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Trial registered on ANZCTR

Registration number

ACTRN12622001290785p

Ethics application status

Submitted, not yet approved

Date submitted

19/09/2022

Date registered

4/10/2022

Date last updated

4/10/2022

Date data sharing statement initially provided

4/10/2022

Type of registration

Prospectively registered

Titles & IDs

Public title

Effect of Probiotic dose escalation on faecal microbiota and gut inflammation in extremely preterm infants – A randomised study

Scientific title

Effect of Probiotic dose escalation on faecal microbiota and gut inflammation in extremely preterm infants – A randomised study

Secondary ID [1]

Nil known

Universal Trial Number (UTN)

Trial acronym

Linked study record

Health condition

Health condition(s) or problem(s) studied:

Preterm gut inflammation

Necrotising enterocolitis

Condition category

Condition code

Inflammatory and Immune System

Other inflammatory or immune system disorders

Oral and Gastrointestinal

Other diseases of the mouth, teeth, oesophagus, digestive system including liver and colon

Intervention/exposure

Study type

Interventional

Description of intervention(s) / exposure

Probiotic mixture containing B. breve M-16V, B. longum subsp. infantis M-63, and B. longum subsp. longum BB536 (Total 3 x 10^9 colony forming unit (cfu) (Guaranteed) in one-gram sachets, Manufactured and supplied by Morinaga Milk Industries, Japan).The single dose (1.5×10^9 cfu/day) will be given via the feeding tube until reaching feeds of 50 mL/kg/day. It will be increased thereafter to 3×10^9 (given twice a day in divided doses), 6×10^9 (given twice a day in divided doses) or 9 ×10^9 cfu (given twice a day in divided doses) based on the allocation status of the enrolled infants, once feeds exceed 50 mL/kg/day. Probiotics will be continued until term equivalent age.
Contents of one sachet will be diluted with 2mL sterile water to make a final volume of 3mL reconstituted solution. This reconstituted solution should be administered immediately after reconstitution. This can be given orally or via tube at any time with regards to feeds.
Probiotics dose is charted in the drug chart for administration

Intervention code [1]

Prevention

Comparator / control treatment

Group of preterm neonates receiving 3×10^9 (given twice a day in divided doses) shall be the controls.

Control group

Dose comparison

Outcomes

Primary outcome [1]

1) Faecal Calprotectin levels.

Timepoint [1]

T1: As soon as possible after birth
T2: Four weeks after supplementation

Primary outcome [2]

2) Faecal microbiota analysis (composite outcome):(A) Faecal microbiota assessed using 16S ribosomal RNA gene sequencing. (B) Next-generation sequencing.

Timepoint [2]

T1: As soon as possible after birth
T2: Four weeks after supplementation

Secondary outcome [1]

Time to full feeds of 150 ml/kg/day assessed by medical records

Timepoint [1]

Before discharge

Secondary outcome [2]

Mental and psychomotor development indices at 24 months of age using Bayley Scale of Infant and Toddler Development (Bayley-III) assessment

Timepoint [2]

Developmental assessment: 18 to 24 months

Secondary outcome [3]

Necrotising enterocolitis >stage II assessed by radiology and/or intraoperative and/or histopathology finding

Timepoint [3]

Before discharge

Secondary outcome [4]

All-cause mortality: Data shall be collected from medical records.

Timepoint [4]

Before discharge

Secondary outcome [5]

Late onset sepsis as assessed by blood culture

Timepoint [5]

Before discharge

Secondary outcome [6]

Duration of parenteral nutrition: Data shall be collected from medical records

Timepoint [6]

Before discharge

Secondary outcome [7]

Hospital stay: Data shall be collected from medical records

Timepoint [7]

Before discharge

Secondary outcome [8]

Anthropometry (Weight, length and head circumference) at discharge. Nurses routinely measure weight, head circumference and length and enter in to medical record. Anthropometry data shall be collected from medical record.

