histopathologic findings demonstrating pseudomembranous colitis (34). Frequency and plans for auditing trial conduct {23} An external audit committee will be established and convened upon the completion of patient enrollment to systematically assess data quality, identify potential issues, and ensure that the statistical analysis remains unbiased. This committee will conduct a thorough review of the dataset, evaluate protocol adherence, and verify the absence of systematic errors or inconsistencies that could impact the integrity of the study findings. In addition to external auditing, the trial will undergo a strictly scheduled internal monitoring framework. Local investigator meetings, involving site investigators and clinical department staff, will be held every two months to review study progress, discuss protocol adherence, verify compliance with VAP prevention bundles, and address operational challenges. Furthermore, formal inter-center meetings, including all principal investigators, trial coordinators, and the lead statistician, will be conducted every three months to assess site performance, identify protocol deviations, and ensure consistency in trial implementation across study centers. Any significant concerns or findings from these meetings will be reported to the trial steering committee and, if necessary, escalated to the external audit committee for further evaluation and corrective action. This structured approach guarantees continuous oversight, standardization across sites, and a rigorous evaluation of study integrity, ensuring the highest scientific and ethical standards throughout the trial. Plans for communicating important protocol amendments to relevant parties (e.g. trial participants, ethical committees) {25} Any protocol amendments will first be discussed at the investigators' meeting. If a change is required, it will be submitted to the ethical committee of the coordinating center for approval before implementation. Dissemination plans {31a} The results of this study will be communicated to the scientific community and other stakeholders through peer-reviewed journal publications, presentations at international conferences, and via sites such as ClinicalTrials.gov and professional networks. Results may also be posted on institutional and academic social media accounts to reach a wider audience. Discussion The PROACT trial is a multicenter, randomized, double-blind, placebo-controlled study designed to rigorously evaluate the efficacy of a multi-strain probiotic formulation in the prevention of VAP among critically ill brain-injured patients. The focus on brain-injured patients reflects their increased risk of developing VAP due to prolonged mechanical ventilation ( 35 ), impaired airway protection associated with altered consciousness ( 36 ), and the absence of other primary infectious processes at admission ( 37 ). Furthermore, in this population, the overall prognosis is predominantly determined by the severity of neurological injury, making the prevention of secondary infections such as VAP particularly relevant to clinical outcomes ( 38 ). Therefore preventing VAP is essential to minimize the duration of mechanical ventilation, reduce ICU and hospital length of stay, prevent additional systemic complications, and support functional recovery. Although VAP may not directly worsen neurological injury, it substantially contributes to ICU morbidity and may impair rehabilitation potential, especially in borderline cases. Conducting a clinical trial of this complexity within the ICU environment entails several methodological and operational challenges, particularly regarding patient recruitment, study product administration, data standardization, regulatory compliance, and inter-center variability in clinical practices—especially antimicrobial stewardship. One of the most significant logistical and ethical challenges is the recruitment of critically ill patients in the ICU setting, where patients often lack decision-making capacity. In such cases, informed consent must be obtained from legally authorized representatives, frequently under urgent and emotionally charged circumstances. Given the acute nature of ICU admissions, timely and ethically sound consent can be difficult to secure. To address this, PROACT incorporates a streamlined consent pathway across all participating sites. Trained investigators are available around the clock to facilitate early enrolment, in accordance with international ethical standards for emergency research. This proactive consent strategy ensures that eligible patients are enrolled as early as possible without compromising ethical integrity. The administration of the investigational product, e.g. probiotics or placebo, twice daily via NGT and oropharyngeal application presents considerable logistical complexity in a high-acuity ICU environment. Proper timing, preparation, and administration must be precisely coordinated with routine care. To ensure protocol fidelity, PROACT has developed a detailed standard operating procedure for study product administration, including specific guidance for patients with altered anatomy (e.g., PEG tubes) or mucosal injury. To minimize protocol deviations, all ICU staff involved in the intervention will undergo structured, site-specific training prior to patient enrolment. In addition, a centralized monitoring system will track administration compliance, and any deviations will be logged, reviewed, and discussed during scheduled investigator meetings. This continuous oversight promotes uniformity across centres and enhances the reliability of the intervention data. Ensuring data integrity and consistency across multiple centres is a core methodological priority. Variations in clinical documentation, ventilator management practices, and microbiological sampling techniques could otherwise introduce significant bias. To mitigate these risks, PROACT utilizes a centralized eCRF with embedded logic checks, range controls, and real-time data validation. All respiratory specimens, including tracheal aspirates and BAL samples, will be processed using standardized protocols and sent to a single reference microbiology laboratory in Greece for molecular and culture-based analyses. This centralized approach ensures uniform diagnostic criteria, eliminates inter-laboratory variability, and enhances diagnostic precision for the primary outcome. One of the most complex challenges in a multi-center international trial is heterogeneity in antimicrobial prescribing practices, driven by differences in local epidemiology, resistance patterns, and institutional protocols. Variability in the timing, type, and escalation of antibiotic therapy can significantly impact VAP incidence and may confound outcome assessment. PROACT directly addresses this issue by implementing comprehensive daily data collection on all antimicrobial agents administered, including timing, dosing, spectrum, and clinical indications. Microbiological culture results are recorded in real time to link antibiotic use with pathogen-specific data. This granular level of antibiotic surveillance enables standardized comparisons across centres, allowing for robust adjustment in the statistical analysis and minimizing the confounding effects of antimicrobial variability. Given the multicenter, international nature of the trial, regulatory compliance presents another substantial challenge. Ethical approval must be obtained from multiple IRBs and ECs, each with unique documentation standards and approval timelines. To streamline this process, the coordinating center provides centralized regulatory support, prepares standardized ethics submission templates, and ensures alignment with international guidelines including International Council for Harmonisation (ICH)-GCP, the Declaration of Helsinki, and relevant national regulations. A notable strength of the PROACT trial is its methodologically robust approach to VAP diagnosis, a historically challenging endpoint due to variability in clinical interpretation and diagnostic criteria. To reduce misclassification bias, PROACT employs a strict and objective diagnostic algorithm incorporating several diagnostic factors. All diagnoses are adjudicated using predefined criteria, ensuring diagnostic consistency and reproducibility across all sites. To preserve blinding and minimize bias, the trial employs strict allocation concealment using pre-randomized, identically packaged study products. Investigators, bedside clinicians, patients, and outcome assessors remain fully blinded throughout the trial. Oversight is maintained by a Trial Steering Committee, responsible for supervising study conduct, protocol adherence, and site performance. Additionally, an independent pharmacovigilance expert monitors for safety signals, reviews adverse events, and ensures the integrity of the study database. Regular monitoring visits and data audits are conducted to maintain high standards of compliance. Despite the considerable methodological and operational complexities inherent in conducting an ICU-based, multicenter, double-blind randomized trial, the PROACT study is uniquely positioned to generate high-quality, clinically meaningful evidence on the role of probiotics in VAP prevention. Through rigorous diagnostic criteria, centralized microbiological analysis, standardized antimicrobial tracking, and robust blinding procedures, PROACT is expected to contribute valuable insights into infection prevention strategies in critically ill populations. If successful, the findings may influence future clinical practice guidelines, support microbiome-targeted interventions, and inform public health policy aimed at reducing the burden of nosocomial infections in ICUs worldwide. Trial status Protocol version: 2.0 February 28, 2025. Ethical approval: September 25, 2023 (Project-ID: 1336/ CE -PROACT) Recruitment initiation: Dec 1, 2023 Anticipated recruitment finalization: March 31, 2026 Abbreviations APACHE , Acute Physiology and Chronic Health Evaluation BAL, Bronchoalveolar Lavage CFU, Colony Forming Unit CPIS, Clinical Pulmonary Infection Score CRBSI, Catheter-Related Bloodstream Infection DTP, Differential Time to Positivity DVT, Deep Venous Thrombosis EC, Ethics Committees eCRF, Electronic Case Report Form FiO2, Fraction of Inspired Oxygen ICU, Intensive Care Unit ITT, Intention-to-Treat IRB, Institutional Review Board LOS, Length of Stay GCP, Good Clinical Practice GM, Gut Microbiota mITT, modified Intention-to-Treat MAR, Missing at Random NGT, Nasogastric Tube OF, Oropharyngeal OM, Oral Microbiota PEEP, Positive End-Expiratory Pressure PEG, Percutaneous Endoscopic Gastrostomy SAE, Serious Adverse Event SAPS II, Simplified Acute Physiology Score SCFAs, Short-Chain Fatty Acids SOFA, Sequential Organ Failure Assessment STEAEs, Serious Treatment-Emergent Adverse Events TBI, Traumatic Brain Injury TEAEs, Treatment-Emergent Adverse Events VAP, Ventilator-Associated Pneumonia VTE, Venous thromboembolism Declarations Acknowledgements We would like to express our gratitude to all the hospital personnel involved in this study, including doctors, nurses, residents, and all other supporting staff. Without their dedication and hard work, this trial would not have been possible. Their commitment to patient care and research has been essential for the successful conduct of this study. In addition, the authors wish to thank Prof. Evangelos Giamarellos-Bourboulis for his invaluable advice and contributions during the design and conduct of the study. In addition, the authors wish to thank Uni-Pharma, Greece for kindly providing the sachets of probiotic formula and placebo. Authors' contributions {31b} Author Contributions: Conceptualization: AC, RS, MTG and FP; Methodology: AC, MTG, RS, and FST; Validation: AC, RS, MTG, FP, FST, MF, and MC; Investigation: AC, RS, CP, NDT, SG, MR, AM, GM, AG, RA, RG, AMo, MF, MC, and VMR; Resources: FP and VMR; Writing—original draft preparation: AC and RS; Writing—review and editing: AC, RS, MTG, FP, FST, MF, and VMR; Supervision: FP, FST, and VMR; Project administration: AC and FP. All authors have read and agreed to the published version of the manuscript. Funding {4} This study did not receive any external funding. Availability of data and materials {29} The datasets generated and analyzed during this study will be available from the corresponding author upon reasonable request, in compliance with ethical and regulatory guidelines. Due to data protection regulations, individual participant data will not be publicly accessible. Ethics approval and consent to participate {24} The PROACT Trial protocol and informed consent forms will be approved by each hospital’s Research Ethics Board. Researchers will carry out this study in line with the GCP and the Declaration of Helsinki. Before randomization, patients or their legal representatives shall provide written informed consent. Consenting individuals will be made aware that if they choose to decline enrollment or withdraw from the study, it will not impact their care. Coded identification, password-protected documents and webpages, lockable filing cabinets and offices will all be used to guarantee confidentiality. Consent for publication {32} No identifiable personal data or images of participants will be published. As part of the informed consent process, participants or their legally authorized representatives will provide consent for the use of anonymized study data in scientific publications and presentations. Competing interests {28} The authors declare that they have no competing interests. No financial compensation was provided to any personnel involved in the study for their contributions to this work. References Torres A, Ewig S, Lode H, Carlet J. Hospital-acquired pneumonia in Europe. Eur Respir J. 2009 May 1;33(5):951–2. Koulenti D, Tsigou E, Rello J. Nosocomial pneumonia in 27 ICUs in Europe: perspectives from the EU-VAP/CAP study. Eur J Clin Microbiol Infect Dis. 2017 Nov 1;36(11):1999–2006. Hellyer TP, Ewan V, Wilson P, Simpson AJ. 