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Protocol summary for the randomised, placebo-controlled restoration of gut microflora in critical illness trial (ROCIT)

Journal: BMJ Open ManuscriptFor ID peerbmjopen-2019-035930 review only Article Type: Protocol

Date Submitted by the 25-Nov-2019 Author:

Complete List of Authors: Litton, Edward; University of Western Australia, Anstey, Matthew; Sir Charles Gairdner Hospital, ICU Broadhurst, David; Edith Cowan University Chapman, Andy; Royal Perth Hospital Currie, Andrew; Murdoch University Ferrier, Janet; St John of God Health Care, ICU Gummer, Joel; Murdoch University Higgins, Alisa; Monash University Lim, Jolene; Fiona Stanley Hospital Manning, Laurens; University of Western Australia Myer, Erina; Sir Charles Gairdner Hospital, ICU Orr, Katrina; Fiona Stanley Hospital Palermo, Anne-Marie; Fiona Stanley Hospital paparini, andrew; Murdoch University http://bmjopen.bmj.com/ Pellicano, Susan; Fiona Stanley Hospital, ICU Raby, Ed; Fiona Stanley Hospital, Infectious Diseases Rammohan, Anu; University of Western Australia, Economics Regli, Adrian; St John of God Health Care Richter, Bernhard; Medical University of Vienna, Division of Cardiology Salman, Sam; University of Western Australia Strunk, Tobias; King Edward Memorial Hospital for Women Perth, Neonatal Directorate Waterson, Sharon; Royal Perth Hospital, ICU Wibrow, Brad; Sir Charles Gairdner Hospital, ICU on October 1, 2021 by guest. Protected copyright. Wood, Fiona; Fiona Stanley Hospital; University of Western Australia

Adult intensive & critical care < ANAESTHETICS, Diagnostic microbiology Keywords: < INFECTIOUS DISEASES, Adult intensive & critical care < INTENSIVE & CRITICAL CARE

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1 2 3 4 5 BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 6 7 8 9 I, the Submitting Author has the right to grant and does grant on behalf of all authors of the Work (as defined 10 in the below author licence), an exclusive licence and/or a non-exclusive licence for contributions from authors 11 who are: i) UK Crown employees; ii) where BMJ has agreed a CC-BY licence shall apply, and/or iii) in accordance 12 with the terms applicable for US Federal Government officers or employees acting as part of their official 13 duties; on a worldwide, perpetual, irrevocable, royalty-free basis to BMJ Publishing Group Ltd (“BMJ”) its 14 licensees and where the relevant Journal is co-owned by BMJ to the co-owners of the Journal, to publish the 15 Work in this journal and any other BMJ products and to exploit all rights, as set out in our licence. 16 17 The Submitting Author accepts and understands that any supply made under these terms is made by BMJ to 18 the Submitting Author Forunless you peer are acting as review an employee on behalf only of your employer or a postgraduate 19 student of an affiliated institution which is paying any applicable article publishing charge (“APC”) for Open 20 Access articles. Where the Submitting Author wishes to make the Work available on an Open Access basis (and 21 intends to pay the relevant APC), the terms of reuse of such Open Access shall be governed by a Creative 22 Commons licence – details of these licences and which Creative Commons licence will apply to this Work are set 23 out in our licence referred to above. 24 25 Other than as permitted in any relevant BMJ Author’s Self Archiving Policies, I confirm this Work has not been 26 accepted for publication elsewhere, is not being considered for publication elsewhere and does not duplicate 27 material already published. I confirm all authors consent to publication of this Work and authorise the granting 28 of this licence. 29 30 31 32 33 34 35 36

37 http://bmjopen.bmj.com/ 38 39 40 41 42 43 44

45 on October 1, 2021 by guest. Protected copyright. 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 Protocol summary for the randomised, placebo-controlled restoration of gut microflora in 4 5 critical illness trial (ROCIT) BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 6 7 Edward Litton, Consultant, ICU, Fiona Stanley Hospital, Perth, Western Australia, Research 8 Fellow School of Medicine, University of Western Australia, Perth, Western Australia, 9 [email protected] 10 11 Matt Anstey Consultant, ICU, Sir Charles Gairdner Hospital, Perth, Western Australia, Clinical 12 Senior Lecturer, University of Western Australia, Perth, Western Australia 13 [email protected] 14 David Broadhurst, Professor, School of Science, Edith Cowan University, Joondalup 15 16 [email protected] 17 Andy Chapman, Consultant ICU, Royal Perth Hospital, Perth, Western Australia 18 [email protected] peer review only 19 Andrew Currie, Senior Lecturer, Medical, Molecular & Forensic Sciences, Murdoch 20 21 University, Perth, Western Australia [email protected] 22 Janet Ferrier, Research Coordinator, Intensive Care Unit, St John of God Hospital Subiaco 23 Perth, Western Australia [email protected] 24 Joel Gummer, Research Fellow, Health Futures Institute, Murdoch University, Perth, 25 26 Western Australia, [email protected] 27 Alisa Higgins, Research Fellow, Australian and New Zealand Intensive Care Research Centre, 28 Monash University, Melbourne, Australia [email protected] 29 Jolene Lim, Registrar, ICU, Fiona Stanley Hospital, Perth, Western Australia, 30 31 [email protected] 32 Laurens Manning, Infectious Diseases Physician, Fiona Stanley Hospital 33 University of Western Australia, [email protected] 34 Erina Myers, Research coordinator, Intensive Care Unit, Sir Charles Gairdner Hospital, Perth, 35 WA, [email protected] 36 Katrina Orr, Supervisor Pharmacist Investigational Drugs, Fiona Stanley Hospital, Murdoch, 37 http://bmjopen.bmj.com/ 38 WA 6150, [email protected] 39 Anna Palermo, Research Manager, ICU, Fiona Stanley Hospital, Perth, Western Australia 40 [email protected] 41 42 Andrew Paparini, Adjunct Senior Research Fellow, Murdoch University; Director, Chief 43 Scientist, Econumerics Pty Ltd Consultants, Perth WA, [email protected] 44 Susan Pellicano, Research Manager and ROCIT study manager, ICU, Fiona Stanley Hospital,

45 Perth, WA [email protected] on October 1, 2021 by guest. Protected copyright. 46 Ed Raby, Consultant, Infectious Diseases, Fiona Stanley Hospital, Perth, Western Australia, 47 48 [email protected] 49 Anu Rammohan, Professor, Department of Economics, University of Western Australia, 50 [email protected] 51 Adrian Regli, Consultant, ICU, St John of God Hospital, Murdoch, WA 6150 52 Clinical Associate Professor, University of Western Australia, [email protected] 53 54 Bernhard Richter, Consultant, Division of Cardiology, Medical University of Vienna, 55 Waehringer Guertel 18-20, Vienna, Austria [email protected] 56 Sam Salman, Clinical Senior Lecturer, Medical School, University of Western Australia, 57 Crawley, WA, 6009, [email protected] 58 59 Tobias Strunk, Consultant Neonatologist, Neonatal Directorate, King Edward Memorial 60 Hospital; Telethon Kids Institute, Perth, WA [email protected]

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1 2 3 4 5 Sharon Waterson, Research coordinator, Royal Perth Hospital, Perth, WA BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 6 [email protected] 7 Brad Wibrow, Intensivist, Sir Charles Gairdner Hospital, Perth, WA 8 [email protected] 9 Fiona Wood, Director of the Burns Service FSH and Burn Injury Research unit UWA 10 11 [email protected] 12 13 Corresponding Author: 14 15 16 Edward Litton FCICM, Intensive Care Unit, Fiona Stanley Hospital, Perth, WA 17 18 [email protected] peer review only 19 20 [email protected] 21 22 23 Tel: +61415 293 281 24 25 Word Count: 2072 26 27 Key Words: Gut microbiome, critical illness, probiotics 28 29 30 31 32 33 34 35 36

37 http://bmjopen.bmj.com/ 38 39 40 Abstract 41 42 Introduction 43 44 The effect of early and sustained administration of daily probiotic therapy on patients 45 on October 1, 2021 by guest. Protected copyright. 46 47 admitted to the intensive care unit (ICU) remains uncertain. 48 49 Methods and analysis 50 51 52 The restoration of gut microflora in critical illness trial (ROCIT) study is a multicentre, 53 54 randomised, placebo-controlled, parallel-group, two-sided superiority trial that will enrol 55 56 57 220 patients in five ICUs. Adult patients within 48 hours of admission to an ICU and 58 59 expected to require intensive care beyond the next calendar day will be randomised in a 1:1 60

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1 2 3 ratio to receive early and sustained Lactobacillus plantarum 299v probiotic therapy in 4 5 BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 6 addition to usual care or placebo in addition to usual care. The primary endpoint is days 7 8 alive and out of hospital to Day 60 (DAOH60). 9 10 11 Ethics and dissemination 12 13 ROCIT has been approved by the South Metropolitan Health Service Human Research Ethics 14 15 16 Committee (ref: RGS00000004) and the St John of God Health Care Human Research Ethics 17 18 Committee (ref: 1183).For The trialpeer results willreview be submitted foronly publication in a peer reviewed 19 20 journal. 21 22 23 Registration details 24 25 The trial was prospectively registered on the Australian and New Zealand Clinical Trials 26 27 28 Registry (ANZCTR 12617000783325). 29 30 31 32 33 Strengths and limitations of this study 34 35 Strengths 36

37 http://bmjopen.bmj.com/ 38 - Early and sustained administration of study drug until determination of the primary 39 40 outcome (Day-60) 41 42 - Pragmatic study design including broad eligibility criteria and administration in a 43 44

45 usual care setting on October 1, 2021 by guest. Protected copyright. 46 47 - Sample size calculation informed by consumers and local baseline data 48 49 50 - Blinded adjudication of outcomes including nosocomial infection 51 52 Limitations 53 54 55 - A requirement to deliver and assess delivery of study drug beyond ICU and hospital 56 57 discharge 58 59 60

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1 2 3 Introduction 4 5 BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 6 Patients admitted to the intensive care unit (ICU) commonly develop dysbiosis, an 7 8 imbalance in intestinal commensal microflora characterised by a decrease in the diversity of 9 10 11 commensal gut bacteria and an overgrowth of pathogenic species that is associated with 12 13 increased morbidity and mortality 1-5. Probiotics are live microorganisms that, when 14 15 6 16 administered in adequate amounts, confer a beneficial effect on the health of the host . 17 18 Probiotic therapy mayFor reduce peer the incidence review of surgical-site only infections and other post- 19 20 operative complications in patients undergoing surgery 7. In patients admitted to the ICU, 21 22 23 probiotic therapy may reduce the risk of nosocomial infections and reduce the hospital 24 25 length of stay 8-11. A recent meta-analysis of 30 randomised controlled trials (RCTs) 26 27 28 concluded that probiotic therapy was associated with a significant reduction in infection, 29 30 but no significant effect on mortality 12. However, study design heterogeneity and risk of 31 32 33 bias have precluded strong recommendations for the use of probiotics in current critical 34 35 care nutrition guidelines 13 14. Furthermore, existing RCTs have generally not addressed the 36

37 http://bmjopen.bmj.com/ 38 attributable risk of nosocomial infection, morbidity and mortality that persists after 39 40 discharge from an index ICU admission. 41 42 Amongst available probiotic strains, Lp299v is a strong candidate therapy to improve 43 44

45 outcomes in critically ill patients. Administration results in intestinal colonisation and on October 1, 2021 by guest. Protected copyright. 46 47 survival of the probiotic through the entire gastrointestinal tract, regardless of gastric pH 15- 48 49 17 50 . In otherwise healthy smokers, Lp299v therapy decreases markers of inflammation and 51 52 oxidative stress 18. In a recent landmark trial, Lp299v therapy reduced sepsis and death in 53 54 19 55 rural Indian newborns . In critically ill patients admitted to the ICU, Lp299v therapy 56 57 exhibits similar suppression of oropharyngeal colonisation with pathogenic bacteria as 58 59 60 chlorhexidine, reduces colonic colonisation with Clostridiodes difficile and attenuates

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1 2 3 markers of systemic inflammation 20-22. The possibility of specific benefit from Lp299v 4 5 BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 6 therapy in patients admitted to the ICU is supported a meta-analysis reporting that whilst 7 8 probiotic therapy appears to reduce nosocomial infection in critical illness, a significant 9 10 12 11 benefit is only evident in trials administering Lp299v . 12 13 The restoration of gut microflora in critical illness trial (ROCIT), was designed to assess 14 15 16 whether, in adult patients admitted to the ICU, early and sustained daily administration of 17 18 probiotic therapy usingFor Lactobacillus peer plantarum review 299v (Lp299v), only compared with placebo, is 19 20 associated with an increase in days alive and out of hospital to Day 60 (DAOH ). 21 60 22 23 This report describes the ROCIT protocol and statistical analysis plan. 24 25 26 27 28 Methods and analysis 29 30 Trial design 31 32 33 ROCIT is a multicentre, placebo-controlled, parallel-group, two-sided superiority trial that 34 35 will randomly allocate patients admitted to the ICU in a 1:1 ratio. Participants will receive 36

37 http://bmjopen.bmj.com/ 38 probiotics in addition to usual care, or placebo in addition to usual care. ROCIT has been 39 40 designed with reference to the Standard Protocol Items: Recommendations for Interval 41 42 Trials checklist and is informed by consumer consultation (Consumer and Community Health 43 44 23

45 Research Network, University of Western Australia, WA, 6009 . The trial was prospectively on October 1, 2021 by guest. Protected copyright. 46 47 registered on the Australian and New Zealand Clinical Trials Registry (ANZCTR 48 49 50 12617000783325). 51 52 Setting and participants 53 54 55 ROCIT will enrol 220 participants from five study sites in Western Australia. Eligible patients 56 57 are those within 48 hours of ICU admission and who are expected to remain in the ICU 58 59 60 beyond the next calendar day. ICU admission includes admission to a high-dependency area,

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1 2 3 defined as an area capable of providing invasive monitoring and a nursing ratio of no greater 4 5 BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 6 than 1:2. Patients who will be excluded include those with an absolute contraindication to 7 8 receiving medication via the enteral route, and those with one or more risk factors for 9 10 11 treatment-associated adverse effects including recent or ongoing immunosuppressive 12 13 therapy 24. The complete inclusion and exclusion criteria are provided in Table 1. The first 14 15 16 patient was enrolled on 28 July 2017 and recruitment to the planned sample size is 17 18 expected to be completedFor in peer early 2020. review only 19 20 Randomisation and blinding 21 22 23 Eligible participants are identified by members of the study and clinical teams at 24 25 participating sites. The variable-block randomisation algorithm is stratified by site and has 26 27 28 been generated using a web-based randomisation interface by an unblinded pharmacist 29 30 with no direct involvement in patient care, data collection or analysis 25. Allocation 31 32 33 concealment is maintained by assigning a unique number to each bottle of study drug 34 35 (Figure 1). The randomisation list is kept by the unblinded pharmacist who is also available 36

37 http://bmjopen.bmj.com/ 38 for unblinding at the request of the patient or treating team. After trial enrolment, the 39 40 participant is assigned the next available subject number, corresponding to the unique, 41 42 consecutively numbered bottle of study drug. 43 44

45 The active study drug, and the placebo are prepared in identically packaged capsules and on October 1, 2021 by guest. Protected copyright. 46 47 bottles by a certified facility (Health World Ltd, 741 Nudgee Road, Northgate, Qld, 4013). All 48 49 50 treating team members, participants, study staff and outcome adjudicators are blinded to 51 52 the treatment allocation. 53 54 55 Study treatments 56 57 Immediately after enrolment, a dose of study drug is administered. A single capsule of the 58 59 60 study drug is then prescribed daily, beginning the next calendar day. Instructions are

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1 2 3 provided to continue once daily administration, including after index ICU and hospital 4 5 BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 6 discharge, until Day 60, (i.e. the completion of the 60-capsule bottle). A standard operating 7 8 procedure (SOP) is provided to bedside clinical staff for the preparation and administration 9 10 11 of study drug and contains instructions for nasogastric tube administration for participants 12 13 unable to swallow capsules (see supplementary appendix). At the time of hospital discharge, 14 15 16 participants are provided with a study diary to record daily study drug administration (see 17 18 supplementary appendix).For Participants peer arereview asked to return only the completed diary along with 19 20 the study drug bottle and any remaining capsules at Day 60. 21 22 23 Participants randomly allocated to the active study arm receive a daily capsule with 20x109 24 25 colony forming units (CFUs) of Lp299v. Participants randomly allocated to the placebo arm 26 27 28 receive an identical-looking capsule of maltodextrin. Independent batch testing of the study 29 30 drug conducted by members of the study team and provided by the unblinded pharmacist 31 32 9 33 confirmed >20x10 Lp299v CFU and unrecordable Lp299v CFU in the active and placebo 34 35 capsules, respectively. 36

37 http://bmjopen.bmj.com/ 38 Study drug is transported under controlled and recorded refrigerated conditions from the 39 40 manufacturer to study sites and stored under refrigerated and monitored conditions during 41 42 the hospital stay. A cool bag is provided to patients for transport of the study drug on 43 44