Timepoint [8]

Before discharge

Secondary outcome [9]

Sepsis due to the administered bifidobacterial strains (Blood culture)

Timepoint [9]

Before discharge

Secondary outcome [10]

Abdominal distension: Data shall be collected from medical records.

Timepoint [10]

Before discharge

Secondary outcome [11]

Diarrhoea and vomiting: Data shall be collected fro medical records.

Timepoint [11]

Before discharge

Secondary outcome [12]

Faecal SCFA levels assessed by modified gas chromatography–mass spectrometry.

Timepoint [12]

T1: As soon as possible after birth
T2: 4 weeks after probiotics supplementation

Eligibility

Key inclusion criteria

: (1) gestation of <28 weeks, (2) readiness to commence on feeds or on feeds for <12 hours and (3) informed parental consent.

Minimum age

0 Days

Maximum age

14 Weeks

Sex

Both males and females

Can healthy volunteers participate?

Key exclusion criteria

(1) congenital malformations, (2) chromosomal aberrations, (3) not ready for feeds/on feeds for more than equal to 12 hours.

Study design

Purpose of the study

Prevention

Allocation to intervention

Randomised controlled trial

Procedure for enrolling a subject and allocating the treatment (allocation concealment procedures)

Opaque, sealed and coded envelopes will be used for randomisation. Allocation concealment will be optimised by prescribing allocation only after obtaining informed parental consent and recording basic neonatal data. The clinical trial pharmacist will supply the randomisation sequence and the sachets containing three-strain (B. breve M-16V, B. longum subsp. infantis M-63 and B. longum subsp. longum BB536; 1×10^9 of each strain/g sachet) probiotic manufactured by Morinaga Milk Industry Co., Japan, to the nursing staff.

Methods used to generate the sequence in which subjects will be randomised (sequence generation)

Group allocation will be based on computer-generated randomisation sequence in random block sizes of 2 and 4.

Masking / blinding

Blinded (masking used)

Who is / are masked / blinded?

The people assessing the outcomes
The people analysing the results/data

Intervention assignment

Parallel

Other design features

This will be an open label study. However, all outcome assessors, including microbiology experts assessing the laboratory-based outcomes, and developmental paediatricians assessing growth and neurodevelopment at 24 months of age using Bayley III will be masked to the allocation status of the enrolled infants.

Phase

Not Applicable

Type of endpoint/s

Safety/efficacy

Statistical methods / analysis

Primary outcome
Based on a mean reduction in calprotectin from baseline to 3–4 weeks post-supplementation of 51 µg/g (standard deviation: 133 µg/g) in 13 extremely preterm neonates in the live three-strain probiotic group of a small trial conducted in our unit, a total sample of 75 participants, 25 in each of three dosage groups, achieves 80% power to detect a difference of approximately 27 µg/g between group mean changes from baseline, while adjusting for baseline calprotectin levels. The calculation also allows for a dropout rate of approximately 10%. The sample size calculation was performed with Power Analysis and Sample Size (PASS 2019. NCSS, LLC. Kaysville, Utah, USA).
The microbiome will be compared between the three doses using the methods outlined in the included appendix. Samples from 25 children in each group (50,000 16S reads; ~50M shotgun reads) would have ~80% power to detect the compositional taxonomic difference of gut microbiome at 5% a level. Most of the parametric and non-parametric test (Wilcoxon Rank-Sum Tests) will have 80% power for individual metagenomics markers (e.g. abundance of particular species) given a non-negligible effect size (fold change or Cohen's d) at sample size 25. T-test will be preferred in our analysis by converting the raw variables into normal-distribution variables.
The power of detecting compositional difference between the groups at a given level of type 1 error (5%) depends on two factors— the sample size and the number of reads. The reads number of 50,000 is one of the common throughputs of 16S rRNA sequencing (it corresponds to around 5Gb shotgun metagenomic sequencing). We have used this fixed number of reads in our power evaluation. The number of samples in each group will influence the type 2 error (and power) detecting the compositional differences between two groups based on metagenomic simulations. The number of Monte-Carlo experiments was set to 5000 for each simulated sample.
The approach to bioinformatics analysis will be based on the principle of intention to treat.
Type of variables: (1) Categorical: Presence/absence of particular species or pathways; the clusters based on unsupervised clustering of gut microbial profile, etc. (2) Continuous: Alpha-diversity, richness, abundance of particular species or pathways, or the difference of these numeric variables (treatment vs baseline) etc.
Odds ratio (OR) vs relative risk (RR): The R package "sjstats" and “logisticRR” can be used to calculate the OR and the RR between binary outcomes (e.g. presence/absence of a particular species or a pathway).
Secondary outcomes
Continuous variables will be compared using the t test for normally distributed data and Wilcoxon rank sum test for skewed data. Categorical variables will be compared using the fisher’s exact test. Logistic regression analysis will be used for analysis of binary outcomes (primary outcome) to derive relative risk and 95% confidence intervals (CI). Linear regression analysis will be used for continuous outcomes to derive regression coefficients and respective CI. A p value <0.05 will be considered statistically significant.