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Hämostaseologie. 2024 Apr;44(02):097–110. Bagdasarian N, Rao K, Malani PN. Diagnosis and Treatment of Clostridium difficile in Adults: A Systematic Review. JAMA. 2015 Jan 27;313(4):398–408. Asehnoune K, Rooze P, Robba C, Bouras M, Mascia L, Cinotti R, et al. Mechanical ventilation in patients with acute brain injury: a systematic review with meta-analysis. Crit Care. 2023 Jun 6;27(1):221. Asehnoune K, Roquilly A, Cinotti R. Respiratory Management in Patients with Severe Brain Injury. Crit Care. 2018 Mar 20;22:76. Khan TA, Kamm S. State of the Globe: Traumatic Brain Injury and Infections: The Two-Hit Insult. J Glob Infect Dis. 2023 Nov 30;15(4):135–6. Robba C, Rebora P, Banzato E, Wiegers EJA, Stocchetti N, Menon DK, et al. Incidence, Risk Factors, and Effects on Outcome of Ventilator-Associated Pneumonia in Patients With Traumatic Brain Injury: Analysis of a Large, Multicenter, Prospective, Observational Longitudinal Study. Chest. 2020 Dec;158(6):2292–303. Supplementary Files CHECKLISTPROACTSPIRIT.docx Cite Share Download PDF Status: Published Journal Publication published 10 Nov, 2025 Read the published version in Trials → Version 1 posted Reviewers agreed at journal 17 Aug, 2025 Reviewers invited by journal 14 Jul, 2025 Editor assigned by journal 29 Jun, 2025 First submitted to journal 26 Jun, 2025 Editorial decision: Minor revision 25 Jun, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6813480","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":484873542,"identity":"7a703ca7-197d-4abd-98af-b344edeca65c","order_by":0,"name":"Alberto Corriero","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCUlEQVRIiWNgGAWjYDACdh4kTgIQ84MZBXi0MPNAlcK0SDaAGAbEagEBgwNgErcO/mbeg48rf9gw8Lf3PvvwoOKenPH51YkfHhgwyPOLHcCqReIwX7LhmYQ0Bokzx41nJJwpNja78XazBNBhhjNnJ2DVwnCYx0yyIeEwg4FEGjNDYltC4rYbZzeAtCQY3MauRf4wj/lPhJZ/CYmbZ5zd/AOfFgOgLYwILQ0JiRv4e7fhtcUQ6BfJhrQ0Hokzx5gZEo4lGEvc4N1mkWAggdMvcsd7D35ssLGR429vY2b8UZMgx99/dvPNHxU28vzSOLwPBUhpQAKsUgKvcjTAf4AU1aNgFIyCUTACAAD4G1kbLElKrgAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-9760-9394","institution":"Università degli Studi di Bari Aldo Moro: Universita degli Studi di Bari Aldo Moro","correspondingAuthor":true,"prefix":"","firstName":"Alberto","middleName":"","lastName":"Corriero","suffix":""},{"id":484873543,"identity":"a5c695e1-5451-46d2-9e3d-c59e3b62e4b1","order_by":1,"name":"Rossana Soloperto","email":"","orcid":"","institution":"ULB: Universite Libre de 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Miulli","correspondingAuthor":false,"prefix":"","firstName":"Giovanna","middleName":"","lastName":"Magnesa","suffix":""},{"id":484873553,"identity":"94abf79d-2aee-4994-bd3c-cc3ed1f61c90","order_by":11,"name":"Angelo Giacomucci","email":"","orcid":"","institution":"University of Perugia: Universita degli Studi di Perugia","correspondingAuthor":false,"prefix":"","firstName":"Angelo","middleName":"","lastName":"Giacomucci","suffix":""},{"id":484873554,"identity":"06a1b342-6d34-4be8-982e-2312527aebea","order_by":12,"name":"Riccardo Accattoli","email":"","orcid":"","institution":"University of Perugia: Universita degli Studi di Perugia","correspondingAuthor":false,"prefix":"","firstName":"Riccardo","middleName":"","lastName":"Accattoli","suffix":""},{"id":484873555,"identity":"5646aa11-b640-456c-ba50-87641ce38d63","order_by":13,"name":"Raffaella Maria Gadaleta","email":"","orcid":"","institution":"University of Bari: Universita degli Studi di Bari Aldo Moro","correspondingAuthor":false,"prefix":"","firstName":"Raffaella","middleName":"Maria","lastName":"Gadaleta","suffix":""},{"id":484873556,"identity":"75e69bb0-3459-480c-840b-65d12b0362a6","order_by":14,"name":"Marilina Florio","email":"","orcid":"","institution":"University of Bari Aldo Moro: Universita degli Studi di Bari Aldo Moro","correspondingAuthor":false,"prefix":"","firstName":"Marilina","middleName":"","lastName":"Florio","suffix":""},{"id":484873557,"identity":"4dd00445-1c39-4701-8c5d-4f2f5500a264","order_by":15,"name":"Marica Cariello","email":"","orcid":"","institution":"University of Bari Aldo Moro: Universita degli Studi di Bari Aldo Moro","correspondingAuthor":false,"prefix":"","firstName":"Marica","middleName":"","lastName":"Cariello","suffix":""},{"id":484873558,"identity":"ad5e7604-d82b-4b59-b303-0485709fde9d","order_by":16,"name":"Antonio Moschetta","email":"","orcid":"","institution":"University of Bari Aldo Moro: Universita degli Studi di Bari Aldo Moro","correspondingAuthor":false,"prefix":"","firstName":"Antonio","middleName":"","lastName":"Moschetta","suffix":""},{"id":484873559,"identity":"55c51f7a-576e-4963-9432-748020016ef7","order_by":17,"name":"Filomena Puntillo","email":"","orcid":"","institution":"University of Bari Aldo Moro: Universita degli Studi di Bari Aldo Moro","correspondingAuthor":false,"prefix":"","firstName":"Filomena","middleName":"","lastName":"Puntillo","suffix":""},{"id":484873560,"identity":"c4751bda-409e-4727-b11e-3ee66f7066df","order_by":18,"name":"Fabio Silvio Taccone","email":"","orcid":"","institution":"ULB: Universite Libre de Bruxelles","correspondingAuthor":false,"prefix":"","firstName":"Fabio","middleName":"Silvio","lastName":"Taccone","suffix":""},{"id":484873561,"identity":"17af6185-eff1-465d-9b90-c14a08e79cf4","order_by":19,"name":"Vito Marco Ranieri","email":"","orcid":"","institution":"University of Bari Aldo Moro: Universita degli Studi di Bari Aldo Moro","correspondingAuthor":false,"prefix":"","firstName":"Vito","middleName":"Marco","lastName":"Ranieri","suffix":""}],"badges":[],"createdAt":"2025-06-03 16:54:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6813480/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6813480/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13063-025-09230-w","type":"published","date":"2025-11-10T15:58:37+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":87034744,"identity":"d8440da5-4ecc-4d8a-bc6d-39da0ce2c80d","added_by":"auto","created_at":"2025-07-18 13:11:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":379281,"visible":true,"origin":"","legend":"\u003cp\u003eDesign of the PROACT Study. NG: nasogastric, OF: oropharyngeal\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6813480/v1/1e07cd9235a7a5c9e2d12798.png"},{"id":87034741,"identity":"593da310-e2b1-4ac0-9456-1ad7c296548c","added_by":"auto","created_at":"2025-07-18 13:11:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":245912,"visible":true,"origin":"","legend":"\u003cp\u003eThe table shows in accordance with SPIRIT guidelines, the timetable and schedule for enrolled patients.\u003c/p\u003e","description":"","filename":"Figure2Trials.png","url":"https://assets-eu.researchsquare.com/files/rs-6813480/v1/2c87e98698831cc8318d09ed.png"},{"id":96105787,"identity":"28decd2e-1993-4899-bd81-68ade52201b9","added_by":"auto","created_at":"2025-11-17 16:11:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2543985,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6813480/v1/c19b13ef-24a6-4cfb-9e96-01679b1b228b.pdf"},{"id":87034746,"identity":"91407083-30d2-4989-a727-b58f303712f5","added_by":"auto","created_at":"2025-07-18 13:11:14","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":41093,"visible":true,"origin":"","legend":"","description":"","filename":"CHECKLISTPROACTSPIRIT.docx","url":"https://assets-eu.researchsquare.com/files/rs-6813480/v1/a4cd5ea7c92533fa2a25fd9a.docx"}],"financialInterests":"","formattedTitle":"Probiotics to Reduce Ventilator-Associated Pneumonia: Study Protocol for a Double-Blind Multicenter Randomized International Clinical Trial (PROACT)","fulltext":[{"header":"Administrative information","content":"\u003ctable border=\"1\" cellpadding=\"0\" align=\"left\" width=\"661\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eTitle {1}\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePROACT - Probiotics in ICU to Reduce Ventilator-Associated Pneumonia: Study Protocol for a Double-Blind Multicentre Randomized \u0026nbsp; \u0026nbsp; International Clinical Trial\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eTrial registration {2a and 2b}.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eClinicalTrials.gov Registry (\u0026nbsp;NCT06092554).\u0026nbsp;\u0026nbsp;Registered on 2023-10-15.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eProtocol version {3}\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e28\u003csup\u003eth\u003c/sup\u003e of \u0026nbsp;February 2025 (3.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eFunding {4}\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eThis study is not funded.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAuthor details {5a}\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026sup1; Department of Interdisciplinary Medicine \u0026ndash; ICU Section, University of Bari Aldo Moro, Piazza G. Cesare 11, 70124, Bari, Italy; [email protected] (AC); [email protected] (MTG); [email protected] (FP)\u003c/p\u003e\n \u003cp\u003e2 Department of Precision and Regenerative Medicine and Jonian Area \u0026ndash; ICU Section, University of Bari Aldo Moro, Bari, Italy; [email protected] (VMR); [email protected] (SG); [email protected] (MR)\u003c/p\u003e\n \u003cp\u003e3 Department of Intensive Care, H\u0026ocirc;pital Universitaire de Bruxelles (HUB), Universit\u0026eacute; Libre de Bruxelles (ULB), Route de Lennik 808, 1070, Brussels, Belgium; [email protected] (FST); [email protected] (MS); [email protected] (RS)\u003c/p\u003e\n \u003cp\u003e4 Department of Interdisciplinary Medicine \u0026ndash; Medical Statistics Section, University of Bari Aldo Moro, Piazza G. Cesare 11, 70124, Bari, Italy; [email protected] (PT)\u003c/p\u003e\n \u003cp\u003e5 \u0026nbsp;Department of Interdisciplinary Medicine \u0026ndash; Microbiology Section, University of Bari Aldo Moro, Piazza G. Cesare 11, 70124, Bari, Italy; [email protected] (AM)\u003c/p\u003e\n \u003cp\u003e6 UOC Anesthesia and Intensive Care, Ospedale \u0026quot;Di Venere\u0026quot;, ASL Bari, Via Ospedale Di Venere 1, 70131 Bari, Italy; [email protected] (CP); [email protected] (NDT)\u003c/p\u003e\n \u003cp\u003e7 ICU Section, Ospedale Miulli, Acquaviva delle Fonti, Bari, Italy; [email protected] (GM)\u003c/p\u003e\n \u003cp\u003e8 Intensive Care Unit, Azienda Ospedaliera di Perugia, Piazzale G. Menghini 6/8, 06156 Perugia, Italy; [email protected] (AG); [email protected] (RA)\u003c/p\u003e\n \u003cp\u003e9 Department di Medicine and Surgery, Anaesthetics, Analgesia and Intensive Care Section, University of Perugia, P.le Lucio Severi, 1, 06132 Perugia, Italy\u003c/p\u003e\n \u003cp\u003e10 Department of Interdisciplinary Medicine, University of Bari Aldo Moro, Piazza G. Cesare 11, 70124, Bari, Italy; [email protected] (AMo); [email protected] (RMG); [email protected] (MF); [email protected] (MC)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eName and contact information for the trial sponsor {5b}\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eUNI-PHARMA S.A. Greece\u0026nbsp;\u0026nbsp;Tel: +30 210 8072512.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eRole of sponsor {5c}\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eGenerates \u003cstrong\u003ecenter-specific randomization lists\u003c/strong\u003e\u003cstrong\u003e,\u0026nbsp;\u003c/strong\u003eprepares \u003cstrong\u003epre-randomized study product packages\u003c/strong\u003e\u003cstrong\u003e,\u0026nbsp;\u003c/strong\u003eand ensures\u003cstrong\u003e\u0026nbsp;\u003cstrong\u003eidentical appearance of placebo and probiotic\u003c/strong\u003e.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Introduction","content":"\u003cp\u003e\u003cstrong\u003eBackground and Rationale {6a}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVentilator-associated pneumonia (VAP)\u0026nbsp;remains a significant burden in the Intensive Care Unit (ICU), with reported mortality rates approaching 50% (1,2). To mitigate this risk, a VAP prevention care bundle, comprising a set of evidence-based, high-impact interventions, has been widely adopted. These strategies include (3\u0026ndash;6): a) elevation of the head of the bed (to reduce the risk of tracheal micro-aspiration); b) daily sedation interruption and assessment of readiness to extubate (to shorten mechanical ventilation duration); c) subglottic secretion drainage; d) avoidance of scheduled ventilator circuit changes; e) peptic ulcer prophylaxis (to reduce stress-related mucosal damage and subsequent complications); and f) venous thromboembolism (VTE) prophylaxis (to prevent immobility-related complications).\u003c/p\u003e\n\u003cp\u003eDespite growing interest, probiotic administration is not currently included among these standard recommendations. However, several studies have demonstrated promising results in reducing VAP incidence, particularly in high-risk populations, such as patients with traumatic brain injury. In a randomized controlled trial (7), the use of a four-strain probiotic preparation significantly reduced VAP incidence in patients with head trauma, from 28.3% in the placebo group to 11.9% in the intervention group. The same probiotic formulation was also evaluated in patients with multiple trauma, where it was associated with a reduction in surgical site infections, suggesting a broader prophylactic potential beyond respiratory infections (8). Additionally, a separate study assessing the use of a dual-strain probiotic preparation in patients with acute stroke demonstrated a reduction in VAP incidence in the probiotic group (9). Notably, patients with stroke and those with traumatic brain injury may share overlapping pathophysiological mechanisms and similar risk for VAP (10). Recent systematic reviews and meta-analyses (11,12) support the potential role of probiotics in reducing VAP incidence. Available data suggest that the prophylactic benefits of probiotics may be most pronounced in patients without active infections at baseline. However, the quality of evidence remains variable, and further high-quality randomized controlled trials with low risk of bias are warranted.