45 hospital discharge and patients are advised to refrigerate the study drug as soon as they on October 1, 2021 by guest. Protected copyright. 46 47 arrive home. A clinical trials notification for ROCIT has been lodged with the Australian 48 49 50 Government Therapeutic Goods Administration (ref: CT-2017-CTN-03603-1). 51 52 Concomitant therapies 53 54 55 Participants are requested to refrain from initiating any probiotic treatment other than the 56 57 study treatment during the 60 days of study participation. Probiotics are not on the hospital 58 59 60

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1 2 3 formulary of any of the five study sites participating ROCIT. All other care is at the discretion 4 5 BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 6 of the treating teams. 7 8 Outcomes 9 10 11 The flow of participants in the study will be reported according to CONSORT criteria (Figure 12 26 13 2) . The primary outcome is days alive and out of hospital (DAOH60). DAOH is a validated 14 15 16 measure that includes death, length of stay (LOS) in hospital, need for ongoing rehabilitation 17 18 and the occurrenceFor and duration peer of hospital review readmission 27-30only. Days spent in a rehabilitation 19 20 facility or high-level nursing facility to Day 60 are considered as days in hospital. Participants 21 22 31 23 who die prior to Day 60 will be recorded as having zero DAOH60 . 24 25 Secondary endpoints include the occurrence of specified nosocomial infections (hospital- 26 27 28 acquired pneumonia, ventilator-associated pneumonia, C. difficile-associated diarrhoea, 29 30 surgical site infection, urinary tract infection, and blood stream infection) defined according 31 32 32 33 to Centre for Disease Control (CDC) criteria (supplementary appendix) . Screening for 34 35 nosocomial infection will occur by identifying each episode of initiation or change of 36

37 http://bmjopen.bmj.com/ 38 antibiotic to Day-60 and will then be assessed independently by two blinded infectious 39 40 diseases specialist clinicians by review of the medical records. Any disagreement will be 41 42 resolved by consensus. Other secondary endpoints include antibiotic-free days to Day 60, 43 44

45 ICU and hospital LOS, and ICU, hospital and 60-day mortality. Quality of life will be assessed on October 1, 2021 by guest. Protected copyright. 46 47 using the EQ 5D-5L at Day 60, administered via telephone by blinded research staff at each 48 49 50 study site (Table 2). 51 52 Data collection and management 53 54 55 Trained research coordinators will collect data at each site using a study-specific case report 56 57 form. Study data are entered into a REDCap database, a secure, web-based software 58 59 33 60 platform . Assessment of the primary outcome will include direct phone contact with

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1 2 3 participants on or shortly after Day 60 where participants are not known to be hospitalised 4 5 BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 6 or have died. Details of the occurrence and duration of an hospital readmissions will be 7 8 collected during this phone call and cross-checked against hospital medical records, and, if 9 10 11 required, general practitioner records. To ensure the accuracy and completeness of data 12 13 there will be pre-specified automatic checks and on-site data monitoring by the project 14 15 16 manager, including 100% source data verification for the primary endpoint. Screening, 17 18 baseline, daily, outcome,For adverse peer event andreview protocol deviation only data are provided in Table 2. 19 20 Sample size and power 21 22 23 A difference of four days in DAOH60 is considered meaningful by a specially convened forum 24 25 of consumers including ICU survivors and next-of-kin (Consumer and Community Health 26 27 28 Research Network, University of Western Australia, WA, 6009). Baseline DAOH60 has been 29 30 calculated using contemporary data from participating hospitals. From these data, a 31 32 33 baseline of 37 DAOH60, a standard deviation (SD) of nine, and a two-tailed 훼 =0.05, a trial of 34 35 162 participants has 80% power to detect a difference in DAOH60 of four days. After 36

37 http://bmjopen.bmj.com/ 38 inflation for non-normal distribution (20%), withdrawn consent (5%) and loss to follow up 39 40 (5%), the final sample size is 220 participants. 41 42 Statistical analysis plan 43 44

45 The primary analysis will be the intention-to-treat population, defined as all eligible and on October 1, 2021 by guest. Protected copyright. 46 47 randomised study participants, except for those who do not consent to use of the data 48 49 50 necessary to determine the primary outcome. There will be no imputation for missing data. 51 52 Normally distributed data will be presented as mean (SD), and non-normally distributed 53 54 55 data as median (interquartile range [IQR]). Comparisons will be performed using Fisher’s 56 57 exact test for categorical data, and Student t test or Wilcoxon rank sum test for normally 58 59 60 and non-normally distributed data respectively. The primary outcome (DAOH60), will be

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1 2 3 analysed using Wilcoxon rank sum test with results presented as a comparison of medians 4 5 BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 6 (IQR). A two-sided P-value of <0.05 will be considered statistically significant. Heterogeneity 7 8 between pre-specified subgroups, identified at baseline, will be assessed by fitting an 9 10 11 interaction term between treatment and subgroup. 12 13 The three subgroup pairs will be: patients with sepsis versus those without sepsis; 14 15 16 emergency versus elective ICU admissions; and surgical versus medical admissions. A per- 17 18 protocol analysis willFor be conducted peer including review all participants only with reported adherence to the 19 20 study medication for >80% of their total study duration. Planned sub-studies include 21 22 23 longitudinal evaluation of faecal microbiome and blood metabolome, and, if there is 24 25 statistically significant difference in the primary outcome, an economic evaluation of the 26 27 28 cost-effectiveness of the intervention. All analyses will be conducted using STATA/SE 13 29 30 (College Station, TX, USA). 31 32 33 Patient and public involvement 34 35 The primary outcome was chosen on the basis of published evidence of the importance 36

37 34 http://bmjopen.bmj.com/ 38 placed by patients on days spent at home . Consideration of additional outcome measures 39 40 was made in conjunction with an ICU consumer forum convened from the Consumer and 41 42 Community Health Research Network (University of Western Australia, WA, 6009). Study 43 44

45 participants are offered the opportunity to have the published study results supplied to on October 1, 2021 by guest. Protected copyright. 46 47 them directly and to be unblinded after final determination of all study outcome measures. 48 49 50 The published manuscript of the primary outcome will be made available to the Consumer 51 52 and Community Health Research Network for dissemination amongst stakeholders. 53 54 55 Data monitoring committee 56 57 The data monitoring committee (DMC) has expertise in critical care, infectious diseases and 58 59 60 trial design but are not otherwise involved in the care of study participants. The members

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1 2 3 are Nolan McDonnell BHB MBChB FANZCA MClinRes, Claire Italiano MBBS FRACP MPHTM, 4 5 BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 6 and Ravi Sonowane MBBS FCICM MPH. The DMC has reviewed and approved the study 7 8 protocol and will review all serious adverse events as they occur. The ROCIT management 9 10 11 committee will inform the DMC of any accumulating external evidence of relevance to the 12 13 ongoing conduct of the study as soon as practicable. No interim analyses are planned but 14 15 16 the DMC will reserve the right to conduct an interim analysis, or advise suspension or 17 18 termination of ongoingFor enrolment peer to the review study. only 19 20 Adverse events 21 22 23 Events that are a part of the natural history of the primary disease process or expected 24 25 complication of critical illness will not be reported as serious adverse events 35. All adverse 26 27 28 events considered to be potentially causally related to the trial will be reported. 29 30 31 32 33 Ethics and dissemination 34 35 Ethics approval 36

37 http://bmjopen.bmj.com/ 38 ROCIT has been approved by the South Metropolitan Health Service Human Research Ethics 39 40 Committee (ref: RGS00000004) and the St John of God Health Care Human Research Ethics 41 42 Committee (ref: 1183). The approved consent pathways included prospective participant 43 44

45 consent for study-eligible patients with capacity, and prospective Person Responsible on October 1, 2021 by guest. Protected copyright. 46 47 acknowledgement with deferred consent for patients who lacked capacity. 48 49 50 Dissemination 51 52 The study results will be submitted for publication in a peer reviewed journal. Study data 53 54 55 and statistical code can be accessed by contacting the corresponding author. Requests for 56 57 access will be reviewed by the named authors on a case-by-case basis. 58 59 60

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1 2 3 4 5 BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 6 7 8 9 10 11 12 13 References 14 15 16 1. Petersen C, Round JL. Defining dysbiosis and its influence on host immunity and disease. 17 Cell Microbiol 2014;16(7):1024-33. doi: 10.1111/cmi.12308 [published Online First: 18 2014/05/07]For peer review only 19 2. Xu R, Tan C, Zhu J, et al. Dysbiosis of the intestinal microbiota in neurocritically ill patients 20 and the risk for death. Critical care 2019;23(1) doi: 10.1186/s13054-019-2488-4 21 22 3. Shimizu K, Ogura H, Goto M, et al. Altered gut flora and environment in patients with 23 severe SIRS. The Journal of trauma 2006;60(1):126-33. doi: 24 10.1097/01.ta.0000197374.99755.fe 25 4. Ojima M, Motooka D, Shimizu K, et al. Metagenomic Analysis Reveals Dynamic Changes of 26 27 Whole Gut Microbiota in the Acute Phase of Intensive Care Unit Patients. Dig Dis Sci 28 2016;61(6):1628-34. doi: 10.1007/s10620-015-4011-3 [published Online First: 29 2015/12/31] 30 5. McDonald D, Ackermann G, Khailova L, et al. Extreme Dysbiosis of the Microbiome in 31 Critical Illness. mSphere 2016;1(4) doi: 10.1128/mSphere.00199-16 [published Online 32 33 First: 2016/09/08] 34 6. Food and Agriculture Organization of the United Nations, and World Health 35 Organization. Report of a Joint FAO/WHO Expert Consultation on evaluation 36 of health and nutritional properties of probiotics in food including powder milk

37 http://bmjopen.bmj.com/ 38 with live lactic acid bacteria. Cordoba, Argentina: FAO/WHO, 2001. 2001 39 7. Skonieczna-Zydecka K, Kaczmarczyk M, Loniewski I, et al. A Systematic Review, Meta- 40 Analysis, and Meta-Regression Evaluating the Efficacy and Mechanisms of Action of 41 Probiotics and Synbiotics in the Prevention of Surgical Site Infections and Surgery- 42 Related Complications. J Clin Med 2018;7(12) doi: 10.3390/jcm7120556 [published 43 44 Online First: 2018/12/19]

45 8. Kotzampassi K, Giamarellos-Bourboulis EJ, Voudouris A, et al. Benefits of a synbiotic on October 1, 2021 by guest. Protected copyright. 46 formula (Synbiotic 2000Forte) in critically Ill trauma patients: early results of a 47 randomized controlled trial. World J Surg 2006;30(10):1848-55. doi: 48 49 10.1007/s00268-005-0653-1 50 9. Barraud D, Blard C, Hein F, et al. Probiotics in the critically ill patient: a double blind, 51 randomized, placebo-controlled trial. Intensive care medicine 2010;36(9):1540-7. 52 doi: 10.1007/s00134-010-1927-0 53 10. Petrof EO, Dhaliwal R, Manzanares W, et al. Probiotics in the critically ill: a systematic 54 55 review of the randomized trial evidence. Critical care medicine 2012;40(12):3290- 56 302. doi: 10.1097/CCM.0b013e318260cc33 57 11. Mahmoodpoor A, Hamishehkar H, Asghari R, et al. Effect of a Probiotic Preparation on 58 Ventilator-Associated Pneumonia in Critically Ill Patients Admitted to the Intensive 59 60

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1 2 3 Care Unit: A Prospective Double-Blind Randomized Controlled Trial. Nutr Clin Pract 4 5 2019;34(1):156-62. doi: 10.1002/ncp.10191 [published Online First: 2018/08/09] BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 6 12. Manzanares W, Lemieux M, Langlois PL, et al. Probiotic and synbiotic therapy in critical 7 illness: a systematic review and meta-analysis. Critical care 2016;20(1):262. doi: 8 10.1186/s13054-016-1434-y 9 13. McClave SA, Taylor BE, Martindale RG, et al. Guidelines for the Provision and 10 11 Assessment of Nutrition Support Therapy in the Adult Critically Ill Patient: Society of 12 Critical Care Medicine (SCCM) and American Society for Parenteral and Enteral 13 Nutrition (A.S.P.E.N.). JPEN J Parenter Enteral Nutr 2016;40(2):159-211. doi: 14 10.1177/0148607115621863 15 16 14. Dhaliwal R, Cahill N, Lemieux M, et al. The Canadian critical care nutrition guidelines in 17 2013: an update on current recommendations and implementation strategies. Nutr 18 Clin Pract 2014;29(1):29-43.For peer doi: 10.1177/0884533613510948review only 19 15. Goossens D, Jonkers D, Russel M, et al. Survival of the probiotic, L. plantarum 299v and 20 its effects on the faecal bacterial flora, with and without gastric acid inhibition. Dig 21 22 Liver Dis 2005;37(1):44-50. [published Online First: 2005/02/11] 23 16. Johansson ML, Molin G, Jeppsson B, et al. Administration of different Lactobacillus 24 strains in fermented oatmeal soup: in vivo colonization of human intestinal mucosa 25 and effect on the indigenous flora. Appl Environ Microbiol 1993;59(1):15-20. 26 27 [published Online First: 1993/01/01] 28 17. Klarin B, Johansson ML, Molin G, et al. Adhesion of the probiotic bacterium Lactobacillus 29 plantarum 299v onto the gut mucosa in critically ill patients: a randomised open 30 trial. Critical care 2005;9(3):R285-93. doi: 10.1186/cc3522 [published Online First: 31 2005/07/01] 32 33 18. Naruszewicz M, Johansson ML, Zapolska-Downar D, et al. Effect of Lactobacillus 34 plantarum 299v on cardiovascular disease risk factors in smokers. The American 35 journal of clinical nutrition 2002;76(6):1249-55. doi: 10.1093/ajcn/76.6.1249 36 [published Online First: 2002/11/27]

37 http://bmjopen.bmj.com/ 38 19. Panigrahi P, Parida S, Nanda NC, et al. A randomized synbiotic trial to prevent sepsis 39 among infants in rural India. Nature 2017;548(7668):407-12. doi: 40 10.1038/nature23480 41 20. Klarin B, Wullt M, Palmquist I, et al. Lactobacillus plantarum 299v reduces colonisation 42 of Clostridium difficile in critically ill patients treated with antibiotics. Acta 43 44 Anaesthesiol Scand 2008;52(8):1096-102. doi: 10.1111/j.1399-6576.2008.01748.x

45 [published Online First: 2008/10/09] on October 1, 2021 by guest. Protected copyright. 46 21. Klarin B, Adolfsson A, Torstensson A, et al. Can probiotics be an alternative to 47 chlorhexidine for oral care in the mechanically ventilated patient? A multicentre, 48 49 prospective, randomised controlled open trial. Critical care 2018;22(1):272. doi: 50 10.1186/s13054-018-2209-4 [published Online First: 2018/10/29] 51 22. McNaught CE, Woodcock NP, Anderson ADG, et al. A prospective randomised trial of 52 probiotics in critically ill patients. Clinical Nutrition 2005;24(2):211-19. doi: 53 https://doi.org/10.1016/j.clnu.2004.08.008 54 55 23. Chan AW, Tetzlaff JM, Gotzsche PC, et al. SPIRIT 2013 explanation and elaboration: 56 guidance for protocols of clinical trials. Bmj 2013;346:e7586. doi: 57 10.1136/bmj.e7586 [published Online First: 2013/01/11] 58 24. Kochan P, Chmielarczyk A, Szymaniak L, et al. Lactobacillus rhamnosus administration 59 60 causes sepsis in a cardiosurgical patient--is the time right to revise probiotic safety

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1 2 3 guidelines? Clin Microbiol Infect 2011;17(10):1589-92. doi: 10.1111/j.1469- 4 5 0691.2011.03614.x [published Online First: 2011/08/19] BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 6 25. sealedenvelope.com: Clerkenwell Workshops, London EC1R 0AT, UK; [accessed 7 11/10/2019 2019. 8 26. Schulz KF, Altman DG, Moher D, et al. CONSORT 2010 statement: updated guidelines for 9 reporting parallel group randomised trials. Bmj 2010;340:c332. doi: 10 11 10.1136/bmj.c332 12 27. Ariti CA, Cleland JG, Pocock SJ, et al. Days alive and out of hospital and the patient 13 journey in patients with heart failure: Insights from the candesartan in heart failure: 14 assessment of reduction in mortality and morbidity (CHARM) program. Am Heart J 15 16 2011;162(5):900-6. doi: 10.1016/j.ahj.2011.08.003 [published Online First: 17 2011/11/19] 18 28. Myles PS, ShulmanFor MA, Heritierpeer S, et reviewal. Validation of daysonly at home as an outcome 19 measure after surgery: a prospective cohort study in Australia. BMJ Open 20 2017;7(8):e015828. doi: 10.1136/bmjopen-2017-015828 [published Online First: 21 22 2017/08/20] 23 29. Bell M, Eriksson LI, Svensson T, et al. Days at Home after Surgery: An Integrated and 24 Efficient Outcome Measure for Clinical Trials and Quality Assurance. 25 EClinicalMedicine 2019;11:18-26. doi: 10.1016/j.eclinm.2019.04.011 [published 26 27 Online First: 2019/07/19] 28 30. Jerath A, Austin PC, Wijeysundera DN. Days Alive and Out of Hospital: Validation of a 29 Patient-centered Outcome for Perioperative Medicine. Anesthesiology 30 2019;131(1):84-93. doi: 10.1097/ALN.0000000000002701 [published Online First: 31 2019/05/17] 32 33 31. Burns KE, Jacob SK, Aguirre V, et al. Stakeholder Engagement in Trial Design: Survey of 34 Visitors to Critically Ill Patients Regarding Preferences for Outcomes and Treatment 35 Options during Weaning from Mechanical Ventilation. Ann Am Thorac Soc 36 2016;13(11):1962-68. doi: 10.1513/AnnalsATS.201606-445OC [published Online

37 http://bmjopen.bmj.com/ 38 First: 2016/09/07] 39 32. CDC. National Healthcare Safety Network (NHSN) Patient Safety Component Manual. 40 Chapter 17: CDC/NHSN Surveillance Definitions for Specific Types of Infections 2019 41 33. Harris PA, Taylor R, Thielke R, et al. Research electronic data capture (REDCap)--a 42 metadata-driven methodology and workflow process for providing translational 43 44 research informatics support. J Biomed Inform 2009;42(2):377-81. doi:

45 10.1016/j.jbi.2008.08.010 on October 1, 2021 by guest. Protected copyright. 46 34. Groff AC, Colla CH, Lee TH. Days Spent at Home - A Patient-Centered Goal and Outcome. 47 The New England journal of medicine 2016;375(17):1610-12. doi: 48 49 10.1056/NEJMp1607206 [published Online First: 2016/10/27] 50 35. Cook D, Lauzier F, Rocha MG, et al. Serious adverse events in academic critical care 51 research. CMAJ : Canadian Medical Association journal = journal de l'Association 52 medicale canadienne 2008;178(9):1181-4. doi: 10.1503/cmaj.071366 53 54 55 56 57 58 Author contribution 59 60

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1 2 3 Edward Litton has made a substantial contribution to the design of the work and drafting 4 5 BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 6 the work and has given final approval for the work and agrees to be accountable for all 7 8 aspects of the work, Matt Anstey has made a substantial contribution to the design of the 9 10 11 work and revising the work and has given final approval for the work and agrees to be 12 13 accountable for all aspects of the work, David Broadhurst has made a substantial 14 15 16 contribution to the design of the work and revising the work and has given final approval for 17 18 the work and agreesFor to be accountable peer forreview all aspects of theonly work, Andy Chapman has made 19 20 a substantial contribution to the design of the work and revising the work and has given 21 22 23 final approval for the work and agrees to be accountable for all aspects of the work, Andrew 24 25 Currie has made a substantial contribution to the design of the work and revising the work 26 27 28 and has given final approval for the work and agrees to be accountable for all aspects of the 29 30 work, Janet Ferrier has made a substantial contribution to the design of the work and 31 32 33 revising the work and has given final approval for the work and agrees to be accountable for 34 35 all aspects of the work, Joel Gummer has made a substantial contribution to the design of 36

37 http://bmjopen.bmj.com/ 38 the work and revising the work and has given final approval for the work and agrees to be 39 40 accountable for all aspects of the work, Alisa Higgins has made a substantial contribution to 41 42 the design of the work and revising the work and has given final approval for the work and 43 44

45 agrees to be accountable for all aspects of the work, Jolene Lim has made a substantial on October 1, 2021 by guest. Protected copyright. 46 47 contribution to the design of the work and revising the work and has given final approval for 48 49 50 the work and agrees to be accountable for all aspects of the work, Laurens Manning has 51 52 made a substantial contribution to the design of the work and revising the work and has 53 54 55 given final approval for the work and agrees to be accountable for all aspects of the work, 56 57 Erina Myers has made a substantial contribution to the design of the work and revising the 58 59 60 work and has given final approval for the work and agrees to be accountable for all aspects

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1 2 3 of the work, Katrina Orr has made a substantial contribution to the design of the work and 4 5 BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 6 revising the work and has given final approval for the work and agrees to be accountable for 7 8 all aspects of the work, Anne-Marie Palermo has made a substantial contribution to the 9 10 11 design of the work and revising the work and has given final approval for the work and 12 13 agrees to be accountable for all aspects of the work, Andrea Paparini has made a substantial 14 15 16 contribution to the design of the work and revising the work and has given final approval for 17 18 the work and agreesFor to be accountable peer forreview all aspects of theonly work, Susan Pellicano has made 19 20 a substantial contribution to the design of the work and revising the work and has given 21 22 23 final approval for the work and agrees to be accountable for all aspects of the work , Ed 24 25 Raby has made a substantial contribution to the design of the work and revising the work 26 27 28 and has given final approval for the work and agrees to be accountable for all aspects of the 29 30 work , Anu Rammohan has made a substantial contribution to the design of the work and 31 32 33 revising the work and has given final approval for the work and agrees to be accountable for 34 35 all aspects of the work , Adrian Regli has made a substantial contribution to the design of 36

37 http://bmjopen.bmj.com/ 38 the work and revising the work and has given final approval for the work and agrees to be 39 40 accountable for all aspects of the work, Bernhard Richter has made a substantial 41 42 contribution to the design of the work and revising the work and has given final approval for 43 44

45 the work and agrees to be accountable for all aspects of the work, Sam Salman has made a on October 1, 2021 by guest. Protected copyright. 46 47 substantial contribution to the design of the work and revising the work and has given final 48 49 50 approval for the work and agrees to be accountable for all aspects of the work , Tobias 51 52 Strunk has made a substantial contribution to the design of the work and revising the work 53 54 55 and has given final approval for the work and agrees to be accountable for all aspects of the 56 57 work, Sharon Waterson has made a substantial contribution to the design of the work and 58 59 60 revising the work and has given final approval for the work and agrees to be accountable for

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1 2 3 all aspects of the work, Brad Wibrow has made a substantial contribution to the design of 4 5 BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 6 the work and revising the work and has given final approval for the work and agrees to be 7 8 accountable for all aspects of the work and Fiona Wood has made a substantial contribution 9 10 11 to the design of the work and revising the work and has given final approval for the work 12 13 and agrees to be accountable for all aspects of the work. 14 15 16 17 18 AcknowledgementsFor peer review only 19 20 The authors would like to acknowledge and thank all participants of the ROCIT study and the 21 22 23 consumers from the Consumer and Community Health Research Network (University of 24 25 Western Australia, WA, 6009) for their contribution to the study design. 26 27 28 29 30 Funding 31 32 33 ROCIT is funded by grants from the Department of Health, Government of Western 34 35 Australia Research Translation Projects, the St John of God Hospital Foundation and the 36

37 http://bmjopen.bmj.com/ 38 Fiona Wood Foundation. Study drug was supplied by Health World Ltd. The funding bodies 39 40 and Health World Ltd had no input into the design or conduct of the trial, and will have no 41 42 input into the analysis or reporting of the trial results. The study sponsor is the Fiona Stanley 43 44

45 Fremantle Hospital Group, South Metropolitan Health Service, Western Australia. on October 1, 2021 by guest. Protected copyright. 46 47 48 49 50 Competing interest 51 52 The authors declare no competing interests. 53 54 55 56 57 Table 1 Trial eligibility criteria 58 59 60 Inclusion criteria 1. Adult patient within 48 hours of admission to an ICU

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1 2 3 2. Expected to require ICU-level care beyond the next calendar 4 5 day BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 6 7 8 Exclusion criteria 1. <18 years of age 9 10 2. Absolute contraindication to receiving medication via the 11 enteral route 12 3. Known to be receiving probiotic therapy at the time of index 13 hospitalisation 14 4. Acute pancreatitis as a cause or complication of current 15 16 admission 17 5. Immunosuppression (defined as chemotherapy within the 18 For peerpreceding fourreview weeks, or receiving only ≧ 1.5mg/kg 19 methylprednisolone daily or equivalent) 20 ≦ 9 21 6. Neutropenia (neutrophil count 1x10 /L) 22 7. Prosthetic heart valve or permanent pacemaker 23 8. Death is deemed to be inevitable as a result of the current 24 acute illness AND either the treating clinician, the patient or 25 the substitute decision-maker, are not committed to full 26 27 active treatment 28 9. Enrolment is not considered in the patient’s best interest 29 10. Previously enrolled in ROCIT 30 11. Unlikely to be residing near or visiting a study centre in 60 31 32 days 33 12. Participating in a competing interventional study 34 13. Pregnancy 35 14. Admitted to hospital from a high-level nursing facility or 36 rehabilitation facility

37 http://bmjopen.bmj.com/ 38 39 ICU intensive care unit, ROCIT restoration of gut microflora in critical illness trial 40 Table 2. Study data to be collected 41 42 43 Time point Study data 44 Screening - Date of screening

45 - Inclusion and exclusion criteria on October 1, 2021 by guest. Protected copyright. 46 - Reason, if not enrolled 47 48 - Study number, patient initials, for enrolled participants 49 50 Baseline - Date and time of randomisation 51 - Date and time of ICU admission 52 - Demographic data 53 54 - ICU admission source and category 55 - Nutrition, acid suppressive therapy and antibiotics 56 - Admission APACHE II score, diagnostic code and co- 57 morbidities 58 59 - SOFA Score and components 60 - Mechanical ventilation

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1 2 3 - Vasoactive medication 4 5 - Renal replacement therapy BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 6 7 Daily during index - Patient location (ICU/HDA or ward) 8 hospitalisation - Received study drug 9 10 - Days of mechanical ventilation, vasoactive medication and 11 renal replacement therapy 12 - Days of antibiotic, antiviral and antifungal medication 13 - New infection diagnosed 14 15 16 Outcome (Day 60) - Hospital length of stay 17 - Nosocomial infection (hospital-acquired pneumonia, 18 For peerventilator-associated review pneumonia, only Clostridium difficile- 19 associated diarrhoea, surgical site infection, urinary tract 20 21 infection, and blood stream infection)* 22 - ICU length of stay 23 - ICU mortality 24 - Hospital mortality 25 - EQ-5D-5L 26 27 28 Adverse events - Description, timing, causality and resolution of adverse 29 events from randomisation to Day 60 30 31 32 Protocol deviations - Randomisation of ineligible patients, failure to comply with 33 the study protocol 34 35 ICU intensive care unit, HDA High dependency area, APACHE acute physiology and chronic 36 health evaluation, SOFA Sequential organ failure score, EQ-5D-5L five level EuroQol five- 37 http://bmjopen.bmj.com/ 38 dimension questionnaire. 39 *The pre-specified nosocomial infections will be identified according to the centre for 40 disease control definitions and are provided in the supplementary appendix 41 42 43 44 Figure legends

45 on October 1, 2021 by guest. Protected copyright. 46 Figure 1. Study drug bottle labelling 47 48 49 Figure 2. Proposed reporting of flow of trial participants 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 4 5 BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 6 7 8 9 10 11 12 13 14 15 16 17 18 For peer review only 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36

37 http://bmjopen.bmj.com/ 38 39 40 41 42 43 44

45 on October 1, 2021 by guest. Protected copyright. 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Excluded (n= x) BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 1 Reasons: 2 - Age <18 years (n=x) 3 - Absolute contraindication to enteral medication (n= x) 4 5 - Already receiving probiotics (n=x) 6 - Acute pancreatitis (n=x) 7 - Immunosuppressed (n=x) 8 - Neutropaenia (n= x) 9 - Prosthetic heart valve or permanent pacemaker (n=x) 10 - Death is deemed inevitable AND not committed to active treatment (n=x) 11 - Enrolment not in the patients’ best interest (n=x) 12 - Previously enrolled in ROCIT 13 - Unlikely to residing near or visiting a study centre in 60 days (n=x) 14 15 - Participating in a competing interventional study (n=x) 16 For peer- Pregnancy review (n=x) only 17 - Admitted to hospital from a high-level nursing or rehabilitation facility (n= x) 18 - Eligible but missed (n=x) 19 - Did not consent (n=x) 20 21 22 Eligible (n= x) 23 Eligible but not enrolled 24 Reasons: 25 - Enrolment overlooked/Clinician Unaware (n= x) 26 - Patient or next- of-kin declined to participate (n= x) 27 - Previously enrolled in this study (n= x) Randomised (n= x) 28 - Other (n= x)

29 30 31 32 33 http://bmjopen.bmj.com/ 34 35 Probiotic therapy (n=x) Placebo (n=x) 36 ¨ Received allocated intervention (n=x) ¨ Received allocated intervention (n=x) 37 38 ¨ Did not receive allocated intervention (n=x) ¨ Did not receive allocated intervention (n= x) 39 - Consent withdrawn (n=x) - Consent withdrawn (n=x) 40 - Patient was ineligible (n=x) - Patient was ineligible (n=x)

41 - on October 1, 2021 by guest. Protected copyright. 42 43 44 45 Lost to follow-up (n=x) Lost to follow-up (n=x) 46 47 Discontinued intervention (n=x) Discontinued intervention (n=x) 48 49 Consent withdrawn (n=x) Consent withdrawn (n=x) 50 Unable to locate patient (n=x) Unable to locate patient (n=x) 51 52 Re 53 54 55 Analysed (n=x) Analysed (n=x) 56 Excluded from analysis (n=x) 57 Excluded from analysis (n=x) 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 23 of 25 BMJ Open

1 2 3 ROCIT Protocol Supplementary Appendix 4 5 BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 6 7 Content Page 8 9 10 Standard Operating Procedure- nasogastric administration ………………………. 1 11 12 Nosocomial infection definitions ………………………. 2-3 13 14 Study drug diary ………………………. 4 15 16 17 18 For peer review only 19 Figure 1 Standard Operating Procedure- nasogastric administration 20 21 22 23 ROCIT Study SOP NG Admin v2.0 20171807 24 25 Nasogastric Administration of ROCIT Study Drug 26 This SOP applies only for participants in the ROCIT Study in whom there is a 27 contraindication to swallowing tablets. 28 For participants who are able to swallow tablets, the single daily ROCIT Study Drug 29 capsule can be administered as a capsule by mouth.

30 For participants who are unable to swallow tablets and in whom a nasogastric tube is present, please follow the instructions below: 31 32 1. Check nasally/orally inserted gastric tube placement and patency. 33 2. Don a pair of disposable glove and open one single capsule of ROCIT study drug 34 into an empty disposable cup. 35 3. Pour 10ml of room temperature water into the disposable cup. 36 4. Use a new tongue depressor to stir continuously as the water is added. This is key 37 as stirring while adding water will minimise clump formation. http://bmjopen.bmj.com/ 38 5. It is expected that there will be some degree of sedimentation at the bottom of the 39 cup. This is the stabiliser from the capsule and not a fault in the administration process. 40 41 6. Draw up the prepared diluted medication into a 20ml syringe enteral syringe.

42 7. Release the enteral tube cap and administer the medication according to site based protocols. 43 44 8. Flush the enteral tube with 10mL of water then replace the cap

45 9. Dispose of the administration equipment according to infection control policies. on October 1, 2021 by guest. Protected copyright. 46

47 48 49 50 51 52 53 54 55 56 57

58 59 60

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1 2 3 4 5 BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 6 7 Table 1 Summary (abbreviated) of nosocomial infection diagnostic criteria* 8 9 10 Nosocomial infection Criteria 11 12 Surgical site infection Superficial incisional: occurs within 30 days after the 13 operation and the infection involves only skin or 14 subcutaneous tissue of the incision and at least one of the 15 16 following: 17 1. Purulent drainage 18 For peer2. Organisms review isolated only 19 3. At least one of pain, tenderness, swelling, redness, 20 21 heat and superficial incision is deliberately opened by 22 surgeon 23 4. Diagnosis of superficial incisional by surgeon 24 Deep incisional: occurs within 30 days after the operation if no 25 implant or within 1 year if implant and related to the 26 27 operation and infection involves the deep tissues and at least 28 one of the following: 29 1. Purulent drainage from the deep incision, but not from 30 the organ of the surgical site 31 32 2. Deep incision spontaneously dehisces or is deliberately 33 opened by the surgeon 34 3. Abscess or other evidence of infection involving the 35 deep incision 36 4. Diagnosis of deep surgical space infection by the

37 http://bmjopen.bmj.com/ 38 surgeon 39 Organ/space: occurs within 30 days or within one year if 40 implant is in place and related to the operation and infection 41 involves any part of the anatomy other than the incision and: 42 43 1. Purulent drainage from a drain into the organ space 44 2. Organisms isolated aseptically from the organ/space

45 3. Abscess from the organ/space on October 1, 2021 by guest. Protected copyright. 46 4. Diagnosis of an organ/space infection by the surgeon 47 48 49 The CDC criteria for burns infection will be included as a 50 surgical site infection. 51 Blood stream infection Clinical criteria are only required if the organism identified is a 52 common skin contaminant 53 54 Clinical criteria (at least one of): 55 1. Fever 56 2. WBC>10,000 or <3,000 per cubic millimetre 57 3. Hypotension (SBP<90) or >25% drop in SBP 58 59 60

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1 2 3 C. difficile-associated Clinical evidence of at least one of the following: 4 5 diarrhoea 1. Pseudomembranes identified at lower gastrointestinal BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 6 endoscopy 7 2. Pathological confirmation of pseudomembranous 8 colitis 9 10 3. C difficile toxin detected in the stool 11 12 Urinary tract infection Clinical criteria of a and b must be satisfied within a two-day 13 period 14 a) Clinical criteria (at least one of): 15 16 i. fever >38.5 C 17 ii. WBC>10,000 or <3,000 per cubic millimetre 18 For peeriii. Urgency review only 19 iv. Dysuria 20 21 v. Suprapubic tenderness 22 b) Bacterial confirmation 23 vi. >105 organisms per ml of urine 24 25 26 Pneumonia Criteria a-c must be satisfied within a 48-hour period 27 a) Radiologic criteria 28 i. New infiltrate that persists for at least 24 hours 29 b) Clinical criteria (one of) 30 i. Temperature >38.5 or <35 C 31 32 ii. WBC>10,000 or <3,000 per cubic millimetre 33 c) Bacterial confirmation (at least one of) 34 i. BAL >103 CFU/ml 35 ii. Histopathological examination of lung tissue 36 iii. Positive blood culture for bacterial pathogen 37 http://bmjopen.bmj.com/ 38 identified in sputum 39 iv. Positive pleural culture with same organism in 40 sputum 41 v. Positive gram stain 42 43 vi. Heavy or moderate growth of one type of 44 pathogenic bacteria on semi-quantitative

45 sputum culture on October 1, 2021 by guest. Protected copyright. 46 Ventilator-associated pneumonia occurs where a patient is on 47 48 mechanical ventilation for > two calendar days on the date of 49 the event, with day of ventilator placement being day one, 50 and the ventilator was in place on the date of the event or the 51 day before. 52 53 54 55 WBC white blood cell, SBP systolic blood pressure, BAL bronchoalveolar lavage, CFU colony 56 forming units. *For full criteria and wording see CDC. National Healthcare Safety Network 57 (NHSN) Patient Safety Component Manual. Chapter 17: CDC/NHSN Surveillance Definitions 58 59 for Specific Types of Infections. 2019. Where any difference occurs between this summary 60 and the CDC definitions, the CDC definitions will take priority.