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe underlying\u0026nbsp;mechanisms\u0026nbsp;of probiotic efficacy in VAP prevention are not yet fully elucidated, but several hypotheses have been proposed. One involves modulation of the\u0026nbsp;gut microbiota (GM), oral microbiota (OM), and lung microbiota via the\u0026nbsp;gut\u0026ndash;lung\u0026nbsp;and\u0026nbsp;gut\u0026ndash;bone marrow\u0026ndash;lung axes\u0026nbsp;(13,14). Importantly, short-chain fatty acids (SCFAs), including butyrate, acetate, and propionate, produced by colonic bacterial fermentation of dietary fiber may exert anti-inflammatory effects and contribute to the maintenance of the respiratory epithelium\u0026apos;s integrity (15). In critically ill patients,\u0026nbsp;dysbiosis, defined as a disruption of the normal gut microbial balance, develops rapidly, often within six hours of ICU admission (16). This leads to a marked decrease in microbial diversity and the overgrowth of opportunistic, potentially pathogenic organisms (17,18). Interrupting this process by restoring eubiosis through\u0026nbsp;early and targeted probiotic administration\u0026nbsp;could help maintain the functionality of the gut-lung axis and reduce secondary infectious complications such as VAP.\u003c/p\u003e\n\u003cp\u003eNevertheless, a recent large-scale randomized controlled trial conducted in Canada failed to demonstrate a reduction in VAP incidence with probiotic therapy (19). However, a substantial proportion of patients in that study were already receiving antibiotics for non-specific infections at enrolment, and the protocol did not mandate microbiological confirmation for VAP diagnosis, potentially confounding the results.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eObjectives {7}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study aims to address key uncertainties that currently limit the incorporation of probiotic therapy into clinical guidelines and public health policies for VAP prevention in the intensive care setting. This randomized controlled trial will enroll critically ill patients with primary non-anoxic brain injury; patients with suspected pulmonary aspiration or pre-existing pulmonary infection at the time of enrolment will be excluded to minimize confounding.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;The primary objective is to evaluate whether a four-strain probiotic formulation (\u003cem\u003eLactobacillus acidophilus\u003c/em\u003e LA-5, \u003cem\u003eLactobacillus plantarum\u003c/em\u003e, \u003cem\u003eBifidobacterium lactis\u003c/em\u003e BB12, and \u003cem\u003eSaccharomyces boulardii\u003c/em\u003e; Lactolevure\u0026reg;, Unipharma, Greece) can reduce the incidence of VAP and improve 30-day all-cause mortality.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTrial design {8}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe PROACT study is a randomized, double-blind, placebo-controlled, multicenter trial designed to evaluate the efficacy of probiotic therapy in preventing VAP in critically ill patients. The study design is illustrated in Figure 1. A total of 186 eligible ICU patients will be randomized in a 1:1 ratio to receive either a four-strain probiotic preparation (intervention group) or placebo (control group). The intervention (e.g. probiotics or placebo) will be administered twice daily for up to 30 days via nasogastric tube and oropharyngeal application. The study employs a double-blind methodology, ensuring that both participants and treating clinicians remain blinded to group allocation.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;The primary outcome analysis will be conducted on an intention-to-treat (ITT) basis. The trial is powered to detect a reduction in VAP incidence from 28.3% to 11.9%, based on effect size estimates observed in the PROVAP study (7).\u003c/p\u003e"},{"header":"Methods: Participants, interventions and outcomes","content":"\u003cp\u003e\u003cstrong\u003eStudy setting {9}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis will be a prospective, randomized, double-blind, placebo-controlled clinical trial. The study protocol will be submitted for approval to the Ethics Committees (EC) and Institutional Review Boards (IRB) as per participating study site regulations.\u003c/p\u003e\n\u003cp\u003eThe study shall be governed by: the international standards for Good Clinical Practice (GCP) developed by the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use; the directive 2001/20/EC for Clinical trials; general Data Protection Regulation 679/2016 (EC).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEligibility Criteria {10}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePatients eligible for enrolment are admitted to the hospital from the emergency department or transferred from another ICU. A legal representative will be asked for written informed consent once a patient is classified as having acute traumatic brain injury (TBI), ischemic stroke or hemorrhagic stroke (e.g. including intracerebral hemorrhage and subarachnoid hemorrhage). Therefore, eligibility screening can start, with exclusion criteria assessed first. If the patient does not meet any exclusion criteria, he will also be considered for inclusion criteria. If the patient meets all the inclusion criteria, he can be successfully enrolled in the study. Inclusion and exclusion criteria are shown in (Table 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e Inclusion and exclusion criteria of the PROACT study.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"663\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eInclusion Criteria\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eExclusion Criteria\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ea) Adults aged 18\u0026ndash;80 years\u003cbr\u003e\u0026nbsp;b) Recent trauma (including TBI with \u0026ge;1 other organ injury) OR stroke/brain hemorrhage without aspiration/lung infection\u003cbr\u003e\u0026nbsp;c) Intubation and mechanical ventilation initiated immediately after the event\u003cbr\u003e\u0026nbsp;d) Expected mechanical ventilation \u0026ge;6 days (treating physician judgement)\u003cbr\u003e\u0026nbsp;e) Written informed consent (patient or legal representative)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ea) Mechanical ventilation \u0026gt;72 hours before enrollment\u003cbr\u003e\u0026nbsp;b) Pregnancy or lactation\u003cbr\u003e\u0026nbsp;c) High infection risk (immunosuppression, HIV \u0026lt;200 CD4+, recent chemotherapy, neutropenia)\u003cbr\u003e\u0026nbsp;d) Critical gastrointestinal conditions (severe pancreatitis, ischemic bowel)\u003cbr\u003e\u0026nbsp;e) Inability to receive enteral medications\u003cbr\u003e\u0026nbsp;f) Intent to withdraw advanced life support\u003cbr\u003e\u0026nbsp;g) Endovascular infection risk *\u0026nbsp;\u003cbr\u003e\u0026nbsp;h) Sepsis and/or septic shock\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eTBI: Traumatic Brain Injury; ED: Emergency Department; HIV: Human Immunodeficiency Virus; CD4+: Cluster of Differentiation 4 positive cells.\u003c/p\u003e\n\u003cp\u003e* Includes: previously documented rheumatic heart disease, congenital valve disease, surgically repaired congenital heart disease, unrepaired cyanotic congenital heart disease, any intracardiac repair with prosthetic material, previous or current endocarditis, permanent endovascular devices such as inferior vena cava filters, dialysis vascular grafts, tunnelled (not short-term) hemodialysis catheters, pacemakers or defibrillators.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWho will take informed consent? {26a}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBefore enrollment, a trained investigator or a designated research team member at each participating center will obtain written informed consent from patients or their legal representatives. Given the critical condition of eligible patients, consent will typically be sought from the patient\u0026rsquo;s legally authorized representative in accordance with local regulations and ethical guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional consent provisions for collection and use of participant data and biological specimens {26b}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe main informed consent document includes consent for collecting, storing and analyzing biological specimens (blood, urine, and stool samples). By signing the consent form, participants or their legal representatives agree to the use of these samples for microbiota analysis and related research on ventilator-associated pneumonia. All data and specimens will be anonymized and handled according to ethical and regulatory guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInterventions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExplanation for the choice of comparators {6b}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA placebo was selected as the comparator to ensure blinding and to isolate the specific effects of probiotic administration on the incidence of VAP. The placebo consists of an inert glucose polymer powder, which has no known impact on gut microbiota composition or host immune function. This approach enables a rigorous assessment of probiotic efficacy while preserving methodological integrity within the double-blind, randomized study design. The choice of this placebo is consistent with prior clinical trials evaluating probiotics in critically ill populations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIntervention description {11a}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eParticipants in both study arms will receive two capsules twice daily for up to 30 days: one capsule will be administered via nasogastric tube (NGT) and the other applied to the oropharyngeal mucosa. The NGT capsule will be dissolved in 100 mL of tap water, while the oropharyngeal capsule will be mixed with a sterile, water-based surgical lubricant for application. In cases where oropharyngeal mucosal injury is present, both capsules will be administered via the NGT. For patients with a percutaneous endoscopic gastrostomy (PEG), the NGT capsule will be delivered through the PEG tube. All administrations will occur at least 30 minutes after oral antiseptic care to minimize any potential interference with probiotic activity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIntervention group\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eParticipants in the intervention group will receive a four-strain probiotic preparation containing \u003cem\u003eLactobacillus acidophilus\u003c/em\u003e LA-5 [1.75 \u0026times; 10⁹ Colony Forming Units (CFU)], \u003cem\u003eLactobacillus plantarum\u003c/em\u003e [0.5 \u0026times; 10⁹ CFU], \u003cem\u003eBifidobacterium lactis\u003c/em\u003e BB12 [1.75 \u0026times; 10⁹ CFU], and \u003cem\u003eSaccharomyces boulardii\u003c/em\u003e [1.5 \u0026times; 10⁹ CFU]. The probiotic capsules will be indistinguishable in appearance from the placebo capsules.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eControl group\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eParticipants in the control group will receive identical placebo capsules containing an inert glucose polymer with no probiotic activity. The placebo will be administered following the same procedure as the probiotic intervention to maintain blinding.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCriteria for discontinuing or modifying allocated interventions {11b}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eParticipants will discontinue the allocated intervention if they experience a serious treatment-emergent adverse event attributable to the study product, such as probiotic-associated infections, severe hypersensitivity reactions, or significant gastrointestinal complications. The intervention will also be terminated in cases where probiotic strains are isolated from a normally sterile site, suggesting potential microbial translocation or infection.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Additional criteria for discontinuation include withdrawal of informed consent by the participant or their legally authorized representative, or the inability to continue enteral administration due to prolonged ileus, bowel obstruction, or exclusive reliance on parenteral nutrition.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Importantly, participants who discontinue the intervention will remain in the study for outcome assessment, and their data will be included in the intention-to-treat (ITT) analysis to preserve the integrity of the trial\u0026rsquo;s primary endpoint evaluation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStrategies to improve adherence to interventions {11c}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo ensure adherence to the allocated intervention, trained research staff will oversee the administration of probiotics or placebo at each study site, following standardized protocols. The administration schedule will be integrated into routine ICU workflows, with clear documentation in patient charts to ensure timely and consistent delivery. Regular training sessions will be conducted for ICU staff to reinforce protocol adherence, including proper handling and administration techniques. Study monitors will perform periodic compliance checks, reviewing records and addressing any deviations. In cases where enteral administration becomes temporarily challenging, alternative administration routes, such as PEG, will be used whenever feasible to maintain adherence.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRelevant concomitant care permitted or prohibited during the trial {11d}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePer institutional guidelines, all participants will receive standard ICU care, including VAP prevention measures. Routine oral care with antiseptics (e.g., chlorhexidine) is permitted but must be administered at least 30 minutes before probiotic or placebo administration to prevent potential neutralization. The use of other probiotics, prebiotics, or symbiotics outside the study protocol is strictly prohibited. Patients receiving systemic antifungal or antibiotic therapy will remain in the study, as these treatments are part of routine ICU management, but their potential impact on study outcomes will be recorded and analyzed. The study investigators will document and review any deviation from the protocol regarding concomitant care.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProvisions for post-trial care {30}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRegardless of the assigned intervention, participants will continue to receive standard ICU care after the study\u0026apos;s conclusion. All adverse events will be monitored and handled in the routine clinical practice, and if needed, appropriate medical treatment will be provided. Any serious adverse reaction that a participant may have towards the study intervention will get the proper follow-up care at no extra charge. The study team will ensure that all the relevant findings are conveyed to the treating physicians and that participants or their legal representatives are informed of any clinically significant results that may have implications for further care.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOutcomes {12}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study will evaluate the effect of probiotic administration on VAP incidence and other clinical outcomes in critically ill patients. Outcomes will be assessed in both the ITT and modified ITT (mITT) populations, with detailed microbiological and clinical evaluations performed throughout the study period.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePrimary Outcome\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe primary outcome is VAP incidence in the ITT population, defined as the first episode of lower respiratory tract infection occurring more than 48 hours after initiation of mechanical ventilation, meeting predefined clinical, radiological and microbiological criteria. VAP is defined as the first episode of a lower respiratory tract infection that meets all the following criteria: (a) onset \u0026gt;48 hours after initiation of mechanical ventilation; (b) increase in Sequential Organ Failure Assessment (SOFA) score by \u0026ge;1 point; (c) new infiltrate in chest X-ray or chest computed tomography; (d) core temperature \u0026gt;38\u0026deg;C; (e) purulent tracheobronchial secretions; (f) clinical pulmonary infection score (CPIS) \u0026gt;6; (g) a sustained increase in the daily minimum fraction of inspired oxygen (FiO₂) of \u0026ge;0.20 and/or in the daily minimum positive end-expiratory pressure (PEEP) of \u0026ge;3 cmH₂O, both lasting for at least 2 consecutive days; (h) and isolation of a pathogen from bronchoalveolar lavage (BAL) fluid (\u0026ge;1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cfu/mL) or tracheal aspirate (\u0026ge;1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cfu/mL) (20,21).VAP incidence will be calculated as follows: (Number of cases with VAP/Total number of patients in each group) = VAP rate per 100 patients.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSecondary Outcomes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSecondary outcomes include\u003cstrong\u003e\u0026nbsp;\u003cstrong\u003eVAP incidence in the mITT population\u003c/strong\u003e,\u0026nbsp;\u003c/strong\u003edefined as patients without pre-existing lung colonization at baseline, as well as other relevant ICU outcomes:\u003cstrong\u003e\u0026nbsp;\u003cstrong\u003ecatheter-related bloodstream infections (CRBSI), sepsis, septic shock, 30-day mortality, ICU length of stay, and duration of mechanical ventilation\u003c/strong\u003e\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eSepsis is defined as a new infection with at least a 2-point increase in the total SOFA score (22). Septic shock is defined as sepsis aggravated by hypotension and serum lactate level \u0026gt;2 mmol/L (18 mg/dL) that requires vasopressor administration despite adequate volume resuscitation (22). CRBSI, according to the Infectious Diseases Society of America, is defined when one of the following criteria is met: 1) isolation of the same pathogen from a quantitative blood culture drawn through the central line and from a peripheral vein with the single bacterial colony count at least threefold higher in the sample from the central line as compared to that obtained from a peripheral vein; 2) same organism recovered from percutaneous blood culture and from quantitative (\u0026gt;15 colony-forming units) culture of the catheter tip; 3) a shorter time to positive culture (\u0026gt;2 hours earlier) in the central line sample than the peripheral sample (differential time to positivity [DTP])(23).Mechanical ventilation days are the total days during which the patient underwent mechanical ventilation. All secondary endpoints will be analyzed for both the ITT and mITT populations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTertiary Outcome\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe agreement rate between investigator-assigned and adjudicated VAP diagnoses will be analyzed as a tertiary outcome, to assess the consistency and robustness of clinical endpoint adjudication across participating centers.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eParticipant timeline {13}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEach participant will follow a structured timeline from enrollment to final follow-up, ensuring consistent data collection and adherence to the study protocol (Figure 2).\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003e\u003cem\u003eScreening, Randomization, and Enrollment (Day 0)\u003c/em\u003e\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eEligible ICU patients will be identified based on the inclusion and exclusion criteria. Informed consent will be obtained from the participant or their legally authorized representative. Participants will then be assigned to a pre-randomized list, ensuring allocation concealment. Baseline data will be collected before intervention initiation, including demographic information, clinical scores, and microbiological samples.\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003e\u003cem\u003eIntervention Period (Day 1-30)\u003c/em\u003e\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eParticipants will receive the allocated intervention twice daily for up to 30 days or until extubation. If a tracheostomy is present, administration will continue until permanent disconnection from the ventilator. Daily assessments will include the following: vital signs, presence of sepsis or septic shock, CRBSI, catecholamine support, enteral feeding, parenteral feeding, combined enteral and parenteral feeding, use of prokinetics, antibiotics (including beta-lactamase inhibitors, second- and third-generation cephalosporins, piperacillin/tazobactam, metronidazole, clindamycin, amikacin, and glycopeptides), microbiological results and antibiogram (if available), Glasgow Coma Scale (GCS) score, SOFA score, presence of diarrhea (Bristol stool chart type 6/7), mechanical ventilation status, peripheral capillary oxygen saturation (SpO₂), PEEP, culture results and adverse events.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eVAP screening will be ensured every day by the attending physician and the research team. The screening protocol begins with clinical suspicion. The presence of new or progressive radiographic infiltrates and at least two of three clinical criteria (24) (e.g. fever/hypothermia, leukocytosis/leukopenia and/or purulent secretions) represent the protocol trigger. Leukocytosis is defined as an elevation in the absolute White Blood Cell (WBC) count (\u0026gt;10,000 cells/\u0026mu;L), while leukopenia is defined as a reduction in the WBC count below 3500 cells/\u0026mu;L (25). Hypothermia is defined as a core body \u0026nbsp;temperature of \u0026lt;35.0 \u0026deg;C (95.0 \u0026deg;F) (26), while fever is defined as a core body temperature of \u0026nbsp;\u0026ge;38.3\u0026deg;C (101\u0026deg; F) (27). Once the clinical suspicion is present, samples of respiratory secretions, e.g. tracheal aspirate and BAL, are obtained. Samples will be quantitatively cultured at the local hospital laboratory. Part of the samples will be stored at \u0026nbsp;-80 \u0026deg;C and shipped by air transport to the Laboratory of Immunology of Infectious Diseases of the 4\u003csup\u003eth\u003c/sup\u003e Department of Internal Medicine of ATTIKON University General Hospital (124 62 Athens, Greece) for molecular testing. A VAP diagnosis will be established if a quantitative culture yields \u0026ge;10\u003csup\u003e5\u003c/sup\u003e CFU/mL of one pathogen in a tracheal aspirate, or if \u0026ge;10\u003csup\u003e4\u003c/sup\u003e copies of a pathogen are detected in BAL through molecular testing (in addition to meeting all the criteria for VAP diagnosis listed in the study endpoints section). The volume to be taken for tracheal aspirates and BAL is 3 mL. For patients with low secretions, perform tracheobronchial tree lavage with 5 ml of 0.9 % saline, specifying in the notes that this is a diluted sample. In all cases, the total quantity must not exceed the indicated volume of 3mL.\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003e\u003cem\u003eFollow-up and Outcome Assessment (Day 30 or 72h post-extubation)\u003c/em\u003e\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eThe data collection will continue for at least 30 days unless the patient is successfully extubated or disconnected from the ventilator if a tracheostomy is present, interrupting mechanical ventilation. In case of extubation, the data collection and the probiotic administration will continue for 72 hours after successful weaning to pass the time window for a late VAP diagnosis. In case of tracheostomy and disconnection from the ventilator, the data collection and the probiotic administration will continue for 72 hours: during this time frame, if the patient is reconnected to the ventilator and the tracheostomy cuff has never been deflated, then the protocol will resume until the completion of the total 30 days. The data collection will continue for 3 additional days if a patient is still in the ICU after the thirtieth day, although the intervention stops. If VAP is diagnosed, the patient will continue the probiotic administration for up to 30 days as per the study protocol since there may still be a beneficial effect on the secondary outcomes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSample size {14}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study is powered to detect a statistically significant reduction in the incidence of VAP between the probiotic and placebo groups. The sample size was calculated using MedCalc\u0026reg; Statistical Software version 19.6.3 (MedCalc Software Ltd, Ostend, Belgium) to compare two independent proportions (28). Sample size calculations were based on findings from the PROVAP trial, which reported a VAP incidence of 28.3% in the placebo group and 11.9% in the probiotic group (7). Using a two-sided alpha level of 0.05 and a beta of 0.20 (corresponding to 80% power), the required sample size was determined to be 186 participants, with 93 patients per group. To account for potential losses to follow-up and unusable data, we considered a 10% oversampling rate , resulting in a final sample size of 206.7 \u0026nbsp; patients, rounded to 208 to ensure equal sample sizes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRecruitment {15}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the current study, participants will be recruited from various ICU centers using consecutive enrollment, where every potentially eligible patient is considered for the study. Recruitment will be consecutive until the required number of 186 participants is obtained. The research team at each site will be responsible for identifying the newly admitted ICU patients, obtaining informed consent, and for verifying eligibility before assigning the patients to the pre-randomized list. The enrollment rate will be monitored regularly. If it falls below expectations, additional centers may be included to ensure timely study completion.\u0026nbsp;\u003cstrong\u003e\u003cbr\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssignment of interventions: allocation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSequence generation {16a}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eParticipants will be randomized 1:1 to the probiotic or placebo group using a computer-generated randomization list stratified by study center. Each participating center will receive its own pre-randomized list, ensuring a balance between groups within each site. Randomization will follow a blocked design to prevent significant imbalances and will be concealed until allocation. Each participant will be assigned to the next available allocation on the site-specific pre-randomized list upon enrollment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConcealment mechanism {16b}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEach participating center will receive pre-randomized study product packages based on a centrally generated randomization list specific to that center, created by the study sponsor. The probiotic and placebo products will be identical in appearance to ensure blinding. Investigators, clinicians, participants, and outcome assessors will remain fully blinded to treatment allocation, as the study products will be pre-assigned before arrival at each center. The allocation sequence will not be disclosed to study personnel at the sites, and strict allocation concealment will be maintained throughout the trial.