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1 2 3 Figure 2. Study drug diary 4 5 BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 6 7 8 9 10 11 12 13 14 15 16 17 18 For peer review only 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36

37 http://bmjopen.bmj.com/ 38 39 40 41 42 43 44

45 on October 1, 2021 by guest. Protected copyright. 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from

Study protocol for the safety and efficacy of probiotic therapy on days alive and out of hospital in adult ICU patients: The multi-centre, randomised, placebo-controlled restoration of gut microflora in critical illness trial (ROCIT).

ForJournal: peerBMJ Open review only Manuscript ID bmjopen-2019-035930.R1

Article Type: Protocol

Date Submitted by the 06-Feb-2020 Author:

Complete List of Authors: Litton, Edward; University of Western Australia, Anstey, Matthew; Sir Charles Gairdner Hospital, ICU Broadhurst, David; Edith Cowan University Chapman, Andy; Royal Perth Hospital Currie, Andrew; Murdoch University Ferrier, Janet; St John of God Health Care, ICU Gummer, Joel; Murdoch University Higgins, Alisa; Monash University Lim, Jolene; Fiona Stanley Hospital Manning, Laurens; University of Western Australia Myer, Erina; Sir Charles Gairdner Hospital, ICU Orr, Katrina; Fiona Stanley Hospital http://bmjopen.bmj.com/ Palermo, Anne-Marie; Fiona Stanley Hospital paparini, andrew; Murdoch University Pellicano, Susan; Fiona Stanley Hospital, ICU Raby, Ed; Fiona Stanley Hospital, Infectious Diseases Rammohan, Anu; University of Western Australia, Economics Regli, Adrian; St John of God Health Care Richter, Bernhard; Medical University of Vienna, Division of Cardiology Salman, Sam; University of Western Australia

Strunk, Tobias; King Edward Memorial Hospital for Women Perth, on October 1, 2021 by guest. Protected copyright. Neonatal Directorate Waterson, Sharon; Royal Perth Hospital, ICU Wibrow, Brad; Sir Charles Gairdner Hospital, ICU Wood, Fiona; Fiona Stanley Hospital; University of Western Australia

Primary Subject Intensive care Heading:

Secondary Subject Heading: Infectious diseases

Adult intensive & critical care < ANAESTHETICS, Diagnostic microbiology Keywords: < INFECTIOUS DISEASES, Adult intensive & critical care < INTENSIVE & CRITICAL CARE

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33 http://bmjopen.bmj.com/ 34 35 36 37 38 39 40

41 on October 1, 2021 by guest. Protected copyright. 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 2 of 39

1 2 3 4 5 BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 6 7 8 9 I, the Submitting Author has the right to grant and does grant on behalf of all authors of the Work (as defined 10 in the below author licence), an exclusive licence and/or a non-exclusive licence for contributions from authors 11 who are: i) UK Crown employees; ii) where BMJ has agreed a CC-BY licence shall apply, and/or iii) in accordance 12 with the terms applicable for US Federal Government officers or employees acting as part of their official 13 duties; on a worldwide, perpetual, irrevocable, royalty-free basis to BMJ Publishing Group Ltd (“BMJ”) its 14 licensees and where the relevant Journal is co-owned by BMJ to the co-owners of the Journal, to publish the 15 Work in this journal and any other BMJ products and to exploit all rights, as set out in our licence. 16 17 The Submitting Author accepts and understands that any supply made under these terms is made by BMJ to 18 the Submitting Author Forunless you peer are acting as review an employee on behalf only of your employer or a postgraduate 19 student of an affiliated institution which is paying any applicable article publishing charge (“APC”) for Open 20 Access articles. Where the Submitting Author wishes to make the Work available on an Open Access basis (and 21 intends to pay the relevant APC), the terms of reuse of such Open Access shall be governed by a Creative 22 Commons licence – details of these licences and which Creative Commons licence will apply to this Work are set 23 out in our licence referred to above. 24 25 Other than as permitted in any relevant BMJ Author’s Self Archiving Policies, I confirm this Work has not been 26 accepted for publication elsewhere, is not being considered for publication elsewhere and does not duplicate 27 material already published. I confirm all authors consent to publication of this Work and authorise the granting 28 of this licence. 29 30 31 32 33 34 35 36

37 http://bmjopen.bmj.com/ 38 39 40 41 42 43 44

45 on October 1, 2021 by guest. Protected copyright. 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 Study protocol for the safety and efficacy of probiotic therapy on days alive and out of 4 5 hospital in adult ICU patients: The multi-centre, randomised, placebo-controlled restoration BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 6 of gut microflora in critical illness trial (ROCIT). 7 8 Edward Litton, Consultant, ICU, Fiona Stanley Hospital, Perth, Western Australia, Research 9 Fellow, St John of God Hospital, Subiaco, Perth, Western Australia, 10 11 [email protected] 12 Matthew Anstey Consultant, ICU, Sir Charles Gairdner Hospital, Perth, Western Australia, 13 Clinical Senior Lecturer, University of Western Australia, Perth, Western Australia 14 [email protected] 15 16 David Broadhurst, Professor, School of Science, Edith Cowan University, Joondalup 17 [email protected] 18 Andy Chapman, ConsultantFor ICU,peer Royal Perth review Hospital, Perth, only Western Australia 19 [email protected] 20 21 Andrew Currie, Senior Lecturer, Medical, Molecular & Forensic Sciences, Murdoch 22 University, Perth, Western Australia [email protected] 23 Janet Ferrier, Research Coordinator, Intensive Care Unit, St John of God Hospital Subiaco 24 Perth, Western Australia [email protected] 25 26 Joel Gummer, Research Fellow, Health Futures Institute, Murdoch University, Perth, 27 Western Australia, [email protected] 28 Alisa Higgins, Research Fellow, Australian and New Zealand Intensive Care Research Centre, 29 Monash University, Melbourne, Australia [email protected] 30 Jolene Lim, Registrar, ICU, Fiona Stanley Hospital, Perth, Western Australia, 31 32 [email protected] 33 Laurens Manning, Infectious Diseases Physician, Fiona Stanley Hospital 34 University of Western Australia, [email protected] 35 Erina Myer, Research coordinator, Intensive Care Unit, Sir Charles Gairdner Hospital, Perth, 36 WA, [email protected] 37 http://bmjopen.bmj.com/ 38 Katrina Orr, Supervisor Pharmacist Investigational Drugs, Fiona Stanley Hospital, Murdoch, 39 WA 6150, [email protected] 40 Anne-Marie Palermo, Research Manager, ICU, Fiona Stanley Hospital, Perth, Western 41 42 Australia [email protected] 43 Andrew Paparini, Adjunct Senior Research Fellow, Murdoch University; Director, Chief 44 Scientist, Econumerics Pty Ltd Consultants, Perth WA, [email protected]

45 Susan Pellicano, Research Manager and ROCIT study manager, ICU, Fiona Stanley Hospital, on October 1, 2021 by guest. Protected copyright. 46 Perth, WA [email protected] 47 48 Ed Raby, Consultant, Infectious Diseases, Fiona Stanley Hospital, Perth, Western Australia, 49 [email protected] 50 Anu Rammohan, Professor, Department of Economics, University of Western Australia, 51 [email protected] 52 Adrian Regli, Consultant, ICU, St John of God Hospital, Murdoch, WA 6150 53 54 Clinical Associate Professor, University of Western Australia, [email protected] 55 Bernhard Richter, Consultant, Division of Cardiology, Medical University of Vienna, 56 Waehringer Guertel 18-20, Vienna, Austria [email protected] 57 Sam Salman, Clinical Senior Lecturer, Medical School, University of Western Australia, 58 59 Crawley, WA, 6009, [email protected] 60

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1 2 3 Tobias Strunk, Consultant Neonatologist, Neonatal Directorate, King Edward Memorial 4 5 Hospital; Telethon Kids Institute, Perth, WA [email protected] BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 6 7 Sharon Waterson, Research coordinator, Royal Perth Hospital, Perth, WA 8 [email protected] 9 10 Brad Wibrow, Intensivist, Sir Charles Gairdner Hospital, Perth, WA 11 [email protected] 12 Fiona Wood, Director of the Burns Service FSH and Burn Injury Research unit UWA 13 [email protected] 14 15 16 Corresponding Author: 17 18 Edward Litton FCICM,For Intensive peer Care Unit, review Fiona Stanley Hospital, only Perth, WA 19 20 [email protected] 21 22 23 [email protected] 24 25 Tel: +61415 293 281 26 27 28 Word Count: 2072 29 30 Key Words: Gut microbiome, critical illness, probiotics 31 32 33 34 35 36

37 http://bmjopen.bmj.com/ 38 39 40 41 42 Abstract 43 44 Introduction 45 on October 1, 2021 by guest. Protected copyright. 46 47 The effect of early and sustained administration of daily probiotic therapy on patients 48 49 50 admitted to the intensive care unit (ICU) remains uncertain. 51 52 Methods and analysis 53 54 The restoration of gut microflora in critical illness trial (ROCIT) study is a multicentre, 55 56 57 randomised, placebo-controlled, parallel-group, two-sided superiority trial that will enrol 58 59 220 patients in five ICUs. Adult patients within 48 hours of admission to an ICU and 60

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1 2 3 expected to require intensive care beyond the next calendar day will be randomised in a 1:1 4 5 BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 6 ratio to receive early and sustained Lactobacillus plantarum 299v probiotic therapy in 7 8 addition to usual care or placebo in addition to usual care. The primary endpoint is days 9 10 11 alive and out of hospital to Day 60 (DAOH60). 12 13 Ethics and dissemination 14 15 16 ROCIT has been approved by the South Metropolitan Health Service Human Research Ethics 17 18 Committee (ref: RGS00000004)For peer and the Streview John of God Health only Care Human Research Ethics 19 20 Committee (ref: 1183). The trial results will be submitted for publication in a peer reviewed 21 22 23 journal. 24 25 Registration details 26 27 28 The trial was prospectively registered on the Australian and New Zealand Clinical Trials 29 30 Registry (ANZCTR 12617000783325). 31 32 33 34 35 Strengths and limitations of this study 36

37 http://bmjopen.bmj.com/ 38 Strengths 39 40 - Early and sustained administration of study drug until determination of the primary 41 42 outcome (Day-60) 43 44

45 - Pragmatic study design including broad eligibility criteria and administration in a on October 1, 2021 by guest. Protected copyright. 46 47 usual care setting 48 49 50 - Sample size calculation informed by consumers and local baseline data 51 52 - Blinded adjudication of outcomes including nosocomial infection 53 54 55 Limitations 56 57 - A requirement to deliver and assess delivery of study drug beyond ICU and hospital 58 59 60 discharge

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1 2 3 4 5 BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 6 Introduction 7 8 Patients admitted to the intensive care unit (ICU) commonly develop dysbiosis, an 9 10 11 imbalance in intestinal commensal microflora characterised by a decrease in the diversity of 12 13 commensal gut bacteria and an overgrowth of pathogenic species that is associated with 14 15 1-5 16 increased morbidity and mortality . Probiotics are live microorganisms that, when 17 18 administered in adequateFor amounts, peer confer review a beneficial effect only on the health of the host 6. 19 20 Probiotic therapy may reduce the incidence of surgical-site infections and other post- 21 22 23 operative complications in patients undergoing surgery 7. In patients admitted to the ICU, 24 25 probiotic therapy may reduce the risk of nosocomial infections and reduce the hospital 26 27 8-11 28 length of stay . A recent meta-analysis of 30 randomised controlled trials (RCTs) 29 30 concluded that probiotic therapy was associated with a significant reduction in infection, 31 32 12 33 but no significant effect on mortality . However, study design heterogeneity and risk of 34 35 bias have precluded strong recommendations for the use of probiotics in current critical 36

37 13 14 http://bmjopen.bmj.com/ 38 care nutrition guidelines . Furthermore, existing RCTs have generally not addressed the 39 40 attributable risk of nosocomial infection, morbidity and mortality that persists after 41 42 discharge from an index ICU admission15. 43 44

45 Amongst available probiotic strains, Lp299v is a strong candidate therapy to improve on October 1, 2021 by guest. Protected copyright. 46 47 outcomes in critically ill patients. Administration results in intestinal colonisation and 48 49 16- 50 survival of the probiotic through the entire gastrointestinal tract, regardless of gastric pH 51 52 18. In otherwise healthy smokers, Lp299v therapy decreases markers of inflammation and 53 54 19 55 oxidative stress . In a recent landmark trial, Lp299v therapy reduced sepsis and death in 56 57 rural Indian newborns 20. In critically ill patients admitted to the ICU, Lp299v therapy 58 59 60 exhibits similar suppression of oropharyngeal colonisation with pathogenic bacteria as

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1 2 3 chlorhexidine, reduces colonic colonisation with Clostridiodes difficile and attenuates 4 5 BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 21-23 6 markers of systemic inflammation . The possibility of specific benefit from Lp299v 7 8 therapy in patients admitted to the ICU is supported by a meta-analysis reporting that whilst 9 10 11 probiotic therapy appears to reduce nosocomial infection in critical illness, a significant 12 13 benefit is only evident in trials administering Lp299v 12. However, recent evidence suggests 14 15 16 that probiotic Lactobacilli strains can directly cause bacteraemia when administered to 17 18 patients in ICU and Forthe safety peer and efficacy review of Lp229v in adult only patients admitted to the ICU 19 20 remains uncertain24. 21 22 23 The restoration of gut microflora in critical illness trial (ROCIT), was designed to assess 24 25 whether, in adult patients admitted to the ICU, early and sustained daily administration of 26 27 28 probiotic therapy using Lactobacillus plantarum 299v (Lp299v), compared with placebo, is 29 30 associated with an increase in days alive and out of hospital to Day 60 (DAOH60). 31 32 33 This report describes the ROCIT protocol and statistical analysis plan. 34 35 36

37 http://bmjopen.bmj.com/ 38 Methods and analysis 39 40 Trial design 41 42 ROCIT is a multicentre, placebo-controlled, parallel-group, two-sided superiority trial that 43 44

45 will randomly allocate patients admitted to the ICU in a 1:1 ratio. Participants will receive on October 1, 2021 by guest. Protected copyright. 46 47 probiotics in addition to usual care, or placebo in addition to usual care. ROCIT has been 48 49 50 designed with reference to the Standard Protocol Items: Recommendations for Interval 51 52 Trials checklist and is informed by consumer consultation (Consumer and Community Health 53 54 25 55 Research Network, University of Western Australia, WA, 6009 . The trial was prospectively 56 57 registered on the Australian and New Zealand Clinical Trials Registry (ANZCTR 58 59 60 12617000783325).