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImplementation {16c}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study sponsor\u0026apos;s designated personnel will prepare and label the packages before shipment, ensuring that the randomization sequence remains concealed from site investigators. Upon enrollment, participants will receive the following available pre-assigned study products at their center. The Principal Investigator at each site will oversee the trial implementation and ensure adherence to the protocol. Investigators, clinicians, and participants will remain blinded, as the probiotic and placebo products appear identical.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssignment of interventions: Blinding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWho will be blinded {17a}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe trial will be double-blind, ensuring that participants, investigators, clinicians, and outcome assessors remain unaware of treatment allocation. To maintain blinding, the probiotic and placebo products will be identical in appearance, packaging, and administration.\u003c/p\u003e\n\u003cp\u003eOnly the designated personnel at the sponsor site responsible for generating the randomization list and preparing the pre-randomized study product packages will be unblinded. These personnel will not be involved in patient care, data collection, or outcome assessment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProcedure for unblinding if needed {17b}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUnblinding will only occur to ensure patient safety, for instance, in the case of a serious adverse event (SAE) that the study intervention may have caused. When unblinding is needed, the Principal Investigator must submit a formal request to the trial\u0026rsquo;s unblinded safety monitor, who will then consult the randomization list kept by the sponsor.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData collection and management\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlans for assessment and collection of outcomes {18a}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll outcomes will be systematically monitored and documented by trained research personnel at each participating center. The following \u0026nbsp;will be assessed clinically on a daily basis: \u0026nbsp;VAP, sepsis, septic \u0026nbsp;shock, CRBSI, length of stay in the ICU, and duration of mechanical ventilation. The diagnosis of VAP will be made according to pre-defined clinical, radiological and microbiological criteria, including CPIS, quantitative cultures of tracheal aspirates or BALn and sustained changes in ventilatory parameters.\u003c/p\u003e\n\u003cp\u003eAll collected data will be entered into a secure electronic Case Report Form (eCRF) (https://data.castoredc.com/) and regularly monitored for accuracy and completeness. Investigators will ensure adherence to standardized sample collection and storage protocols to maintain consistency across all study sites.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlans to promote participant retention and complete follow-up {18b}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo guarantee high retention and complete follow-up, research staff will perform daily patient assessments during the ICU stay and strictly monitor patient compliance with the study protocol. In participants who are extubated before day 30, the follow-up will extend to 72 hours after extubation to include any late-onset VAP. The research team will contact the receiving facility if a participant is transferred to another unit or hospital to get the outcome data. Data quality control and site visits will be performed regularly to prevent missing data and to enforce the protocol.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData management {19}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll study data will be documented in a secure web based eCRF to enhance the quality and comprehensiveness of the data collected per the regulatory requirements. The participants\u0026rsquo; data will be recorded in the study daily by the trained research personnel at each site, and the information will be entered into the eCRF regularly. Data will be collected daily by trained research staff at each participating center and entered into the eCRF in real-time. To maintain data integrity, the study monitors will conduct periodic data audits. At the end of the trial, the final dataset will be locked, and a copy will be securely archived for regulatory compliance. Data handling will follow GCP guidelines, and no personally identifiable information will be included in the dataset.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConfidentiality {27}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll participant data will be handled in strict compliance with confidentiality regulations, including GCP guidelines and the General Data Protection Regulation (GDPR 2016/679). All data will be de-identified before being entered into the eCRF after assigning a unique study ID to each participant. The final dataset will be stored on a password-protected server, with access restricted to authorized personnel only. The final dataset will contain no personally identifiable information and will be stored on a password-protected server that only authorized personnel can access. Any data shared for publication or external analysis will be fully anonymized. During the study, confidentiality will be maintained, and no personal data will be disclosed to anyone outside the research team unless required by law or regulatory authorities.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlans for collection, laboratory evaluation and storage of biological specimens for genetic or molecular analysis in this trial/future use {33}\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBiological specimens will be collected for two distinct purposes: (1) microbiota composition analysis using blood, urine, and stool samples and (2) initial assessment of pathogens in the airways at enrollment/VAP diagnosis using tracheal aspirates and BAL samples. No samples will be retained for future use beyond this trial, and all will be destroyed after analysis is completed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAirways initial assessment and VAP diagnosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTracheal aspirates and BAL will be performed before enrollment and at each suspected VAP episode. All samples will undergo initial microbiological analysis at the local microbiology laboratory, and a portion will be stored at \u0026minus;80\u0026deg;C. At the end of the study, frozen samples will be shipped by air transport to the Laboratory of Immunology of Infectious Diseases of the 4th Department of Internal Medicine of ATTIKON University General Hospital (124 62 Athens, Greece) for molecular testing. For each collection, 3 mL of tracheobronchial secretions will be obtained; if secretions are scarce, 5 mL of saline will be flushed into the airways, and 3 mL will be aspirated for analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiobanking procedures\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBiological samples, including feces, blood, and urine, will be collected pre- and post-treatment (after 30 days or 72 hours after successful extubation) to study how probiotics administration affects GM\u0026rsquo;s composition (11).\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003eFecal samples will be taken pre- and post-treatment at admission and at the end of the probiotic treatment (after 30 days or after successful extubation). They should be placed immediately in the containers provided and refrigerated at +4\u0026deg;C for a maximum of 24 hours before being stored at -80\u0026deg;C until tested.\u003c/li\u003e\n \u003cli\u003eCollection of faces at the patient\u0026rsquo;s bedside.\u003c/li\u003e\n \u003cli\u003eTransfer at least 5/10 teaspoons of material with the help of the scoop placed on the inner face of the stool collection container cap.\u003c/li\u003e\n \u003cli\u003eUse the scoop to break up the fecal material.\u003c/li\u003e\n \u003cli\u003eCap the container tightly.\u003c/li\u003e\n \u003cli\u003eFreeze samples at -80\u0026deg;C.\u003c/li\u003e\n \u003cli\u003eBlood samples will be taken before and after treatment at admission and at the end of the probiotic treatment (after 30 days or 72 hours after successful extubation).\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eFor each blood collection performed:\u003c/p\u003e\n\u003col\u003e\n \u003cli\u003eThe normal aliquot will be sent to the central laboratory for the routinely scheduled laboratory tests.\u003c/li\u003e\n \u003cli\u003eOne aliquot for serum (tubes normally used for serology). The aliquots obtained will be immediately frozen at -80\u0026deg;C.\u003c/li\u003e\n \u003cli\u003eTwo aliquots of 10 ml each (total of 20 ml) will be used to isolate peripheral blood mononuclear cells (PBMCs) (blood collected in tubes containing Lithium Heparin). Isolated PBMCs will be stored at -80\u0026deg;C.\u003c/li\u003e\n \u003cli\u003eOne aliquot of whole blood (tube containing EDTA, normally used for blood counts) will be stored at -80\u0026deg;C.\u003c/li\u003e\n \u003cli\u003eUrine samples will be taken pre- and post-treatment at admission and at the end of the probiotic treatment (after 30 days or 72h after successful extubation). \u0026nbsp;The samples will be collected in urine containers. Once delivered, a portion will be used for routine screening, and the remaining will be aliquoted into 15ml falcons. The urine aliquots will be stored at -80\u0026deg;C.\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical methods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical methods for primary and secondary outcomes {20a}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDescriptive statistical analyses will be conducted. An initial univariate analysis will compare the placebo and probiotic groups across all main variables. Subsequently, a multivariate logistic regression analysis will be performed to identify variables significantly associated with VAP incidence.\u003c/p\u003e\n\u003cp\u003eFor the primary outcome (VAP incidence in the ITT population), a multivariate logistic regression model adjusted for relevant baseline covariates - including age, sex, SOFA score at admission, Charlson Comorbidity Index, baseline GCS, baseline antibiotic use, catecholamine support, nutrition type (enteral, parenteral, or mixed), baseline PEEP levels, smoking history, and hospital center) - will be used to estimate odds ratios (OR) with 95% confidence intervals (CI) comparing the probiotic and placebo groups. The analysis will be performed through a random effect model to account for the center effect.\u0026nbsp;\u003cs\u003eA\u003c/s\u003eThe analysis will be performed on the ITT population, with a sensitivity analysis on the mITT population. The mITT population comprises only patients with less than 10\u0026sup2; cfu/mL of bacterial growth in tracheobronchial secretions before starting the study drug. Commensal bacteria will not be accounted for.\u003c/p\u003e\n\u003cp\u003eFor secondary outcomes, statistical methods will include:\u003c/p\u003e\n\u003cul type=\"disc\"\u003e\n \u003cli\u003e\u003cstrong\u003eVAP incidence in the mITT population:\u003c/strong\u003e Multivariate logistic regression adjusted for baseline covariates (age, sex, SOFA score at admission, Charlson Comorbidity Index, baseline GCS, baseline antibiotic use, catecholamine support, nutrition type, baseline PEEP, smoking history, and hospital center).\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003e30-day mortality:\u003c/strong\u003e Kaplan-Meier survival analysis with log-rank test; hazard ratios (HR) with 95% CI will be estimated using multivariate Cox regression\u0026nbsp;adjusted for baseline covariates.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eSepsis, septic shock, and CRBSI incidence:\u003c/strong\u003e Multivariate logistic regression adjusted for baseline covariates.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eICU length of stay and mechanical ventilation duration:\u003c/strong\u003e Independent samples t-test (if normally distributed) or Mann-Whitney U test (if non-normally distributed); multivariable linear regression models adjusted for baseline covariates may also be applied if appropriate.\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eAll statistical tests will be two-sided, with a significance level of \u0026alpha; = 0.05. Missing data will be handled using multiple imputation techniques where applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInterim analyses {21b}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo interim analyses are pre-planned.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods for additional analyses (e.g. subgroup analyses) {20b}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eStratification Analysis for Antibiotic Use\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGiven the possible variability in antibiotic administration practices among participating centers, despite the full adherence to VAP bundles, a stratification analysis will be conducted to assess the impact of empirical antibiotic use on VAP incidence. Patients will be stratified based on:\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003eEarly Empirical Antibiotic Use: Defined as antibiotic administration within the first 48 hours of mechanical ventilation.\u003c/li\u003e\n \u003cli\u003eLate or No Empirical Antibiotic Use: Defined as antibiotic administration after 48 hours or no antibiotic exposure prior to VAP diagnosis.