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1 2 3 Setting and participants 4 5 BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 6 ROCIT will enrol 220 participants from five study sites in Western Australia (see 7 8 supplementary appendix for the study site list). Eligible patients are those within 48 hours of 9 10 11 ICU admission and who are expected to remain in the ICU beyond the next calendar day. 12 13 ICU admission includes admission to a high-dependency area, defined as an area capable of 14 15 16 providing invasive monitoring and a nursing ratio of no greater than 1:2. Patients who will 17 18 be excluded includeFor those with peer an absolute review contraindication only to receiving medication via the 19 20 enteral route, and those with one or more risk factors for treatment-associated adverse 21 22 23 effects including recent or ongoing immunosuppressive therapy 26. The complete inclusion 24 25 and exclusion criteria are provided in Table 1. The first patient was enrolled on 28 July 2017 26 27 28 and recruitment to the planned sample size is expected to be completed in early 2020. 29 30 Randomisation and blinding 31 32 33 Eligible participants are identified by members of the study and clinical teams at 34 35 participating sites. This pragmatic approach, embedded in clinical care, will maximise 36

37 http://bmjopen.bmj.com/ 38 recruitment. The variable-block randomisation algorithm is stratified by site and has been 39 40 generated using a web-based randomisation interface by an unblinded pharmacist with no 41 42 direct involvement in patient care, data collection or analysis 27. Allocation concealment is 43 44

45 maintained by assigning a unique number to each bottle of study drug (Figure 1). The on October 1, 2021 by guest. Protected copyright. 46 47 randomisation list is kept by the unblinded pharmacist who is also available for unblinding 48 49 50 at the request of the patient or treating team. After trial enrolment, the participant is 51 52 assigned the next available subject number, corresponding to the unique, consecutively 53 54 55 numbered bottle of study drug. 56 57 The active study drug, and the placebo are prepared in identically packaged capsules and 58 59 60 bottles by a certified facility (Health World Ltd, 741 Nudgee Road, Northgate, Qld, 4013). All

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1 2 3 treating team members, participants, study staff and outcome adjudicators are blinded to 4 5 BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 6 the treatment allocation. 7 8 Study treatments 9 10 11 Immediately after enrolment, a dose of study drug is administered. A single capsule of the 12 13 study drug is then prescribed daily, beginning the next calendar day. Instructions are 14 15 16 provided to continue once daily administration, including after index ICU and hospital 17 18 discharge, until DayFor 60, (i.e. peerthe completion review of the 60-capsule only bottle). A standard operating 19 20 procedure (SOP) is provided to bedside clinical staff for the preparation and administration 21 22 23 of study drug and contains instructions for nasogastric tube administration for participants 24 25 unable to swallow capsules (see Supplementary Appendix Figure 1.). At the time of hospital 26 27 28 discharge, participants are provided with a study diary to record daily study drug 29 30 administration (see Supplementary Appendix Figure 2.). Participants are asked to return the 31 32 33 completed diary along with the study drug bottle and any remaining capsules at Day 60. 34 35 Participants randomly allocated to the active study arm receive a daily capsule with 20x109 36

37 http://bmjopen.bmj.com/ 38 colony forming units (CFUs) of Lp299v. Participants randomly allocated to the placebo arm 39 40 receive an identical-looking capsule of maltodextrin. Independent batch testing of the study 41 42 drug conducted by members of the study team and provided by the unblinded pharmacist 43 44 9

45 confirmed >20x10 Lp299v CFU and unrecordable Lp299v CFU in the active and placebo on October 1, 2021 by guest. Protected copyright. 46 47 capsules, respectively. 48 49 50 Study drug is transported under controlled and recorded refrigerated conditions from the 51 52 manufacturer to study sites and stored under refrigerated and monitored conditions during 53 54 55 the hospital stay. A cool bag is provided to patients for transport of the study drug on 56 57 hospital discharge and patients are advised to refrigerate the study drug as soon as they 58 59 60

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1 2 3 arrive home. A clinical trials notification for ROCIT has been lodged with the Australian 4 5 BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 6 Government Therapeutic Goods Administration (ref: CT-2017-CTN-03603-1). 7 8 Concomitant therapies 9 10 11 Participants are requested to refrain from initiating any probiotic treatment other than the 12 13 study treatment during the 60 days of study participation. Probiotics are not on the hospital 14 15 16 formulary of any of the five study sites participating ROCIT. All other care is at the discretion 17 18 of the treating teams.For peer review only 19 20 Discontinuation 21 22 23 Study drug may be discontinued at the request of the participant or treating clinician at any 24 25 stage if the participant or treating clinician suspects an adverse reaction or that continued 26 27 28 participation is not in the best interest of the participant. A suspected or confirmed severe 29 30 adverse drug reaction will result in immediate and permanent discontinuation of the study 31 32 33 medication. Study drug will also be discontinued permanently if Lactobacillus Plantarum is 34 35 grown from a sterile site or is the predominant growth from a non-sterile site. 36

37 http://bmjopen.bmj.com/ 38 Outcomes 39 40 The flow of participants in the study will be reported according to CONSORT criteria (Figure 41 42 2)28. The primary outcome is days alive and out of hospital (DAOH ). DAOH is a validated 43 60 44

45 measure that includes death, length of stay (LOS) in hospital, need for ongoing rehabilitation on October 1, 2021 by guest. Protected copyright. 46 47 and the occurrence and duration of hospital readmission 29-32. Days spent in a rehabilitation 48 49 50 facility or high-level nursing facility to Day 60 are considered as days in hospital. Participants 51 52 33 who die prior to Day 60 will be recorded as having zero DAOH60 . 53 54 55 Secondary endpoints include the occurrence of specified nosocomial infections (hospital- 56 57 acquired pneumonia, ventilator-associated pneumonia, C. difficile-associated diarrhoea, 58 59 60 surgical site infection, urinary tract infection, and blood stream infection) defined according

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1 2 3 to Centre for Disease Control (CDC) criteria (see Supplementary Appendix Table 1.) 34. 4 5 BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 6 Screening for nosocomial infection will occur by identifying each episode of initiation or 7 8 change of antibiotic to Day 60 and will then be assessed independently by two blinded 9 10 11 infectious diseases specialist clinicians by review of the medical records. Any disagreement 12 13 will be resolved by consensus. Other secondary endpoints include antibiotic-free days to 14 15 16 Day 60, ICU and hospital LOS, and ICU, hospital and 60-day mortality. Quality of life will be 17 18 assessed using the Forfive-level peer EuroQol (EQ review 5D-5L) at Day 60, only administered via telephone by 19 20 blinded research staff at each study site (Table 2). 21 22 23 Data collection and management 24 25 Trained research coordinators will collect data at each site using a study-specific case report 26 27 28 form. Study data are entered into a REDCap database, a secure, web-based software 29 30 platform 35. Assessment of the primary outcome will include direct phone contact with 31 32 33 participants on or shortly after Day 60 where participants are not known to be hospitalised 34 35 or have died. Details of the occurrence and duration of an hospital readmissions will be 36

37 http://bmjopen.bmj.com/ 38 collected during this phone call and cross-checked against hospital medical records, and, if 39 40 required, general practitioner records. To ensure the accuracy and completeness of data 41 42 there will be pre-specified automatic checks and on-site data monitoring by the project 43 44

45 manager, including 100% source data verification for the primary endpoint. Screening, on October 1, 2021 by guest. Protected copyright. 46 47 baseline, daily, outcome, adverse event and protocol deviation data are provided in Table 2. 48 49 50 The plans for collecting and storing biological specimens for analysis in ancillary studies are 51 52 provided in the supplementary appendix. 53 54 55 Sample size and power 56 57 A difference of four days in DAOH60 is considered meaningful by a specially convened forum 58 59 60 of consumers including ICU survivors and next-of-kin (Consumer and Community Health

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1 2 3 Research Network, University of Western Australia, WA, 6009). Baseline DAOH has been 4 60 5 BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 6 calculated using contemporary data from participating hospitals. From these data, a 7 8 baseline of 37 DAOH60, a standard deviation (SD) of nine, and a two-tailed 훼 =0.05, a trial of 9 10 11 162 participants has 80% power to detect a difference in DAOH60 of four days. After 12 13 inflation for non-normal distribution (20%), withdrawn consent (5%) and loss to follow up 14 15 16 (5%), the final sample size is 220 participants. 17 18 Statistical analysis planFor peer review only 19 20 The primary analysis will be the intention-to-treat population, defined as all eligible and 21 22 23 randomised study participants, except for those who do not consent to use of the data 24 25 necessary to determine the primary outcome. There will be no imputation for missing data. 26 27 28 Normally distributed data will be presented as mean (SD), and non-normally distributed 29 30 data as median (interquartile range [IQR]). Comparisons will be performed using Fisher’s 31 32 33 exact test for categorical data, and Student t test or Wilcoxon rank sum test for normally 34 35 and non-normally distributed data respectively. The primary outcome (DAOH60), will be 36

37 http://bmjopen.bmj.com/ 38 analysed using Wilcoxon rank sum test with results presented as a comparison of medians 39 40 (IQR). A two-sided P-value of <0.05 will be considered statistically significant. Heterogeneity 41 42 between pre-specified subgroups, identified at baseline, will be assessed by fitting an 43 44

45 interaction term between treatment and subgroup. on October 1, 2021 by guest. Protected copyright. 46 47 The three subgroup pairs will be: patients with sepsis versus those without sepsis; 48 49 50 emergency versus elective ICU admissions; and surgical versus medical admissions. A per- 51 52 protocol analysis will be conducted including all participants with reported adherence to the 53 54 55 study medication for >80% of their total study duration. Planned sub-studies include 56 57 longitudinal evaluation of faecal microbiome and blood metabolome, and, if there is 58 59 60 statistically significant difference in the primary outcome, an economic evaluation of the

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1 2 3 cost-effectiveness of the intervention. All analyses will be conducted using STATA/SE 13 4 5 BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 6 (College Station, TX, USA). 7 8 Patient and public involvement 9 10 11 The primary outcome was chosen on the basis of published evidence of the importance 12 13 placed by patients on days spent at home36. Consideration of additional outcome measures 14 15 16 was made in conjunction with an ICU consumer forum convened from the Consumer and 17 18 Community Health ForResearch peer Network (University review of Western only Australia, WA, 6009). Study 19 20 participants are offered the opportunity to have the published study results supplied to 21 22 23 them directly and to be unblinded after final determination of all study outcome measures. 24 25 The published manuscript of the primary outcome will be made available to the Consumer 26 27 28 and Community Health Research Network for dissemination amongst stakeholders. 29 30 Data monitoring committee 31 32 33 The data monitoring committee (DMC) has expertise in critical care, infectious diseases and 34 35 trial design but are not otherwise involved in the care of study participants and is 36

37 http://bmjopen.bmj.com/ 38 independent from competing interests. The members are Nolan McDonnell BHB MBChB 39 40 FANZCA MClinRes, Claire Italiano MBBS FRACP MPHTM, and Ravi Sonowane MBBS FCICM 41 42 MPH. The DMC has reviewed and approved the study protocol and will review all serious 43 44

45 adverse events as they occur. The ROCIT management committee will inform the DMC of on October 1, 2021 by guest. Protected copyright. 46 47 any accumulating external evidence of relevance to the ongoing conduct of the study as 48 49 50 soon as practicable. No interim analyses are planned but the DMC will reserve the right to 51 52 conduct an interim analysis, or advise suspension or termination of ongoing enrolment to 53 54 55 the study. 56 57 Adverse events 58 59 60

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1 2 3 Events that are a part of the natural history of the primary disease process or expected 4 5 BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 37 6 complication of critical illness will not be reported as serious adverse events . All adverse 7 8 events considered to be potentially causally related to the trial, and all serious adverse 9 10 11 events, will be reported (supplementary appendix Table 2). 12 13 14 15 16 Ethics and dissemination 17 18 Ethics approval For peer review only 19 20 ROCIT has been approved by the South Metropolitan Health Service Human Research Ethics 21 22 23 Committee (ref: RGS00000004) and the St John of God Health Care Human Research Ethics 24 25 Committee (ref: 1183). The approved consent pathways included prospective participant 26 27 28 consent for study-eligible patients with capacity, and prospective Person Responsible 29 30 acknowledgement with deferred consent for patients who lacked capacity. Protocol 31 32 33 modifications will be submitted to Human Research Ethics Committee review prior to 34 35 dissemination and initiation at trial sites. A copy of the consent form is provided in the 36

37 http://bmjopen.bmj.com/ 38 supplementary appendix. 39 40 Dissemination 41 42 The study results will be submitted for publication in a peer reviewed journal. Study data 43 44

45 and statistical code can be accessed by contacting the corresponding author. Requests for on October 1, 2021 by guest. Protected copyright. 46 47 access will be reviewed by the named authors on a case-by-case basis. 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 References 4 5 BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 6 1. Petersen C, Round JL. Defining dysbiosis and its influence on host immunity and disease. 7 Cell Microbiol 2014;16(7):1024-33. doi: 10.1111/cmi.12308 [published Online First: 8 2014/05/07] 9 2. Xu R, Tan C, Zhu J, et al. Dysbiosis of the intestinal microbiota in neurocritically ill patients 10 11 and the risk for death. Critical care 2019;23(1) doi: 10.1186/s13054-019-2488-4 12 3. Shimizu K, Ogura H, Goto M, et al. Altered gut flora and environment in patients with 13 severe SIRS. The Journal of trauma 2006;60(1):126-33. doi: 14 10.1097/01.ta.0000197374.99755.fe 15 16 4. Ojima M, Motooka D, Shimizu K, et al. Metagenomic Analysis Reveals Dynamic Changes of 17 Whole Gut Microbiota in the Acute Phase of Intensive Care Unit Patients. Dig Dis Sci 18 2016;61(6):1628-34.For peerdoi: 10.1007/s10620-015-4011-3 review only [published Online First: 19 2015/12/31] 20 5. McDonald D, Ackermann G, Khailova L, et al. Extreme Dysbiosis of the Microbiome in 21 22 Critical Illness. mSphere 2016;1(4) doi: 10.1128/mSphere.00199-16 [published Online 23 First: 2016/09/08] 24 6. Food and Agriculture Organization of the United Nations, and World Health 25 Organization. Report of a Joint FAO/WHO Expert Consultation on evaluation 26 27 of health and nutritional properties of probiotics in food including powder milk 28 with live lactic acid bacteria. Cordoba, Argentina: FAO/WHO, 2001. 2001 29 7. Skonieczna-Zydecka K, Kaczmarczyk M, Loniewski I, et al. A Systematic Review, Meta- 30 Analysis, and Meta-Regression Evaluating the Efficacy and Mechanisms of Action of 31 Probiotics and Synbiotics in the Prevention of Surgical Site Infections and Surgery- 32 33 Related Complications. J Clin Med 2018;7(12) doi: 10.3390/jcm7120556 [published 34 Online First: 2018/12/19] 35 8. Kotzampassi K, Giamarellos-Bourboulis EJ, Voudouris A, et al. Benefits of a synbiotic 36 formula (Synbiotic 2000Forte) in critically Ill trauma patients: early results of a

37 http://bmjopen.bmj.com/ 38 randomized controlled trial. World J Surg 2006;30(10):1848-55. doi: 39 10.1007/s00268-005-0653-1 40 9. Barraud D, Blard C, Hein F, et al. Probiotics in the critically ill patient: a double blind, 41 randomized, placebo-controlled trial. Intensive care medicine 2010;36(9):1540-7. 42 doi: 10.1007/s00134-010-1927-0 43 44 10. Petrof EO, Dhaliwal R, Manzanares W, et al. Probiotics in the critically ill: a systematic

45 review of the randomized trial evidence. Critical care medicine 2012;40(12):3290- on October 1, 2021 by guest. Protected copyright. 46 302. doi: 10.1097/CCM.0b013e318260cc33 47 11. Mahmoodpoor A, Hamishehkar H, Asghari R, et al. Effect of a Probiotic Preparation on 48 49 Ventilator-Associated Pneumonia in Critically Ill Patients Admitted to the Intensive 50 Care Unit: A Prospective Double-Blind Randomized Controlled Trial. Nutr Clin Pract 51 2019;34(1):156-62. doi: 10.1002/ncp.10191 [published Online First: 2018/08/09] 52 12. Manzanares W, Lemieux M, Langlois PL, et al. Probiotic and synbiotic therapy in critical 53 illness: a systematic review and meta-analysis. Critical care 2016;20(1):262. doi: 54 55 10.1186/s13054-016-1434-y 56 13. McClave SA, Taylor BE, Martindale RG, et al. Guidelines for the Provision and 57 Assessment of Nutrition Support Therapy in the Adult Critically Ill Patient: Society of 58 Critical Care Medicine (SCCM) and American Society for Parenteral and Enteral 59 60