\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003e\u003cstrong\u003eSubgroup Analysis: Trauma vs. Stroke/Brain Hemorrhage\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs the study includes both TBI and stroke patients, a subgroup analysis will be performed to evaluate whether probiotics have a differential effect on VAP incidence between these two groups. The primary and secondary outcomes will be analyzed separately within each subgroup using logistic regression models adjusted for baseline characteristics to determine if the probiotic intervention has varying efficacy based on the underlying condition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSensitivity Analysis Adjusting for ICU Site Effect\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSince this is a multicenter trial, a mixed-effects logistic regression model will be used to adjust for potential variability across participating ICUs. ICU site will be included as a random effect, allowing for the evaluation of potential center-to-center variations in patient management, baseline VAP incidence, and intervention response. This will help ensure that observed effects are not confounded by site-specific differences in ICU practices.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods in analysis to handle protocol non-adherence and any statistical methods to handle missing data {20c}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll analyses will follow the ITT principle, meaning all randomized participants will be analyzed in their initially assigned group, regardless of protocol deviations. As per predefined criteria, a mITT analysis will also be performed, excluding patients with bacterial growth \u0026ge;10\u0026sup2; CFU/mL in tracheal aspirates or BAL at baseline. Missing data will be minimized through real-time data entry monitoring and automated validation checks in the eCRF. They are expected to be primarily related to observed patient characteristics such as age, baseline severity scores - including SOFA and Acute Physiology and Chronic Health Evaluation II (APACHE II) - comorbidities, and ICU type, rather than unobserved outcomes or treatment effects. Furthermore, when identified, missing data will be handled using multiple imputation techniques under the assumption of missing at random (MAR). Analyses will be conducted on the imputed datasets, and results will be pooled according to Rubin\u0026rsquo;s rules. In addition, sensitivity analyses will be performed using complete case analyses to evaluate the robustness of the findings.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlans to give access to the full protocol, participant level-data and statistical code {31c}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDue to the\u003cstrong\u003e\u0026nbsp;\u003cstrong\u003eItalian data protection laws and GDPR regulations\u003c/strong\u003e\u003c/strong\u003e, access to \u003cstrong\u003eparticipant-level data\u003c/strong\u003e is restricted. The full study protocol and statistical analysis plan will be made available \u003cstrong\u003eupon reasonable request\u003c/strong\u003e to authorized regulatory authorities, ethics committees, and academic institutions in compliance with legal and ethical requirements. \u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOversight and monitoring\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eComposition of the coordinating center and trial steering committee {5d}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe coordinating center will oversee the day-to-day management of the trial, including site coordination, regulatory compliance, and data monitoring. Regular meetings will be held to review trial progress and address operational challenges. The Trial Steering Committee will provide scientific oversight, ensuring protocol adherence and evaluating study milestones. \u0026nbsp;A Data Management Team will ensure accurate and timely data collection, with regular quality control checks.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAn Endpoint Adjudication Committee will independently review VAP diagnoses and key clinical outcomes for consistency. The committee will independently confirm or refute the diagnosis of VAP according to pre-specified clinical and microbiological criteria, remaining blinded to the treatment allocation. This adjudication process will serve to validate the primary outcome (VAP incidence) and ensure consistency and objectivity across participating centers. Additionally, the agreement rate between investigator-assigned and adjudicated VAP diagnoses will be analyzed as a tertiary outcome.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eComposition of the data monitoring committee, its role and reporting structure{21a}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data monitoring function of the study is performed by an independent pharmacologist responsible for ongoing safety monitoring, compliance verification, and data integrity assessments. The role of the monitor is independent of that of the trial steering committee and principal investigators. The data monitor has the responsibility of reviewing adverse events and serious adverse events related to the intervention at regular intervals, reviewing VAP incidence and other safety endpoints to detect safety issues and reporting any protocol deviations or significant findings to the trial steering committee. The data monitor makes recommendations for continuation or possible changes to the study if necessary. The steering committee will receive all reports from the data monitor and recommend any needed study changes. Regular monitoring will occur throughout the trial, with additional safety reviews conducted if specific concerns arise.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdverse event reporting and harms {22}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInvestigators will closely monitor the patient daily for any possible treatment-emergent adverse events (TEAEs) and Serious TEAEs (STEAEs). Any unfavorable and unanticipated medical occurrence in a patient receiving a pharmaceutical product that is not necessarily related to the therapy is referred to as a TEAE. The TEAEs might be a symptom, a sign or a laboratory result.\u003c/p\u003e\n\u003cp\u003eIf a TAEs meets any of the following criteria, it is defined STEAEs:\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003eResults in death\u003c/li\u003e\n \u003cli\u003eLife-threatening situation i.e. the patient is at risk of death\u003c/li\u003e\n \u003cli\u003eRequires In-patient hospitalization or prolongation of existing hospitalization\u003c/li\u003e\n \u003cli\u003ePersistent or significant disability/incapacity\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eCongenital anomaly/ birth defect\u003c/li\u003e\n \u003cli\u003ean important medical event that may not result in death, be life-threatening, or require hospitalization may be considered an SAE when, based upon appropriate medical judgment, the event may jeopardize the subject\u0026rsquo;s health and may require medical or surgical intervention to prevent one of the outcomes listed above, e.g. death.\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003e\u003cem\u003eGrading of severity\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe severity of the TEAE shall be graded as follows:\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003eMild: the TEAE is transient and well tolerated by the patient\u003c/li\u003e\n \u003cli\u003eModerate: the TEAE causes discomfort and affects the patient\u0026rsquo;s usual activities.\u003c/li\u003e\n \u003cli\u003eSevere: the TEAE affects the patient\u0026rsquo;s usual activities to an important degree and may cause disability or be life-threatening.\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003e\u003cem\u003eRelationship to the supplement\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe investigator will use the following definitions to assess the relationship between the adverse event and the probiotic administration:\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003eProbably related: strong relationship; another etiology is improbable or clearly less probable.\u003c/li\u003e\n \u003cli\u003ePossibly related: strong relationship; an alternative etiology is as probable or less probable.\u003c/li\u003e\n \u003cli\u003eProbably not related: slight or no time associated with the probiotic administration.\u003c/li\u003e\n \u003cli\u003eUnrelated: no relationship with the probiotic administration, TEAEs due to an underlying or concomitant disease, or another pharmaceutical product.\u0026nbsp;\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eInvestigators will actively look for any TEAEs that are probably related or possibly related to the administration of the probiotics. Specifically, any culture obtained by the ICU and processed by the microbiology laboratory positive for either \u003cem\u003eLactobacillus plantarum, Lactobacillus acidophilus\u003c/em\u003e, \u003cem\u003eBifidobacterium lactis\u003c/em\u003e and \u003cem\u003eSaccharomyces boulardii\u003c/em\u003e will be recorded. If the microorganism is isolated from a sterile site or is the predominant strain in a non-sterile setting, the patient will be withdrawn from the trial, and the intervention will be discontinued. \u0026nbsp;Other TAEs include allergy, abdominal distension, diarrhea, pleural effusion, deep venous thrombosis and \u003cem\u003eClostridioides Difficile\u0026nbsp;\u003c/em\u003einfection.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAbdominal distension is defined as the objective physical manifestation of an increase in abdominal girth (29). The percussion note over the abdomen will be tympanic, and the abdominal X-ray will reveal dilated bowel loops with multiple air-fluid levels (30). Diarrhea is defined as three or more bowel movements per day with loose or watery stools (Bristol chart of 6 or 7) (31).\u003c/p\u003e\n\u003cp\u003ePleural effusion is defined as the excessive accumulation of fluid in the pleural space, identified by chest X-ray or chest ultrasonography (32). Deep venous thrombosis (DVT) is defined as the formation of a \u0026nbsp;blood clot in the body\u0026rsquo;s deep veins, most commonly the leg, but it can also occur in the arms and the mesenteric and cerebral veins. Once clinical (Wells criteria) and laboratory (D-Dimer) suspicions arise, these are confirmed by point-of-care ultrasound (33).\u0026nbsp;DVT will be recorded only if clinically suspected and subsequently confirmed by diagnostic imaging (e.g., point-of-care ultrasound or formal vascular Doppler studies). No routine screening for asymptomatic DVT will be performed.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eClostridium difficile\u003c/em\u003e infection is defined as the presence of BOTH the following criteria: 1) presence of diarrhea (see previous definition), 2) positive stool test for toxigenic C. difficile or its toxins, or colonoscopic/histopathologic findings demonstrating pseudomembranous colitis (34).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFrequency and plans for auditing trial conduct {23}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAn external audit committee will be established and convened upon the completion of patient enrollment to systematically assess data quality, identify potential issues, and ensure that the statistical analysis remains unbiased. This committee will conduct a thorough review of the dataset, evaluate protocol adherence, and verify the absence of systematic errors or inconsistencies that could impact the integrity of the study findings.\u003c/p\u003e\n\u003cp\u003eIn addition to external auditing, the trial will undergo a strictly scheduled internal monitoring framework. Local investigator meetings, involving site investigators and clinical department staff, will be held every two months to review study progress, discuss protocol adherence, verify compliance with VAP prevention bundles, and address operational challenges. Furthermore, formal inter-center meetings, including all principal investigators, trial coordinators, and the lead statistician, will be conducted every three months to assess site performance, identify protocol deviations, and ensure consistency in trial implementation across study centers. Any significant concerns or findings from these meetings will be reported to the trial steering committee and, if necessary, escalated to the external audit committee for further evaluation and corrective action.\u003c/p\u003e\n\u003cp\u003eThis structured approach guarantees continuous oversight, standardization across sites, and a rigorous evaluation of study integrity, ensuring the highest scientific and ethical standards throughout the trial.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePlans for communicating important protocol amendments to relevant parties\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e(e.g. trial participants, ethical committees) {25}\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAny protocol amendments will first be discussed at the investigators\u0026apos; meeting. If a change is required, it will be submitted to the ethical committee of the coordinating center for approval before implementation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDissemination plans {31a}\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results of this study will be communicated to the scientific community and other stakeholders through peer-reviewed journal publications, presentations at international conferences, and via sites such as ClinicalTrials.gov and professional networks. \u0026nbsp;Results may also be posted on institutional and academic social media accounts to reach a wider audience.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe PROACT trial is a multicenter, randomized, double-blind, placebo-controlled study designed to rigorously evaluate the efficacy of a multi-strain probiotic formulation in the prevention of VAP among critically ill brain-injured patients. The focus on brain-injured patients reflects their increased risk of developing VAP due to prolonged mechanical ventilation (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e), impaired airway protection associated with altered consciousness (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e), and the absence of other primary infectious processes at admission (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). Furthermore, in this population, the overall prognosis is predominantly determined by the severity of neurological injury, making the prevention of secondary infections such as VAP particularly relevant to clinical outcomes (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Therefore preventing VAP is essential to minimize the duration of mechanical ventilation, reduce ICU and hospital length of stay, prevent additional systemic complications, and support functional recovery. Although VAP may not directly worsen neurological injury, it substantially contributes to ICU morbidity and may impair rehabilitation potential, especially in borderline cases.\u003c/p\u003e\u003cp\u003eConducting a clinical trial of this complexity within the ICU environment entails several methodological and operational challenges, particularly regarding patient recruitment, study product administration, data standardization, regulatory compliance, and inter-center variability in clinical practices\u0026mdash;especially antimicrobial stewardship.\u003c/p\u003e\u003cp\u003eOne of the most significant logistical and ethical challenges is the recruitment of critically ill patients in the ICU setting, where patients often lack decision-making capacity. In such cases, informed consent must be obtained from legally authorized representatives, frequently under urgent and emotionally charged circumstances. Given the acute nature of ICU admissions, timely and ethically sound consent can be difficult to secure. To address this, PROACT incorporates a streamlined consent pathway across all participating sites. Trained investigators are available around the clock to facilitate early enrolment, in accordance with international ethical standards for emergency research. This proactive consent strategy ensures that eligible patients are enrolled as early as possible without compromising ethical integrity.\u003c/p\u003e\u003cp\u003eThe administration of the investigational product, e.g. probiotics or placebo, twice daily via NGT and oropharyngeal application presents considerable logistical complexity in a high-acuity ICU environment. Proper timing, preparation, and administration must be precisely coordinated with routine care. To ensure protocol fidelity, PROACT has developed a detailed standard operating procedure for study product administration, including specific guidance for patients with altered anatomy (e.g., PEG tubes) or mucosal injury.\u003c/p\u003e\u003cp\u003eTo minimize protocol deviations, all ICU staff involved in the intervention will undergo structured, site-specific training prior to patient enrolment. In addition, a centralized monitoring system will track administration compliance, and any deviations will be logged, reviewed, and discussed during scheduled investigator meetings. This continuous oversight promotes uniformity across centres and enhances the reliability of the intervention data.\u003c/p\u003e\u003cp\u003eEnsuring data integrity and consistency across multiple centres is a core methodological priority. Variations in clinical documentation, ventilator management practices, and microbiological sampling techniques could otherwise introduce significant bias. To mitigate these risks, PROACT utilizes a centralized eCRF with embedded logic checks, range controls, and real-time data validation.\u003c/p\u003e\u003cp\u003eAll respiratory specimens, including tracheal aspirates and BAL samples, will be processed using standardized protocols and sent to a single reference microbiology laboratory in Greece for molecular and culture-based analyses. This centralized approach ensures uniform diagnostic criteria, eliminates inter-laboratory variability, and enhances diagnostic precision for the primary outcome.\u003c/p\u003e\u003cp\u003eOne of the most complex challenges in a multi-center international trial is heterogeneity in antimicrobial prescribing practices, driven by differences in local epidemiology, resistance patterns, and institutional protocols. Variability in the timing, type, and escalation of antibiotic therapy can significantly impact VAP incidence and may confound outcome assessment.\u003c/p\u003e\u003cp\u003ePROACT directly addresses this issue by implementing comprehensive daily data collection on all antimicrobial agents administered, including timing, dosing, spectrum, and clinical indications. Microbiological culture results are recorded in real time to link antibiotic use with pathogen-specific data. This granular level of antibiotic surveillance enables standardized comparisons across centres, allowing for robust adjustment in the statistical analysis and minimizing the confounding effects of antimicrobial variability.\u003c/p\u003e\u003cp\u003eGiven the multicenter, international nature of the trial, regulatory compliance presents another substantial challenge. Ethical approval must be obtained from multiple IRBs and ECs, each with unique documentation standards and approval timelines. To streamline this process, the coordinating center provides centralized regulatory support, prepares standardized ethics submission templates, and ensures alignment with international guidelines including International Council for Harmonisation (ICH)-GCP, the Declaration of Helsinki, and relevant national regulations.\u003c/p\u003e\u003cp\u003eA notable strength of the PROACT trial is its methodologically robust approach to VAP diagnosis, a historically challenging endpoint due to variability in clinical interpretation and diagnostic criteria. To reduce misclassification bias, PROACT employs a strict and objective diagnostic algorithm incorporating several diagnostic factors. All diagnoses are adjudicated using predefined criteria, ensuring diagnostic consistency and reproducibility across all sites.\u003c/p\u003e\u003cp\u003eTo preserve blinding and minimize bias, the trial employs strict allocation concealment using pre-randomized, identically packaged study products. Investigators, bedside clinicians, patients, and outcome assessors remain fully blinded throughout the trial. Oversight is maintained by a Trial Steering Committee, responsible for supervising study conduct, protocol adherence, and site performance. Additionally, an independent pharmacovigilance expert monitors for safety signals, reviews adverse events, and ensures the integrity of the study database. Regular monitoring visits and data audits are conducted to maintain high standards of compliance.\u003c/p\u003e\u003cp\u003eDespite the considerable methodological and operational complexities inherent in conducting an ICU-based, multicenter, double-blind randomized trial, the PROACT study is uniquely positioned to generate high-quality, clinically meaningful evidence on the role of probiotics in VAP prevention. Through rigorous diagnostic criteria, centralized microbiological analysis, standardized antimicrobial tracking, and robust blinding procedures, PROACT is expected to contribute valuable insights into infection prevention strategies in critically ill populations.\u003c/p\u003e\u003cp\u003e If successful, the findings may influence future clinical practice guidelines, support microbiome-targeted interventions, and inform public health policy aimed at reducing the burden of nosocomial infections in ICUs worldwide.\u003c/p\u003e"},{"header":"Trial status","content":"\u003cp\u003eProtocol version: 2.0 February 28, 2025.\u003c/p\u003e\n\u003cp\u003eEthical approval: September 25, 2023 (Project-ID: 1336/ CE -PROACT)\u003c/p\u003e\n\u003cp\u003eRecruitment initiation: Dec 1, 2023\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAnticipated recruitment finalization: March 31, 2026\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAPACHE , Acute Physiology and Chronic Health Evaluation\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBAL, Bronchoalveolar Lavage\u003c/p\u003e\n\u003cp\u003eCFU, Colony Forming Unit\u003c/p\u003e\n\u003cp\u003eCPIS, Clinical Pulmonary Infection Score\u003c/p\u003e\n\u003cp\u003eCRBSI, Catheter-Related Bloodstream Infection\u003c/p\u003e\n\u003cp\u003eDTP, Differential Time to Positivity\u003c/p\u003e\n\u003cp\u003eDVT, Deep Venous Thrombosis\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEC, Ethics Committees \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eeCRF, Electronic Case Report Form\u003c/p\u003e\n\u003cp\u003eFiO2, Fraction of Inspired Oxygen\u003c/p\u003e\n\u003cp\u003eICU, Intensive Care Unit\u003c/p\u003e\n\u003cp\u003eITT, Intention-to-Treat\u003c/p\u003e\n\u003cp\u003eIRB, Institutional Review Board\u003c/p\u003e\n\u003cp\u003eLOS, Length of Stay\u003c/p\u003e\n\u003cp\u003eGCP, Good Clinical Practice\u003c/p\u003e\n\u003cp\u003eGM, Gut Microbiota\u003c/p\u003e\n\u003cp\u003emITT, modified Intention-to-Treat\u003c/p\u003e\n\u003cp\u003eMAR, Missing at Random\u003c/p\u003e\n\u003cp\u003eNGT, Nasogastric Tube\u003c/p\u003e\n\u003cp\u003eOF, Oropharyngeal\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOM, Oral Microbiota\u003c/p\u003e\n\u003cp\u003ePEEP, Positive End-Expiratory Pressure\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePEG, Percutaneous Endoscopic Gastrostomy\u003c/p\u003e\n\u003cp\u003eSAE, Serious Adverse Event\u003c/p\u003e\n\u003cp\u003eSAPS II, Simplified Acute Physiology Score\u003c/p\u003e\n\u003cp\u003eSCFAs, Short-Chain Fatty Acids\u003c/p\u003e\n\u003cp\u003eSOFA, Sequential Organ Failure Assessment\u003c/p\u003e\n\u003cp\u003eSTEAEs, Serious Treatment-Emergent Adverse Events\u003c/p\u003e\n\u003cp\u003eTBI, Traumatic Brain Injury\u003c/p\u003e\n\u003cp\u003eTEAEs, Treatment-Emergent Adverse Events\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eVAP, Ventilator-Associated Pneumonia\u003c/p\u003e\n\u003cp\u003eVTE, Venous thromboembolism\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to express our gratitude to all the hospital personnel involved in this study, including doctors, nurses, residents, and all other supporting staff. Without their dedication and hard work, this trial would not have been possible. Their commitment to patient care and research has been essential for the successful conduct of this study.\u003c/p\u003e\n\u003cp\u003eIn addition, the authors wish to thank Prof. Evangelos Giamarellos-Bourboulis for his invaluable advice and contributions during the design and conduct of the study. In addition, the authors wish to thank Uni-Pharma, Greece for kindly providing the sachets of probiotic formula and placebo.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions {31b}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthor Contributions: Conceptualization: AC, RS, MTG and FP; Methodology: AC, MTG, RS, and FST; Validation: AC, RS, MTG, FP, FST, MF, and MC; Investigation: AC, RS, CP, NDT, SG, MR, AM, GM, AG, RA, RG, AMo, MF, MC, and VMR; Resources: FP and VMR; Writing—original draft preparation: AC and RS; Writing—review and editing: AC, RS, MTG, FP, FST, MF, and VMR; Supervision: FP, FST, and VMR; Project administration: AC and FP. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding {4}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study did not receive any external funding.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials {29}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and analyzed during this study will be available from the corresponding author upon reasonable request, in compliance with ethical and regulatory guidelines. Due to data protection regulations, individual participant data will not be publicly accessible.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate {24}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe PROACT Trial protocol and informed consent forms will be approved by each hospital’s Research Ethics Board. Researchers will carry out this study in line with the GCP and the Declaration of Helsinki. Before randomization, patients or their legal representatives shall provide written informed consent.\u003c/p\u003e\n\u003cp\u003eConsenting individuals will be made aware that if they choose to decline enrollment or withdraw from the study, it will not impact their care. Coded identification, password-protected documents and webpages, lockable filing cabinets and offices will all be used to guarantee confidentiality.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication {32}\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo identifiable personal data or images of participants will be published. As part of the informed consent process, participants or their legally authorized representatives will provide consent for the use of anonymized study data in scientific publications and presentations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests {28}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests. No financial compensation was provided to any personnel involved in the study for their contributions to this work.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eTorres A, Ewig S, Lode H, Carlet J. Hospital-acquired pneumonia in Europe. Eur Respir J. 2009 May 1;33(5):951\u0026ndash;2. \u003c/li\u003e\n\u003cli\u003eKoulenti D, Tsigou E, Rello J. Nosocomial pneumonia in 27 ICUs in Europe: perspectives from the EU-VAP/CAP study. Eur J Clin Microbiol Infect Dis. 2017 Nov 1;36(11):1999\u0026ndash;2006. \u003c/li\u003e\n\u003cli\u003eHellyer TP, Ewan V, Wilson P, Simpson AJ. The Intensive Care Society recommended bundle of interventions for the prevention of ventilator-associated pneumonia. J Intensive Care Soc. 2016 Aug;17(3):238\u0026ndash;43. \u003c/li\u003e\n\u003cli\u003eMastrogianni M, Katsoulas T, Galanis P, Korompeli A, Myrianthefs P. The Impact of Care Bundles on Ventilator-Associated Pneumonia (VAP) Prevention in Adult ICUs: A Systematic Review. Antibiotics. 