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1 2 3 Nutrition (A.S.P.E.N.). JPEN J Parenter Enteral Nutr 2016;40(2):159-211. doi: 4 5 10.1177/0148607115621863 BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 6 14. Dhaliwal R, Cahill N, Lemieux M, et al. The Canadian critical care nutrition guidelines in 7 2013: an update on current recommendations and implementation strategies. Nutr 8 Clin Pract 2014;29(1):29-43. doi: 10.1177/0884533613510948 9 15. Ho KM, Kalgudi S, Corbett JM, et al. Gut microbiota in surgical and critically ill patients. 10 11 Anaesthesia and intensive care 2020:310057X20903732. doi: 12 10.1177/0310057X20903732 [published Online First: 2020/03/07] 13 16. Goossens D, Jonkers D, Russel M, et al. Survival of the probiotic, L. plantarum 299v and 14 its effects on the faecal bacterial flora, with and without gastric acid inhibition. Dig 15 16 Liver Dis 2005;37(1):44-50. [published Online First: 2005/02/11] 17 17. Johansson ML, Molin G, Jeppsson B, et al. Administration of different Lactobacillus 18 strains in fermentedFor peeroatmeal soup: review in vivo colonization only of human intestinal mucosa 19 and effect on the indigenous flora. Appl Environ Microbiol 1993;59(1):15-20. 20 [published Online First: 1993/01/01] 21 22 18. Klarin B, Johansson ML, Molin G, et al. Adhesion of the probiotic bacterium Lactobacillus 23 plantarum 299v onto the gut mucosa in critically ill patients: a randomised open 24 trial. Critical care 2005;9(3):R285-93. doi: 10.1186/cc3522 [published Online First: 25 2005/07/01] 26 27 19. Naruszewicz M, Johansson ML, Zapolska-Downar D, et al. Effect of Lactobacillus 28 plantarum 299v on cardiovascular disease risk factors in smokers. The American 29 journal of clinical nutrition 2002;76(6):1249-55. doi: 10.1093/ajcn/76.6.1249 30 [published Online First: 2002/11/27] 31 20. Panigrahi P, Parida S, Nanda NC, et al. A randomized synbiotic trial to prevent sepsis 32 33 among infants in rural India. Nature 2017;548(7668):407-12. doi: 34 10.1038/nature23480 35 21. Klarin B, Wullt M, Palmquist I, et al. Lactobacillus plantarum 299v reduces colonisation 36 of Clostridium difficile in critically ill patients treated with antibiotics. Acta

37 http://bmjopen.bmj.com/ 38 Anaesthesiol Scand 2008;52(8):1096-102. doi: 10.1111/j.1399-6576.2008.01748.x 39 [published Online First: 2008/10/09] 40 22. Klarin B, Adolfsson A, Torstensson A, et al. Can probiotics be an alternative to 41 chlorhexidine for oral care in the mechanically ventilated patient? A multicentre, 42 prospective, randomised controlled open trial. Critical care 2018;22(1):272. doi: 43 44 10.1186/s13054-018-2209-4 [published Online First: 2018/10/29]

45 23. McNaught CE, Woodcock NP, Anderson ADG, et al. A prospective randomised trial of on October 1, 2021 by guest. Protected copyright. 46 probiotics in critically ill patients. Clinical Nutrition 2005;24(2):211-19. doi: 47 https://doi.org/10.1016/j.clnu.2004.08.008 48 49 24. Yelin I, Flett KB, Merakou C, et al. Genomic and epidemiological evidence of bacterial 50 transmission from probiotic capsule to blood in ICU patients. Nat Med 51 2019;25(11):1728-32. doi: 10.1038/s41591-019-0626-9 [published Online First: 52 2019/11/09] 53 25. Chan AW, Tetzlaff JM, Gotzsche PC, et al. SPIRIT 2013 explanation and elaboration: 54 55 guidance for protocols of clinical trials. Bmj 2013;346:e7586. doi: 56 10.1136/bmj.e7586 [published Online First: 2013/01/11] 57 26. Kochan P, Chmielarczyk A, Szymaniak L, et al. Lactobacillus rhamnosus administration 58 causes sepsis in a cardiosurgical patient--is the time right to revise probiotic safety 59 60

14 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 17 of 39 BMJ Open

1 2 3 guidelines? Clin Microbiol Infect 2011;17(10):1589-92. doi: 10.1111/j.1469- 4 5 0691.2011.03614.x [published Online First: 2011/08/19] BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 6 27. sealedenvelope.com: Clerkenwell Workshops, London EC1R 0AT, UK; [accessed 7 11/10/2019 2019. 8 28. Schulz KF, Altman DG, Moher D, et al. CONSORT 2010 statement: updated guidelines for 9 reporting parallel group randomised trials. Bmj 2010;340:c332. doi: 10 11 10.1136/bmj.c332 12 29. Ariti CA, Cleland JG, Pocock SJ, et al. Days alive and out of hospital and the patient 13 journey in patients with heart failure: Insights from the candesartan in heart failure: 14 assessment of reduction in mortality and morbidity (CHARM) program. Am Heart J 15 16 2011;162(5):900-6. doi: 10.1016/j.ahj.2011.08.003 [published Online First: 17 2011/11/19] 18 30. Myles PS, ShulmanFor MA, Heritierpeer S, et reviewal. Validation of daysonly at home as an outcome 19 measure after surgery: a prospective cohort study in Australia. BMJ Open 20 2017;7(8):e015828. doi: 10.1136/bmjopen-2017-015828 [published Online First: 21 22 2017/08/20] 23 31. Bell M, Eriksson LI, Svensson T, et al. Days at Home after Surgery: An Integrated and 24 Efficient Outcome Measure for Clinical Trials and Quality Assurance. 25 EClinicalMedicine 2019;11:18-26. doi: 10.1016/j.eclinm.2019.04.011 [published 26 27 Online First: 2019/07/19] 28 32. Jerath A, Austin PC, Wijeysundera DN. Days Alive and Out of Hospital: Validation of a 29 Patient-centered Outcome for Perioperative Medicine. Anesthesiology 30 2019;131(1):84-93. doi: 10.1097/ALN.0000000000002701 [published Online First: 31 2019/05/17] 32 33 33. Burns KE, Jacob SK, Aguirre V, et al. Stakeholder Engagement in Trial Design: Survey of 34 Visitors to Critically Ill Patients Regarding Preferences for Outcomes and Treatment 35 Options during Weaning from Mechanical Ventilation. Ann Am Thorac Soc 36 2016;13(11):1962-68. doi: 10.1513/AnnalsATS.201606-445OC [published Online

37 http://bmjopen.bmj.com/ 38 First: 2016/09/07] 39 34. CDC. National Healthcare Safety Network (NHSN) Patient Safety Component Manual. 40 Chapter 17: CDC/NHSN Surveillance Definitions for Specific Types of Infections 2019 41 35. Harris PA, Taylor R, Thielke R, et al. Research electronic data capture (REDCap)--a 42 metadata-driven methodology and workflow process for providing translational 43 44 research informatics support. J Biomed Inform 2009;42(2):377-81. doi:

45 10.1016/j.jbi.2008.08.010 on October 1, 2021 by guest. Protected copyright. 46 36. Groff AC, Colla CH, Lee TH. Days Spent at Home - A Patient-Centered Goal and Outcome. 47 The New England journal of medicine 2016;375(17):1610-12. doi: 48 49 10.1056/NEJMp1607206 [published Online First: 2016/10/27] 50 37. Cook D, Lauzier F, Rocha MG, et al. Serious adverse events in academic critical care 51 research. CMAJ : Canadian Medical Association journal = journal de l'Association 52 medicale canadienne 2008;178(9):1181-4. doi: 10.1503/cmaj.071366 53 54 55 56 57 58 Author contribution 59 60

15 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 18 of 39

1 2 3 Edward Litton has made a substantial contribution to the design of the work and drafting 4 5 BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 6 the work and has given final approval for the work and agrees to be accountable for all 7 8 aspects of the work, Matt Anstey has made a substantial contribution to the design of the 9 10 11 work and revising the work and has given final approval for the work and agrees to be 12 13 accountable for all aspects of the work, David Broadhurst has made a substantial 14 15 16 contribution to the design of the work and revising the work and has given final approval for 17 18 the work and agreesFor to be accountable peer forreview all aspects of theonly work, Andy Chapman has made 19 20 a substantial contribution to the design of the work and revising the work and has given 21 22 23 final approval for the work and agrees to be accountable for all aspects of the work, Andrew 24 25 Currie has made a substantial contribution to the design of the work and revising the work 26 27 28 and has given final approval for the work and agrees to be accountable for all aspects of the 29 30 work, Janet Ferrier has made a substantial contribution to the design of the work and 31 32 33 revising the work and has given final approval for the work and agrees to be accountable for 34 35 all aspects of the work, Joel Gummer has made a substantial contribution to the design of 36

37 http://bmjopen.bmj.com/ 38 the work and revising the work and has given final approval for the work and agrees to be 39 40 accountable for all aspects of the work, Alisa Higgins has made a substantial contribution to 41 42 the design of the work and revising the work and has given final approval for the work and 43 44

45 agrees to be accountable for all aspects of the work, Jolene Lim has made a substantial on October 1, 2021 by guest. Protected copyright. 46 47 contribution to the design of the work and revising the work and has given final approval for 48 49 50 the work and agrees to be accountable for all aspects of the work, Laurens Manning has 51 52 made a substantial contribution to the design of the work and revising the work and has 53 54 55 given final approval for the work and agrees to be accountable for all aspects of the work, 56 57 Erina Myer has made a substantial contribution to the design of the work and revising the 58 59 60 work and has given final approval for the work and agrees to be accountable for all aspects

16 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 19 of 39 BMJ Open

1 2 3 of the work, Katrina Orr has made a substantial contribution to the design of the work and 4 5 BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 6 revising the work and has given final approval for the work and agrees to be accountable for 7 8 all aspects of the work, Anne-Marie Palermo has made a substantial contribution to the 9 10 11 design of the work and revising the work and has given final approval for the work and 12 13 agrees to be accountable for all aspects of the work, Andrew Paparini has made a 14 15 16 substantial contribution to the design of the work and revising the work and has given final 17 18 approval for the workFor and agrees peer to be accountablereview for all only aspects of the work, Susan 19 20 Pellicano has made a substantial contribution to the design of the work and revising the 21 22 23 work and has given final approval for the work and agrees to be accountable for all aspects 24 25 of the work , Ed Raby has made a substantial contribution to the design of the work and 26 27 28 revising the work and has given final approval for the work and agrees to be accountable for 29 30 all aspects of the work , Anu Rammohan has made a substantial contribution to the design 31 32 33 of the work and revising the work and has given final approval for the work and agrees to be 34 35 accountable for all aspects of the work, Adrian Regli has made a substantial contribution to 36

37 http://bmjopen.bmj.com/ 38 the design of the work and revising the work and has given final approval for the work and 39 40 agrees to be accountable for all aspects of the work, Bernhard Richter has made a 41 42 substantial contribution to the design of the work and revising the work and has given final 43 44

45 approval for the work and agrees to be accountable for all aspects of the work, Sam Salman on October 1, 2021 by guest. Protected copyright. 46 47 has made a substantial contribution to the design of the work and revising the work and has 48 49 50 given final approval for the work and agrees to be accountable for all aspects of the work , 51 52 Tobias Strunk has made a substantial contribution to the design of the work and revising the 53 54 55 work and has given final approval for the work and agrees to be accountable for all aspects 56 57 of the work, Sharon Waterson has made a substantial contribution to the design of the work 58 59 60 and revising the work and has given final approval for the work and agrees to be

17 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 20 of 39

1 2 3 accountable for all aspects of the work, Brad Wibrow has made a substantial contribution to 4 5 BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 6 the design of the work and revising the work and has given final approval for the work and 7 8 agrees to be accountable for all aspects of the work and Fiona Wood has made a substantial 9 10 11 contribution to the design of the work and revising the work and has given final approval for 12 13 the work and agrees to be accountable for all aspects of the work. 14 15 16 17 18 AcknowledgementsFor peer review only 19 20 The authors would like to acknowledge and thank all participants of the ROCIT study and the 21 22 23 consumers from the Consumer and Community Health Research Network (University of 24 25 Western Australia, WA, 6009) for their contribution to the study design. 26 27 28 29 30 Funding 31 32 33 ROCIT is funded by grants from the Department of Health, Government of Western 34 35 Australia Research Translation Projects, the St John of God Hospital Foundation and the 36

37 http://bmjopen.bmj.com/ 38 Fiona Wood Foundation. Study drug was supplied by Health World Ltd. The funding bodies 39 40 and Health World Ltd had no input into the design or conduct of the trial, and will have no 41 42 input into the analysis or reporting of the trial results. The study sponsor is the Fiona Stanley 43 44

45 Fremantle Hospital Group, South Metropolitan Health Service, Western Australia. on October 1, 2021 by guest. Protected copyright. 46 47 48 49 50 Competing interest 51 52 The authors declare no competing interests. 53 54 55 56 57 Table 1 Trial eligibility criteria 58 59 60 Inclusion criteria 1. Adult patient within 48 hours of admission to an ICU

18 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 21 of 39 BMJ Open

1 2 3 2. Expected to require ICU-level care beyond the next calendar 4 5 day BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 6 7 8 Exclusion criteria 1. <18 years of age 9 10 2. Absolute contraindication to receiving medication via the 11 enteral route 12 3. Known to be receiving probiotic therapy at the time of index 13 hospitalisation 14 4. Acute pancreatitis as a cause or complication of current 15 16 admission 17 5. Immunosuppression (defined as chemotherapy within the 18 For peerpreceding fourreview weeks, or receiving only ≧ 1.5mg/kg 19 methylprednisolone daily or equivalent) 20 ≦ 9 21 6. Neutropenia (neutrophil count 1x10 /L) 22 7. Prosthetic heart valve or permanent pacemaker 23 8. Death is deemed to be inevitable as a result of the current 24 acute illness AND either the treating clinician, the patient or 25 the substitute decision-maker, are not committed to full 26 27 active treatment 28 9. Enrolment is not considered in the patient’s best interest 29 10. Previously enrolled in ROCIT 30 11. Unlikely to be residing near or visiting a study centre in 60 31 32 days 33 12. Participating in a competing interventional study 34 13. Pregnancy 35 14. Admitted to hospital from a high-level nursing facility or 36 rehabilitation facility

37 http://bmjopen.bmj.com/ 38 39 ICU intensive care unit, ROCIT restoration of gut microflora in critical illness trial 40 Table 2. Study data to be collected 41 42 43 Time point Study data 44 Screening - Date of screening

45 - Inclusion and exclusion criteria on October 1, 2021 by guest. Protected copyright. 46 - Reason, if not enrolled 47 48 - Study number, patient initials, for enrolled participants 49 50 Baseline - Date and time of randomisation 51 - Date and time of ICU admission 52 - Demographic data 53 54 - ICU admission source and category 55 - Nutrition, acid suppressive therapy and antibiotics 56 - Admission APACHE II score, diagnostic code and co- 57 morbidities 58 59 - SOFA Score and components 60 - Mechanical ventilation

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1 2 3 - Vasoactive medication 4 5 - Renal replacement therapy BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 6 7 Daily during index - Patient location (ICU/HDA or ward) 8 hospitalisation - Received study drug 9 10 - Days of mechanical ventilation, vasoactive medication and 11 renal replacement therapy 12 - Days of antibiotic, antiviral and antifungal medication 13 - New infection diagnosed 14 15 16 Outcome (Day 60) - Hospital length of stay 17 - Nosocomial infection (hospital-acquired pneumonia, 18 For peerventilator-associated review pneumonia, only Clostridium difficile- 19 associated diarrhoea, surgical site infection, urinary tract 20 21 infection, and blood stream infection)* 22 - ICU length of stay 23 - ICU mortality 24 - Hospital mortality 25 - EQ-5D-5L 26 27 28 Adverse events - Description, timing, causality and resolution of adverse 29 events from randomisation to Day 60 30 31 32 Protocol deviations - Randomisation of ineligible patients, failure to comply with 33 the study protocol 34 35 ICU intensive care unit, HDA High dependency area, APACHE acute physiology and chronic 36 health evaluation, SOFA Sequential organ failure score, EQ-5D-5L five level EuroQol five- 37 http://bmjopen.bmj.com/ 38 dimension questionnaire. 39 *The pre-specified nosocomial infections will be identified according to the centre for 40 disease control definitions and are provided in the supplementary appendix 41 42 43 44 Figure legends

45 on October 1, 2021 by guest. Protected copyright. 46 Figure 1. Study drug bottle labelling 47 48 49 Figure 2. Proposed reporting of flow of trial participants 50 51 52 53 54 55 56 57 58 59 60

20 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 23 of 39 BMJ Open

1 2 3 4 5 BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 6 7 8 9 10 11 12 13 14 15 16 17 18 For peer review only 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36

37 http://bmjopen.bmj.com/ 38 39 40 41 42 43 44

45 on October 1, 2021 by guest. Protected copyright. 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open Page 24 of 39 Met inclusion criteria (n= x)

Excluded (n= x) BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 1 Reasons: 2 - Age <18 years (n=x) 3 - Absolute contraindication to enteral medication (n= x) 4 5 - Already receiving probiotics (n=x) 6 - Acute pancreatitis (n=x) 7 - Immunosuppressed (n=x) 8 - Neutropaenia (n= x) 9 - Prosthetic heart valve or permanent pacemaker (n=x) 10 - Death is deemed inevitable AND not committed to active treatment (n=x) 11 - Enrolment not in the patients’ best interest (n=x) 12 - Previously enrolled in ROCIT 13 - Unlikely to residing near or visiting a study centre in 60 days (n=x) 14 15 - Participating in a competing interventional study (n=x) 16 For peer- Pregnancy review (n=x) only 17 - Admitted to hospital from a high-level nursing or rehabilitation facility (n= x) 18 - Eligible but missed (n=x) 19 - Did not consent (n=x) 20 21 22 Eligible (n= x) 23 Eligible but not enrolled 24 Reasons: 25 - Enrolment overlooked/Clinician Unaware (n= x) 26 - Patient or next- of-kin declined to participate (n= x) 27 - Previously enrolled in this study (n= x) Randomised (n= x) 28 - Other (n= x)

29 30 31 32 33 http://bmjopen.bmj.com/ 34 35 Probiotic therapy (n=x) Placebo (n=x) 36 ¨ Received allocated intervention (n=x) ¨ Received allocated intervention (n=x) 37 38 ¨ Did not receive allocated intervention (n=x) ¨ Did not receive allocated intervention (n= x) 39 - Consent withdrawn (n=x) - Consent withdrawn (n=x) 40 - Patient was ineligible (n=x) - Patient was ineligible (n=x)

41 - on October 1, 2021 by guest. Protected copyright. 42 43 44 45 Lost to follow-up (n=x) Lost to follow-up (n=x) 46 47 Discontinued intervention (n=x) Discontinued intervention (n=x) 48 49 Consent withdrawn (n=x) Consent withdrawn (n=x) 50 Unable to locate patient (n=x) Unable to locate patient (n=x) 51 52 Re 53 54 55 Analysed (n=x) Analysed (n=x) 56 Excluded from analysis (n=x) 57 Excluded from analysis (n=x) 58 59 60 For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml Page 25 of 39 BMJ Open

1 2 3 ROCIT Protocol Supplementary Appendix 4 5 BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 6 7 Content Page 8 9 10 Figure 1. Standard Operating Procedure- nasogastric administration …………………. 1 11 12 Table 1. Nosocomial infection definitions …………………. 2-3 13 14 Figure 2. Study drug diary …………………. 4 15 16 17 Study sites …………………. 5 18 For peer review only 19 Collection and storage of biological specimens …………………. 5 20 21 22 Consent form …………………. 6-11 23 24 Table 2. Adverse Events ………………… 12 25 26 27 Figure 1 Standard Operating Procedure- Nasogastric Administration 28

29 ROCIT Study SOP NG Admin v2.0 20171807 30

31 Nasogastric Administration of ROCIT Study Drug

32 This SOP applies only for participants in the ROCIT Study in whom there is a contraindication to swallowing tablets. 33 For participants who are able to swallow tablets, the single daily ROCIT Study Drug 34 capsule can be administered as a capsule by mouth.

35 For participants who are unable to swallow tablets and in whom a nasogastric tube is present, please follow the instructions below: 36

37 1. Check nasally/orally inserted gastric tube placement and patency. http://bmjopen.bmj.com/

38 2. Don a pair of disposable glove and open one single capsule of ROCIT study drug into an empty disposable cup. 39 3. Pour 10ml of room temperature water into the disposable cup. 40 4. Use a new tongue depressor to stir continuously as the water is added. This is key 41 as stirring while adding water will minimise clump formation.

42 5. It is expected that there will be some degree of sedimentation at the bottom of the cup. This is the stabiliser from the capsule and not a fault in the administration 43 process.

44 6. Draw up the prepared diluted medication into a 20ml syringe enteral syringe.

45 7. Release the enteral tube cap and administer the medication according to site on October 1, 2021 by guest. Protected copyright. based protocols. 46 47 8. Flush the enteral tube with 10mL of water then replace the cap 48 9. Dispose of the administration equipment according to infection control policies.

49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 Table 1 Summary (abbreviated) of nosocomial infection diagnostic criteria* 4 5 BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 6 Nosocomial infection Criteria 7 8 Surgical site infection Superficial incisional: occurs within 30 days after the 9 10 operation and the infection involves only skin or 11 subcutaneous tissue of the incision and at least one of the 12 following: 13 1. Purulent drainage 14 2. Organisms isolated 15 16 3. At least one of pain, tenderness, swelling, redness, 17 heat and superficial incision is deliberately opened by 18 For peersurgeon review only 19 4. Diagnosis of superficial incisional by surgeon 20 21 Deep incisional: occurs within 30 days after the operation if no 22 implant or within 1 year if implant and related to the 23 operation and infection involves the deep tissues and at least 24 one of the following: 25 1. Purulent drainage from the deep incision, but not from 26 27 the organ of the surgical site 28 2. Deep incision spontaneously dehisces or is deliberately 29 opened by the surgeon 30 3. Abscess or other evidence of infection involving the 31 32 deep incision 33 4. Diagnosis of deep surgical space infection by the 34 surgeon 35 Organ/space: occurs within 30 days or within one year if 36 implant is in place and related to the operation and infection

37 http://bmjopen.bmj.com/ 38 involves any part of the anatomy other than the incision and: 39 1. Purulent drainage from a drain into the organ space 40 2. Organisms isolated aseptically from the organ/space 41 3. Abscess from the organ/space 42 43 4. Diagnosis of an organ/space infection by the surgeon 44

45 The CDC criteria for burns infection will be included as a on October 1, 2021 by guest. Protected copyright. 46 surgical site infection. 47 48 Blood stream infection Clinical criteria are only required if the organism identified is a 49 common skin contaminant 50 Clinical criteria (at least one of): 51 1. Fever 52 2. WBC>10,000 or <3,000 per cubic millimetre 53 54 3. Hypotension (SBP<90) or >25% drop in SBP 55 56 C. difficile-associated Clinical evidence of at least one of the following: 57 diarrhoea 1. Pseudomembranes identified at lower gastrointestinal 58 59 endoscopy 60

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1 2 3 2. Pathological confirmation of pseudomembranous 4 5 colitis BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 6 3. C difficile toxin detected in the stool 7 8 Urinary tract infection Clinical criteria of a and b must be satisfied within a two-day 9 10 period 11 a) Clinical criteria (at least one of): 12 i. fever >38.5 C 13 ii. WBC>10,000 or <3,000 per cubic millimetre 14 iii. Urgency 15 16 iv. Dysuria 17 v. Suprapubic tenderness 18 For peerb) Bacterial reviewconfirmation only 19 vi. >105 organisms per ml of urine 20 21 22 Pneumonia Criteria a-c must be satisfied within a 48-hour period 23 a) Radiologic criteria 24 i. New infiltrate that persists for at least 24 hours 25 26 b) Clinical criteria (one of) 27 i. Temperature >38.5 or <35 C 28 ii. WBC>10,000 or <3,000 per cubic millimetre 29 c) Bacterial confirmation (at least one of) 30 i. BAL >103 CFU/ml 31 32 ii. Histopathological examination of lung tissue 33 iii. Positive blood culture for bacterial pathogen 34 identified in sputum 35 iv. Positive pleural culture with same organism in 36 sputum 37 http://bmjopen.bmj.com/ 38 v. Positive gram stain 39 vi. Heavy or moderate growth of one type of 40 pathogenic bacteria on semi-quantitative 41 sputum culture 42 43 Ventilator-associated pneumonia occurs where a patient is on 44 mechanical ventilation for > two calendar days on the date of

45 the event, with day of ventilator placement being day one, on October 1, 2021 by guest. Protected copyright. 46 and the ventilator was in place on the date of the event or the 47 48 day before. 49 50 51 WBC white blood cell, SBP systolic blood pressure, BAL bronchoalveolar lavage, CFU colony 52 forming units. *For full criteria and wording see CDC. National Healthcare Safety Network 53 54 (NHSN) Patient Safety Component Manual. Chapter 17: CDC/NHSN Surveillance Definitions 55 for Specific Types of Infections. 2019. Where any difference occurs between this summary 56 and the CDC definitions, the CDC definitions will take priority. 57 Figure 2. Study drug diary 58 59 60

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1 2 3 Figure 2. Daily Medication Diary 4 5 BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 6 7 8 9 10 11 12 13 14 15 16 17 18 For peer review only 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36

37 http://bmjopen.bmj.com/ 38 39 40 41 42 43 44

45 on October 1, 2021 by guest. Protected copyright. 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 Study Sites 4 5 BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 6 1. Fiona Stanley Hospital, Robin Warren Drive, Perth, Western Australia 7 2. St John of God Hospital Subiaco, Salvado Road, Perth, Western Australia 8 3. St John of God Hospital Murdoch, Murdoch Drive, Perth, Western Australia 9 10 4. Sir Charles Gairdner Hospital, Nedlands, Perth, Western Australia 11 5. Royal Perth Hospital, Wellington Street, Perth, Western Australia 12 13 14 Collection and storage of biological specimens 15 16 17 Blood (plasma) and faeces will be collected from all study participants at four time points. 18 These time points are:For peer review only 19 20 21 1. Study enrolment (within 48h of admission to ICU or HDA) 22 2. 48h post enrolment or immediately prior to ICU discharge, whichever occurs first 23 3. 7 days post enrolment or immediately prior to hospital discharge, whichever occurs first 24 4. Day 60 post enrolment 25 26 27 Each blood sample will require a single 5ml serum tube. Feaces will be collected using a 28 rectal swab by bedside clinical staff as is routine for patients admitted to the ICU and 29 according to a standard operating procedure. For Day 60 samples in patients no longer 30 remaining in hospital, a swab kit will be provided to the patient with simple standardised 31 32 instructions on how to perform a rectal swab. This technique has been used in previous 33 studies and found to be performed reliably. Samples will all be processed and stored 34 within 2 hours from collection for biobanking, and in accordance with best practice 35 recommendations for studies of the metabolome and 36 microbiome.

37 http://bmjopen.bmj.com/ 38 Identification of the bacterial species of participant faecal samples will be performed 39 using next generation sequencing. Measurement of the metabolome will be performed 40 by liquid chromatography-mass spectrometry and will be targeted towards lipid and 41 fatty acid metabolism (short chain fatty acids and Trimethylamine N-oxide). 42 43 44

45 on October 1, 2021 by guest. Protected copyright. 46 47 48 49 50 51 52

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1 2 3 4 5 BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 6 7 The ROCIT Study 8 9 Patient Information and Consent Form 10 11 1. Introduction 12 You are invited to take part in this research project because you have been admitted 13 14 to the Intensive Care Unit (ICU) or a high dependency area (HDA), and have been 15 identified as potentially eligible to participate in the ROCIT Study. 16 17 This Patient Information and Consent Form tells you about the research project. It 18 explains the proceduresFor involved. peer Knowing review what is involved only will help you decide if you 19 want to take part in the research project. Please read this information carefully. Ask 20 21 questions about anything that you don’t understand or want to know more about. 22 Before deciding whether or not to take part, you might want to talk about it with a 23 relative, friend or healthcare worker. This research project has been approved by the 24 South Metropolitan Health Service Human Research Ethics Committee at Fiona 25 Stanley Hospital. 26 27 Participation in this research project is voluntary. If you don’t wish to take part, you 28 29 don’t have to. You will receive the best possible care whether you take part or not. If 30 you decide you want to take part in the research project, you will be asked to sign the 31 consent section. By signing it you are telling us that you: 32 33 • understand what you have read 34 • consent to take part in the research project 35 36 • consent to participate in the research processes that are described

37 • consent to the use of your personal and health information as described. http://bmjopen.bmj.com/ 38 39 You will be given a copy of this Patient Information and Consent Form to keep. 40 41 42 2. What is the purpose of this research project? 43 The purpose of this research project is to find out if giving probiotic therapy 44 (bacteria that may provide a health benefit) will reduce the risk of hospital acquired

45 infections and lead to more rapid recovery for patients admitted to an ICU or HDA on October 1, 2021 by guest. Protected copyright. 46 compared with current standard care that does not involve giving probiotics to 47 patients in the ICU or HDA. 48 49 50 The probiotic used in this study (Plantarum 299v) is a bacteria found in the 51 gastrointestinal tract of healthy people. Probiotics have several potentially 52 beneficial properties including reducing the risk of growth of bacteria that may 53 cause infection, protecting the lining of the gastrointestinal tract and reducing 54 inflammation. Although there is some evidence that probiotics may be useful in 55 critically ill patients, the data is currently insufficient to guide practice and 56 probiotics are not used in the critical care areas of any of the participating 57 58 institutions. However, the probiotic being studied is registered with the 59 Therapeutic Goods Administration of Australia and is available for purchase ‘over 60 the counter’ in Australian pharmacies. It is not an experimental product.

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1 2 3 4 The ROCIT study will involve 220 patients admitted to critical care areas in several BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 5 6 hospitals in Western Australia. The study has been funded by the State Health 7 Research Advisory Council, Department of Health of Western Australia and has 8 been designed by a collaborative group of clinicians and researchers. 9 10 3. What does participation in this research project involve? 11 12 13 The ROCIT trial team has assessed you as suitable to participate in this research 14 project. If you agree to participate, you will be randomly assigned (like the toss of a 15 coin) to receive either probiotic therapy in addition to standard care or placebo (sugar 16 pill) in addition to standard care. For the purposes of this study and this Information 17 Sheet, the term ‘placebo’ refers to the sugar pill. A placebo is an inactive and harmless 18 ‘treatment’ given For to the patientspeer in review the study who only are randomised not to receive 19 probiotics. Standard care is the care that you have, and will continue to receive, as a 20 21 result of your current condition whether or not you agree to continue to take part in this 22 study. You have a one in two chance of receiving probiotics or placebo in addition to 23 standard care. Participants randomised to probiotics will receive a once daily 24 administration of a single strain probiotic capsule containing 20 billion colony forming 25 units of Lactobacillus plantarum strain 299v. Participants randomised to placebo will 26 receive a once a day placebo capsule. All other treatment will remain at the discretion 27 of the treating team. 28 29 30 The study is ‘blinded’. This means that you, your relative, the researchers and the 31 clinical staff caring for your relative will not know whether you are receiving probiotics 32 or placebo. The blinding and the placebo are used to ensure that the results are a true 33 reflection of the effect of the probiotic therapy. For this study, ‘blinding’ will be 34 conducted by providing the study drug in identical packaging and ensuring that the 35 36 probiotic and placebo capsules also look identical. The researchers will tell your doctors if they need to know which treatment has been received. The techniques of 37 http://bmjopen.bmj.com/ 38 blinding and use of placebo are essential for the conduct of high quality clinical trials. 39 40 Treatment 41 42 If you agree to participate you will receive one capsule daily of either probiotic 43 44 therapy or placebo whilst in the hospital and after discharge from hospital, for a

45 maximum total study period of 60 days. At the time of hospital discharge, you will be on October 1, 2021 by guest. Protected copyright. 46 provided with study medication and, where feasible, sent reminders by text message 47 and phone call till the end of the total study period of 60 days. You will be asked to 48 record your use of study medication daily. You will be visited at day 60 by study staff 49 to collect your used bottles of study medication appointments or alternatively the 50 participant can return it to the pharmacy of the hospital in which they were treated. 51 52 .. At day 60 you will be asked to complete a short (10 minutes or less) questionnaire 53 on how you are feeling and functioning. If you prefer to be followed up at day 60 in 54 hospital, the study team will attempt to arrange this when you are also scheduled to 55 return for other medical The study-related treatments and procedures are as follows: 56 - You will be assigned to receive either probiotics or placebo delivered as a 57 capsule once daily from enrolment in the study to a maximum of 60 days. 58 59 - Whilst in the ICU, hospital ward or after discharge from hospital, a total of four 60 rectal swabs are required (days 1,3,7 & 60). Rectal swabs are routinely taken

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1 2 3 on admission to ICU to screen for infection. The additional swabs for this 4 study will be used to determine the bacteria in your gastrointestinal tract. BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 5 6 Measuring this at four time points will give the study investigators an 7 understanding of changes over time as recovery takes place, and also the 8 potential benefit of the probiotics. 9 - Whilst in the ICU and hospital ward a total of three blood samples will be 10 taken in addition to the standard blood tests required by the medical team 11 caring for you. Each will be a single blood tube of approximately 5 ml (or one 12 13 table spoon) drawn, where available, from an existing intravascular device. 14 The blood tests will be used to measure changes in how metabolism is 15 occurring over time and the influence of the probiotics on this process. An 16 additional fourth blood test at day 60 will be requested from participants who 17 are returning to the hospital for clinical need. Failure to collect this blood 18 sample doesFor not preclude peer participation review in the ROCITonly study. 19 - During the study period, you will be continuously monitored whilst in hospital to 20 21 enable the detection of any problems or complications. You will also be 22 provided with contact details in the case of any concerns after discharge from 23 hospital. 24 - Data will continue to be collected from the hospital medical notes and tests 25 ordered by the clinical team treating you until the end of the study period. If 26 required, we may also, with your permission, contact your GP to request 27 clarification of clinical events related to the study. 28 29 - Samples will be stored at the hospital at which they are taken, then transferred 30 via Harry Perkins FSH for analysis at: 31 Cryptick Lab, 32 Murdoch University 33 90 South Street 34 Murdoch WA 6150 35 36 4. What are the possible benefits? 37 http://bmjopen.bmj.com/ 38 We cannot guarantee or promise that you will receive any benefits from this 39 research; however there may be possible benefits for patients admitted to the ICU 40 or HDA in the future. 41 42 5. What are the possible risks? 43 44 Possible risks are from complications of study treatment. The study has been