2023 Feb;12(2):227. \u003c/li\u003e\n\u003cli\u003e\u0026Aacute;lvarez Lerma F, S\u0026aacute;nchez Garc\u0026iacute;a M, Lorente L, Gordo F, A\u0026ntilde;\u0026oacute;n JM, \u0026Aacute;lvarez J, et al. Guidelines for the prevention of ventilator-associated pneumonia and their implementation. The Spanish \u0026ldquo;Zero-VAP\u0026rdquo; bundle. Med Intensiva Engl Ed. 2014 May 1;38(4):226\u0026ndash;36. \u003c/li\u003e\n\u003cli\u003eMartinez-Reviejo R, Tejada S, Jansson M, Ruiz-Spinelli A, Ramirez-Estrada S, Ege D, et al. Prevention of ventilator-associated pneumonia through care bundles: A systematic review and meta-analysis. J Intensive Med. 2023 Oct 31;3(4):352\u0026ndash;64. \u003c/li\u003e\n\u003cli\u003eTsilika M, Thoma G, Aidoni Z, Tsaousi G, Fotiadis K, Stavrou G, et al. A four-probiotic preparation for ventilator-associated pneumonia in multi-trauma patients: results of a randomized clinical trial. Int J Antimicrob Agents. 2022 Jan 1;59(1):106471. \u003c/li\u003e\n\u003cli\u003eTzikos G, Tsalkatidou D, Stavrou G, Thoma G, Chorti A, Tsilika M, et al. A Four-Probiotic Regime to Reduce Surgical Site Infections in Multi-Trauma Patients. Nutrients. 2022 Jan;14(13):2620. \u003c/li\u003e\n\u003cli\u003eXin J, Ying S, Bei Y, HuiLing W, HaiYan L, YaJing C, et al. The effect of early enteral nutrition supplemented with probiotics on ventilator-associated pneumonia in patients with severe stroke. Zhongguo Weishengtaxixue Zazhi Chin J Microecol. 2019;31(2):174\u0026ndash;8. \u003c/li\u003e\n\u003cli\u003eQu D, Li W, Zhang S, Li R, Wang H, Chen B. Traumatic Brain Injury Is Associated With Both Hemorrhagic Stroke and Ischemic Stroke: A Systematic Review and Meta-Analysis. Front Neurosci [Internet]. 2022 [cited 2023 Jun 30];16. Available from: https://www.frontiersin.org/articles/10.3389/fnins.2022.814684\u003c/li\u003e\n\u003cli\u003eCorriero A, Gadaleta RM, Puntillo F, Inchingolo F, Moschetta A, Brienza N. The central role of the gut in intensive care. Crit Care. 2022 Dec 7;26(1):379. \u003c/li\u003e\n\u003cli\u003eLi C, Lu F, Chen J, Ma J, Xu N. Probiotic Supplementation Prevents the Development of Ventilator-Associated Pneumonia for Mechanically Ventilated ICU Patients: A Systematic Review and Network Meta-analysis of Randomized Controlled Trials. Front Nutr [Internet]. 2022 [cited 2022 Jul 29];9. Available from: https://www.frontiersin.org/articles/10.3389/fnut.2022.919156\u003c/li\u003e\n\u003cli\u003eTrompette A, Gollwitzer ES, Yadava K, Sichelstiel AK, Sprenger N, Ngom-Bru C, et al. Gut microbiota metabolism of dietary fiber influences allergic airway disease and hematopoiesis. Nat Med. 2014 Feb;20(2):159\u0026ndash;66. \u003c/li\u003e\n\u003cli\u003eSchuijt TJ, Lankelma JM, Scicluna BP, de Sousa e Melo F, Roelofs JJTH, de Boer JD, et al. The gut microbiota plays a protective role in the host defence against pneumococcal pneumonia. Gut. 2016 Apr;65(4):575\u0026ndash;83. \u003c/li\u003e\n\u003cli\u003eMaslowski KM, Vieira AT, Ng A, Kranich J, Sierro F, Yu D, et al. Regulation of inflammatory responses by gut microbiota and chemoattractant receptor GPR43. Nature. 2009 Oct 29;461(7268):1282\u0026ndash;6. \u003c/li\u003e\n\u003cli\u003eHayakawa M, Asahara T, Henzan N, Murakami H, Yamamoto H, Mukai N, et al. Dramatic Changes of the Gut Flora Immediately After Severe and Sudden Insults. Dig Dis Sci. 2011 Aug 1;56(8):2361\u0026ndash;5. \u003c/li\u003e\n\u003cli\u003eLankelma JM, van Vught LA, Belzer C, Schultz MJ, van der Poll T, de Vos WM, et al. Critically ill patients demonstrate large interpersonal variation in intestinal microbiota dysregulation: a pilot study. Intensive Care Med. 2017 Jan;43(1):59\u0026ndash;68. \u003c/li\u003e\n\u003cli\u003eMcDonald D, Ackermann G, Khailova L, Baird C, Heyland D, Kozar R, et al. Extreme Dysbiosis of the Microbiome in Critical Illness. mSphere. 2016 Aug;1(4):e00199-16. \u003c/li\u003e\n\u003cli\u003eJohnstone J, Meade M, Lauzier F, Marshall J, Duan E, Dionne J, et al. Effect of Probiotics on Incident Ventilator-Associated Pneumonia in Critically Ill Patients: A Randomized Clinical Trial. JAMA. 2021 Sep 21;326(11):1024\u0026ndash;33. \u003c/li\u003e\n\u003cli\u003eKalil AC, Metersky ML, Klompas M, Muscedere J, Sweeney DA, Palmer LB, et al. Management of Adults With Hospital-acquired and Ventilator-associated Pneumonia: 2016 Clinical Practice Guidelines by the Infectious Diseases Society of America and the American Thoracic Society. Clin Infect Dis Off Publ Infect Dis Soc Am. 2016 Sep 1;63(5):e61\u0026ndash;111. \u003c/li\u003e\n\u003cli\u003eVAE | PSC | NHSN | CDC [Internet]. 2023 [cited 2023 Dec 7]. Available from: https://www.cdc.gov/nhsn/psc/vae/index.html\u003c/li\u003e\n\u003cli\u003eEvans L, Rhodes A, Alhazzani W, Antonelli M, Coopersmith CM, French C, et al. Surviving Sepsis Campaign: International Guidelines for Management of Sepsis and Septic Shock 2021. Crit Care Med. 2021 Nov;49(11):e1063. \u003c/li\u003e\n\u003cli\u003eHaddadin Y, Annamaraju P, Regunath H. Central Line Associated Blood Stream Infections. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 [cited 2023 Jan 4]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK430891/\u003c/li\u003e\n\u003cli\u003eDupont H, Mentec H, Sollet JP, Bleichner G. Impact of appropriateness of initial antibiotic therapy on the outcome of ventilator-associated pneumonia. Intensive Care Med. 2001 Feb;27(2):355\u0026ndash;62. \u003c/li\u003e\n\u003cli\u003eLeukocytosis and Leukopenia | Washington Manual of Medical Therapeutics [Internet]. [cited 2023 Apr 22]. Available from: https://www.unboundmedicine.com/washingtonmanual/view/Washington-Manual-of-Medical-Therapeutics/602479/all/Leukocytosis_and_Leukopenia?refer=true\u003c/li\u003e\n\u003cli\u003eBrown DJA, Brugger H, Boyd J, Paal P. Accidental hypothermia. N Engl J Med. 2012 Nov 15;367(20):1930\u0026ndash;8. \u003c/li\u003e\n\u003cli\u003eLaupland KB. Fever in the critically ill medical patient. Crit Care Med. 2009 Jul;37(7):S273. \u003c/li\u003e\n\u003cli\u003eSchoonjans F. MedCalc. [cited 2025 May 3]. MedCalc\u0026rsquo;s Sample size for Comparison of proportions. Available from: https://www.medcalc.org/calc/sample-size-comparison-of-proportions.php\u003c/li\u003e\n\u003cli\u003eLacy BE, Cangemi D, Vazquez-Roque M. Management of Chronic Abdominal Distension and Bloating. Clin Gastroenterol Hepatol. 2021 Feb 1;19(2):219-231.e1. \u003c/li\u003e\n\u003cli\u003eNoor MohdT, Kochhar R. Acute Abdominal Distension. In: Chawla R, Todi S, editors. ICU Protocols: A stepwise approach [Internet]. India: Springer; 2012 [cited 2023 Nov 14]. p. 305\u0026ndash;9. Available from: https://doi.org/10.1007/978-81-322-0535-7_38\u003c/li\u003e\n\u003cli\u003eDionne JC, Mbuagbaw L. Diarrhea in the critically ill: definitions, epidemiology, risk factors and outcomes. Curr Opin Crit Care. 2023 Apr;29(2):138. \u003c/li\u003e\n\u003cli\u003eBediwy AS, Al-Biltagi M, Saeed NK, Bediwy HA, Elbeltagi R. Pleural effusion in critically ill patients and intensive care setting. World J Clin Cases. 2023 Feb 16;11(5):989\u0026ndash;99. \u003c/li\u003e\n\u003cli\u003eLinnemann B, Beyer-Westendorf J, Espinola-Klein C, M\u0026uuml;hlberg KS, M\u0026uuml;ller OJ, Klamroth R. Management of Deep Vein Thrombosis: An Update Based on the Revised AWMF S2k Guideline. H\u0026auml;mostaseologie. 2024 Apr;44(02):097\u0026ndash;110. \u003c/li\u003e\n\u003cli\u003eBagdasarian N, Rao K, Malani PN. Diagnosis and Treatment of Clostridium difficile in Adults: A Systematic Review. JAMA. 2015 Jan 27;313(4):398\u0026ndash;408. \u003c/li\u003e\n\u003cli\u003eAsehnoune K, Rooze P, Robba C, Bouras M, Mascia L, Cinotti R, et al. Mechanical ventilation in patients with acute brain injury: a systematic review with meta-analysis. Crit Care. 2023 Jun 6;27(1):221. \u003c/li\u003e\n\u003cli\u003eAsehnoune K, Roquilly A, Cinotti R. Respiratory Management in Patients with Severe Brain Injury. Crit Care. 2018 Mar 20;22:76. \u003c/li\u003e\n\u003cli\u003eKhan TA, Kamm S. State of the Globe: Traumatic Brain Injury and Infections: The Two-Hit Insult. J Glob Infect Dis. 2023 Nov 30;15(4):135\u0026ndash;6. \u003c/li\u003e\n\u003cli\u003eRobba C, Rebora P, Banzato E, Wiegers EJA, Stocchetti N, Menon DK, et al. Incidence, Risk Factors, and Effects on Outcome of Ventilator-Associated Pneumonia in Patients With Traumatic Brain Injury: Analysis of a Large, Multicenter, Prospective, Observational Longitudinal Study. Chest. 2020 Dec;158(6):2292\u0026ndash;303. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"trials","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"trls","sideBox":"Learn more about [Trials](http://trialsjournal.biomedcentral.com/)","snPcode":"13063","submissionUrl":"https://www.editorialmanager.com/trls","title":"Trials","twitterHandle":"MedicalEvidence","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Ventilator-associated pneumonia, probiotics, randomized controlled trial, mechanical ventilation, gut-lung axis, microbiota, dysbiosis","lastPublishedDoi":"10.21203/rs.3.rs-6813480/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6813480/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e: Ventilator-associated pneumonia (VAP) remains a significant complication among critically ill patients, with associated mortality approaching 50%. Despite the implementation of established preventive strategies, additional interventions are urgently needed to further reduce the incidence of VAP. Probiotic therapy has emerged as a promising adjunctive approach; the benefits of probiotic therapy may be more pronounced in critically ill patients without pre-existing infections. The PROACT study will evaluate the prophylactic role of probiotics in a critically ill population with acute brain injury to reduce the VAP incidence, while also exploring microbiological endpoints and mortality to refine patient selection criteria.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003cem\u003e: \u003c/em\u003eThe PROACT study is a prospective, randomized, double-blind, placebo-controlled, multicenter trial designed to evaluate the efficacy of probiotic prophylaxis in adult critically ill patients requiring invasive mechanical ventilation following acute brain injury (e.g. head trauma, ischemic or hemorrhagic stroke). Patients with suspected pulmonary aspiration or pre-existing pulmonary infections at enrollment are excluded to reduce confounding. Participants are randomized in a 1:1 ratio to receive either a placebo (glucose polymer) or a probiotic formulation containing \u003cem\u003eLactobacillus acidophilus\u003c/em\u003e LA-5, \u003cem\u003eLactobacillus plantarum\u003c/em\u003e, \u003cem\u003eBifidobacterium lactis\u003c/em\u003e BB12, and \u003cem\u003eSaccharomyces boulardii\u003c/em\u003e. The assigned intervention is administered twice daily via nasogastric tube and oropharyngeal application for up to 30 days or until Intensive Care Unit (ICU) discharge. The primary endpoint is the incidence of VAP, as defined by current international guidelines, in the intention-to-treat (ITT) population. Secondary endpoints include the incidence of VAP in the modified intention-to-treat (mITT) population, catheter-related bloodstream infections, occurrence of sepsis and septic shock, 30-day all-cause mortality, ICU length of stay and duration of mechanical ventilation. The study is powered at 80% (α=0.05) to detect a clinically meaningful difference based on effect size estimates based on the PROVAP study, requiring a total sample size of 186 patients. All participating centers implement standard VAP prevention bundles as part of routine care.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDiscussion\u003c/strong\u003e\u003cem\u003e:\u003c/em\u003e By evaluating the efficacy of probiotic therapy in preventing VAP and the impact on mortality among critically ill brain-injured patients, this trial has the potential to generate high-quality evidence supporting the incorporation of probiotics into standard VAP prevention protocols. The findings may have significant implications for clinical practice guidelines and public health policy related to infection control and microbiome-targeted interventions in the intensive care setting.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTrial Registration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRegistered at ClinicalTrials.gov (identifier: [NCT06092554]). Registered on 2023-10-15.\u003c/p\u003e","manuscriptTitle":"Probiotics to Reduce Ventilator-Associated Pneumonia: Study Protocol for a Double-Blind Multicenter Randomized International Clinical Trial (PROACT)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-18 13:11:09","doi":"10.21203/rs.3.rs-6813480/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-08-17T09:59:45+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-14T05:26:35+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-29T08:44:48+00:00","index":"","fulltext":""},{"type":"submitted","content":"Trials","date":"2025-06-26T12:49:03+00:00","index":"","fulltext":""},{"type":"decision","content":"Minor revision","date":"2025-06-25T05:28:30+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"trials","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"trls","sideBox":"Learn more about [Trials](http://trialsjournal.biomedcentral.com/)","snPcode":"13063","submissionUrl":"https://www.editorialmanager.com/trls","title":"Trials","twitterHandle":"MedicalEvidence","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f3c9d01e-cd38-45b8-8f89-b1ce0fd58aad","owner":[],"postedDate":"July 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-17T16:09:18+00:00","versionOfRecord":{"articleIdentity":"rs-6813480","link":"https://doi.org/10.1186/s13063-025-09230-w","journal":{"identity":"trials","isVorOnly":false,"title":"Trials"},"publishedOn":"2025-11-10 15:58:37","publishedOnDateReadable":"November 10th, 2025"},"versionCreatedAt":"2025-07-18 13:11:09","video":"","vorDoi":"10.1186/s13063-025-09230-w","vorDoiUrl":"https://doi.org/10.1186/s13063-025-09230-w","workflowStages":[]},"version":"v1","identity":"rs-6813480","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6813480","identity":"rs-6813480","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}