45 designed in a manner to ensure that the welfare of participants is protected at all on October 1, 2021 by guest. Protected copyright. 46 times and that respect for patient autonomy is demonstrated through appropriate 47 mechanisms of consent. 48 49 Probiotic therapy is available as over-the-counter medication in Australia and we do 50 not expect adverse events related to the study treatment with probiotics. However, 51 52 adverse events are possible. We will be screening for infection throughout the study 53 period. Any suspected or confirmed infection related to probiotics will result in 54 cessation of study medication. The probiotics being used in the study have been 55 tested and known to be sensitive commonly used antibiotics in the event of 56 suspected probiotic-associated infection. The placebo used in this study contains a 57 standard inert material commonly used in placebo medication. 58 59 60

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1 2 3 As occurs in routine clinical practice, all patients are monitored closely for development 4 of any side effects from treatments which will be immediately treated by amending or BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 5 6 stopping the treatment. There may be additional risks that the researchers do not 7 expect or do not know about. You should tell a member of the research team 8 immediately about any unusual symptoms that you get. 9 10 6. What if new information arises during this research project? 11 During the research project, new information about the risks and benefits of the project 12 13 may become known to the researchers. If this occurs, you will be told about this new 14 information and the researcher will discuss whether this new information affects you. 15 16 7. Are there alternatives to participation? 17 Participation in this research is not your only option. If you do not participate in this 18 study, you will receiveFor standard peer medical review treatment. onlyYou can discuss this with your 19 healthcare worker and any family members or close friends before deciding whether 20 21 or not you should take part in this research project. 22 23 8. Do I have to take part in this research project? 24 Participation in any research project is voluntary. If you do not wish to take part, you 25 do not have to. If you decide to take part and later change your mind, you are free to 26 withdraw from the project at any stage. 27 28 29 Your decision whether you take part or not, or to take part and then withdraw, will not 30 affect your routine treatment or your relationship with those treating you or your 31 relationship with this Fiona Stanley Hospital. 32 33 9. What if I withdraw from this research project? 34 If you decide to withdraw from the project, please notify a member of the research 35 36 team before you withdraw. This notice will allow that person or the research supervisor to inform you if there are any special requirements to withdrawing. The researchers 37 http://bmjopen.bmj.com/ 38 would like to keep the personal and health information about you that have already 39 been collected. This is to help them make sure that the results of the research can be 40 measured properly. 41 42 10. Could this research project be stopped unexpectedly? 43 44 This research project may be stopped for a variety of reasons. These may include

45 reasons such as unacceptable side effects, the treatment being shown not to be on October 1, 2021 by guest. Protected copyright. 46 effective or the treatment being shown to work and not need further investigation. 47 48 11. How will I be informed of the results of this research project? 49 Once the project has been completed, a summary of the results will be available from 50 the Chief Investigator Dr Ed Litton at Fiona Stanley Hospital on request. 51 52 53 12. What else do I need to know? 54 55 What will happen to information about me? 56 The information gathered about you by the investigator or obtained during the trial 57 from blood tests or other procedures will be held by the investigator in strict 58 59 confidence. Information from your medical records is essential to evaluate the results 60 of this study. Information relevant to the research will be recorded on the

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1 2 3 understanding that it will be treated confidentially. Your GP may also be contacted. 4 Your trial records without your name attached will be made available to the study BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 5 6 management committee and through publication in the peer-reviewed medical 7 literature to government regulatory bodies in Australia and overseas. All the people 8 who handle your information will adhere to traditional standards of confidentiality and 9 will also comply with all relevant privacy legislation. In Australia this is the Privacy Act 10 1988. If the results of the trial are published in a medical journal, as is intended, no 11 reader will be able to identify individual patients. 12 13 How can I access my information? 14 In accordance with relevant laws, you have the right to access the information 15 collected and stored by the researchers about you. You also have the right to request 16 that any information, with which you disagree, be corrected. Please contact one of the 17 researchers named below if you would like to access your information. 18 For peer review only 19 Action if an adverse event arises during the trial 20 21 In the event that you suffer an adverse event or a medical accident during this study 22 that arises from your participation in the study, you will be offered all full and necessary 23 treatment by this Hospital. The South Metropolitan Health Service Human Research 24 Ethics Committee has approved this study on the basis (amongst others) that the 25 reported risk of such an event is either small or acceptable in terms of the risk you 26 face as a result of your current illness or the benefit that is possible with the new 27 treatment being tested. No provisions have been made in this trial to offer trial subjects 28 29 who suffer an adverse reaction monetary compensation, but the absence of such a 30 provision does not remove your rights to seek compensation under common law. 31 32 Whom should I contact if I need more information? 33 If you have any questions please contact: 34 35 36 Dr Edward Litton Susan Pellicano Intensive Care Unit Project Coordinator 37 http://bmjopen.bmj.com/ 38 Fiona Stanley Hospital Fiona Stanley Hospital 39 Tel: 08 6152 6547 Tel:08 6152 6546 40 41 42 43 44 If you have questions about your rights as a research Participant, you may contact:

45 the South Metropolitan Health Service Research Ethics and Governance Unit. on October 1, 2021 by guest. Protected copyright. 46 Tel: (08) 6151 1180 47 Email: [email protected] 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 4 5 BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 6 Study - Patient Consent Form 7 8 9 10 Name of Patient ______Date of Birth______11 12 1. I agree voluntarily to take part in Multicentre Study: ROCIT Study. This study has 13 been funded by the State Health Research Advisory Council of W.A. I am 18 years 14 15 of age or over. 16 17 2. I have been given a full explanation of the purpose of this study, of the procedure 18 involved and of whatFor will bepeer expected review of me. The doctor only has explained the possible 19 problems that might arise as a result of my participation in this study. 20 21 3. I agree to inform the supervising doctor of any unexpected or unusual symptoms I 22 23 may experience as soon as possible. 24 25 4. I understand that I am entirely free to withdraw from the study at any time and 26 that this withdrawal will not in any way affect my standard or conventional treatment 27 or medical management. 28 29 5. I understand that the information in my medical records is essential to evaluate 30 31 the results of this study. I agree to the release of this information to the research staff 32 and the clinical staff on the understanding that it will be treated confidentially. 33 34 6. I agree to my General Practitioner (GP) being informed about my participation in 35 this study and to my GP providing clarification of clinical events related to the study. 36

37 http://bmjopen.bmj.com/ 38 7. I understand that I will not be referred to by name in any report concerning this 39 study. In turn, I cannot restrict in any way the use of the results that arise from this 40 study. 41 42 8. I have been given and read a copy of this Consent Form and Information Sheet. 43 44

45 on October 1, 2021 by guest. Protected copyright. 46 47 Signature of Patient...... Signature of Doctor...... 48 49 50 Name of Patient: ...... Name of Doctor...... 51 52 53 Date: ...... Date: ...... 54 55 56 57 58 Time: ...... Time: ...... 59 60

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1 2 3 Table 2. Adverse Events 4 5 BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 6 System Clinical Time of onset Duration Resolution 7 Features 8 9 10 11 12 13 14 15 16 17 18 For peer review only 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36

37 http://bmjopen.bmj.com/ 38 39 40 41 42 43 44

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1 2 3 4 5 BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 6 7 8 SPIRIT 2013 Checklist: Recommended items to address in a clinical trial protocol and 9 related documents* 10 11 Section/item Item Description 12 No 13 14 Administrative information 15 16 Title 1 Descriptive title identifying the study design, population, interventions, 17 and, if applicable, trial acronym – page 1 18 For peer review only 19 Trial registration 2a Trial identifier and registry name. If not yet registered, name of 20 intended registry – page 3&5 21 22 2b All items from the World Health Organization Trial Registration Data 23 24 Set – page 3 25 26 Protocol version 3 Date and version identifier – page 3 27 28 Funding 4 Sources and types of financial, material, and other support – page 18 29 Roles and 5a Names, affiliations, and roles of protocol contributors – page 1,2,15-18 30 31 responsibilities 5b Name and contact information for the trial sponsor – page 2 32 33 5c Role of study sponsor and funders, if any, in study design; collection, 34 35 management, analysis, and interpretation of data; writing of the report; 36 and the decision to submit the report for publication, including whether

37 they will have ultimate authority over any of these activities – page 17- http://bmjopen.bmj.com/ 38 18 39 40 5d Composition, roles, and responsibilities of the coordinating centre, 41 42 steering committee, endpoint adjudication committee, data 43 management team, and other individuals or groups overseeing the 44 trial, if applicable (see Item 21a for data monitoring committee) – page 45 11 for Data Monitoring Committee on October 1, 2021 by guest. Protected copyright. 46 47 48 Introduction 49 50 Background and 6a Description of research question and justification for undertaking the 51 rationale trial, including summary of relevant studies (published and 52 unpublished) examining benefits and harms for each intervention – 53 page 4 54 55 6b Explanation for choice of comparators -page 4-5 56 57 Objectives 7 Specific objectives or hypotheses – page 5 58 59 60

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1 2 Trial design 8 Description of trial design including type of trial (eg, parallel group, 3 crossover, factorial, single group), allocation ratio, and framework (eg, 4

superiority, equivalence, noninferiority, exploratory) – page 5 BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 5 6 7 8 Methods: Participants, interventions, and outcomes 9 10 Study setting 9 Description of study settings (eg, community clinic, academic hospital) 11 and list of countries where data will be collected. Reference to where 12 list of study sites can be obtained – page 6 & Supplementary 13 14 appendix 15 16 Eligibility criteria 10 Inclusion and exclusion criteria for participants. If applicable, eligibility 17 criteria for study centres and individuals who will perform the 18 Forinterventions peer (eg,review surgeons, psychotherapists) only – page 6 and Table 1 19 20 Interventions 11a Interventions for each group with sufficient detail to allow replication, 21 including how and when they will be administered page 7-8 22 23 11b Criteria for discontinuing or modifying allocated interventions for a 24 25 given trial participant (eg, drug dose change in response to harms, 26 participant request, or improving/worsening disease) – page 8 27 28 11c Strategies to improve adherence to intervention protocols, and any 29 procedures for monitoring adherence (eg, drug tablet return, 30 laboratory tests) – page 7&9 31 32 11d Relevant concomitant care and interventions that are permitted or 33 prohibited during the trial – page 8 34 35 Outcomes 12 Primary, secondary, and other outcomes, including the specific 36

37 measurement variable (eg, systolic blood pressure), analysis metric http://bmjopen.bmj.com/ 38 (eg, change from baseline, final value, time to event), method of 39 aggregation (eg, median, proportion), and time point for each 40 outcome. Explanation of the clinical relevance of chosen efficacy and 41 42 harm outcomes is strongly recommended – page 8&9 43 44 Participant 13 Time schedule of enrolment, interventions (including any run-ins and

45 timeline washouts), assessments, and visits for participants. A schematic on October 1, 2021 by guest. Protected copyright. 46 diagram is highly recommended (see Figure) – page 7 47 48 Sample size 14 Estimated number of participants needed to achieve study objectives 49 and how it was determined, including clinical and statistical 50 51 assumptions supporting any sample size calculations – page 9 &10 52 53 Recruitment 15 Strategies for achieving adequate participant enrolment to reach 54 target sample size – page 6 55 56 Methods: Assignment of interventions (for controlled trials) 57 58 Allocation: 59 60

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1 2 Sequence 16a Method of generating the allocation sequence (eg, computer- 3 generation generated random numbers), and list of any factors for stratification. 4

To reduce predictability of a random sequence, details of any planned BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 5 6 restriction (eg, blocking) should be provided in a separate document 7 that is unavailable to those who enrol participants or assign 8 interventions – page 6 9 10 Allocation 16b Mechanism of implementing the allocation sequence (eg, central 11 concealment telephone; sequentially numbered, opaque, sealed envelopes), 12 13 mechanism describing any steps to conceal the sequence until interventions are 14 assigned – page 6 15 16 Implementation 16c Who will generate the allocation sequence, who will enrol participants, 17 and who will assign participants to interventions – page 6 18 For peer review only 19 Blinding 17a Who will be blinded after assignment to interventions (eg, trial 20 (masking) participants, care providers, outcome assessors, data analysts), and 21 how – page 6 22 23 17b If blinded, circumstances under which unblinding is permissible, and 24 25 procedure for revealing a participant’s allocated intervention during 26 the trial – page 6 27 28 Methods: Data collection, management, and analysis 29 30 Data collection 18a Plans for assessment and collection of outcome, baseline, and other 31 methods trial data, including any related processes to promote data quality (eg, 32 duplicate measurements, training of assessors) and a description of 33 34 study instruments (eg, questionnaires, laboratory tests) along with 35 their reliability and validity, if known. Reference to where data 36 collection forms can be found, if not in the protocol – page 9

37 http://bmjopen.bmj.com/ 38 18b Plans to promote participant retention and complete follow-up, 39 including list of any outcome data to be collected for participants who 40 41 discontinue or deviate from intervention protocols – page 9 42 Data 19 Plans for data entry, coding, security, and storage, including any 43 44 management related processes to promote data quality (eg, double data entry;

45 range checks for data values). Reference to where details of data on October 1, 2021 by guest. Protected copyright. 46 management procedures can be found, if not in the protocol – page 9 47 48 Statistical 20a Statistical methods for analysing primary and secondary outcomes. 49 methods Reference to where other details of the statistical analysis plan can be 50 51 found, if not in the protocol – page 9&10 52 53 20b Methods for any additional analyses (eg, subgroup and adjusted 54 analyses) – page 10 55 56 20c Definition of analysis population relating to protocol non-adherence 57 (eg, as randomised analysis), and any statistical methods to handle 58 missing data (eg, multiple imputation) – page 10 59 60

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1 2 Methods: Monitoring 3 4 Data monitoring 21a Composition of data monitoring committee (DMC); summary of its role 5 and reporting structure; statement of whether it is independent from BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 6 the sponsor and competing interests; and reference to where further 7 8 details about its charter can be found, if not in the protocol. 9 Alternatively, an explanation of why a DMC is not needed – page 11 10 11 21b Description of any interim analyses and stopping guidelines, including 12 who will have access to these interim results and make the final 13 decision to terminate the trial – page 10 14 15 Harms 22 Plans for collecting, assessing, reporting, and managing solicited and 16 spontaneously reported adverse events and other unintended effects 17 18 Forof peer trial interventions review or trial conduct only – page 10&11 19 20 Auditing 23 Frequency and procedures for auditing trial conduct, if any, and 21 whether the process will be independent from investigators and the 22 sponsor – page 10 23 24 Ethics and dissemination 25 26 Research ethics 24 Plans for seeking research ethics committee/institutional review board 27 28 approval (REC/IRB) approval – page 12 29 30 Protocol 25 Plans for communicating important protocol modifications (eg, 31 amendments changes to eligibility criteria, outcomes, analyses) to relevant parties 32 (eg, investigators, REC/IRBs, trial participants, trial registries, journals, 33 regulators) – page 12 34 35 Consent or assent 26a Who will obtain informed consent or assent from potential trial 36 participants or authorised surrogates, and how (see Item 32) – page 6 37 http://bmjopen.bmj.com/ 38 26b Additional consent provisions for collection and use of participant data 39 40 and biological specimens in ancillary studies, if applicable – all study 41 procedures were described in the primary consent form. 42 43 Confidentiality 27 How personal information about potential and enrolled participants will 44 be collected, shared, and maintained in order to protect confidentiality

45 on October 1, 2021 by guest. Protected copyright. before, during, and after the trial – page 9&10 46 47 Declaration of 28 Financial and other competing interests for principal investigators for 48 49 interests the overall trial and each study site – page 15-18 50 51 Access to data 29 Statement of who will have access to the final trial dataset, and 52 disclosure of contractual agreements that limit such access for 53 investigators – page 12 54 55 Ancillary and 30 Provisions, if any, for ancillary and post-trial care, and for 56 post-trial care compensation to those who suffer harm from trial participation – n/a 57 58 59 60

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1 2 Dissemination 31a Plans for investigators and sponsor to communicate trial results to 3 policy participants, healthcare professionals, the public, and other relevant 4

groups (eg, via publication, reporting in results databases, or other BMJ Open: first published as 10.1136/bmjopen-2019-035930 on 21 June 2020. Downloaded from 5 6 data sharing arrangements), including any publication restrictions – 7 page 12 8 9 31b Authorship eligibility guidelines and any intended use of professional 10 writers – page 15-18 11 12 31c Plans, if any, for granting public access to the full protocol, participant- 13 level dataset, and statistical code – page 12 14 15 16 Appendices 17 Informed consent 32 Model consent form and other related documentation given to 18 For peer review only 19 materials participants and authorised surrogates – page 12 & Supplementary 20 appendix 21 22 Biological 33 Plans for collection, laboratory evaluation, and storage of biological 23 specimens specimens for genetic or molecular analysis in the current trial and for 24 future use in ancillary studies, if applicable – page 9 & Supplementary 25 26 appendix 27 *It is strongly recommended that this checklist be read in conjunction with the SPIRIT 2013 28 29 Explanation & Elaboration for important clarification on the items. Amendments to the 30 protocol should be tracked and dated. The SPIRIT checklist is copyrighted by the SPIRIT 31 Group under the Creative Commons “Attribution-NonCommercial-NoDerivs 3.0 Unported” 32 license. 33 34 35 36

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