Recruitment

Recruitment status

Not yet recruiting

Date of first participant enrolment

Anticipated

2/02/2023

Actual

Date of last participant enrolment

Anticipated

31/12/2023

Actual

Date of last data collection

Anticipated

31/01/2024

Actual

Sample size

Target

Accrual to date

Final

Recruitment in Australia

Recruitment state(s)

Recruitment hospital [1]

King Edward Memorial Hospital - Subiaco

Recruitment postcode(s) [1]

6008 - Subiaco

Funding & Sponsors

Funding source category [1]

Self funded/Unfunded

Name [1]

Address [1]

Country [1]

Primary sponsor type

Government body

Name

Child and adoloscent health service

Address

Department of Neonatology
King Edward memorial Hospital
374 Bagot Road
Subiaco 6008
Western Australia

Country

Australia

Secondary sponsor category [1]

Individual

Name [1]

Chandra Parakash Rath

Address [1]

Department of Neonatology
374 Bagot Road
Kind Edward Memorial Hospital
Subiaco WA-6008

Country [1]

Australia

Ethics approval

Ethics application status

Submitted, not yet approved

Ethics committee name [1]

Child and adoloscent health service human research ethics committee

Ethics committee address [1]

Office 5E, Perth Children’s Hospital
15 Hospital Avenue, Nedlands WA 6009

Ethics committee country [1]

Australia

Date submitted for ethics approval [1]

29/09/2022

Approval date [1]

Ethics approval number [1]

Summary

Brief summary

Premature babies (born before 8 months of pregnancy) are at risk necrotising enterocolitis (NEC), a potentially serious inflammatory condition of the bowel, as well as hospital acquired infections, and poor nutrition. Complications of prematurity such as these, especially NEC, increase the risk of death, and long-term disability. The risk of these complication is high especially in extremely premature babies born before 28 weeks of pregnancy. Probiotics are beneficial bacteria which have been shown to significantly reduce the risk of death, NEC, and hospital acquired infections whilst facilitating nutrition in very premature babies. Probiotic supplementation for very premature babies is a standard practice in all neonatal intensive care units (NICU) in Australia, New Zealand and many units in other countries.
We currently administer a daily dose of 3 billion probiotic bacteria for premature babies in our unit using a product imported from Japan, under a special permission from the Australian government. This dose is based on our research using this product and studies of other probiotics. This study is designed to assess if a dose higher than 3 billion bacteria per day is more effective whilst being safe in reducing inflammation and improving the balance of beneficial versus potentially harmful bacteria in the gut of extremely premature babies. 
Specifically, the proposed study will compare the dose of 3 billion against 6 and 9 billion bacteria per day in extremely premature babies using the stool (poo) samples. We think that babies who receive higher dose of probiotics will have reduced gut inflammation, more beneficial and less harmful  bacteria in the gut and overall health compared to those who receive lower dose.

Trial website

Trial related presentations / publications

Public notes

Contacts

Principal investigator

Name

Dr Chandra Prakash Rath

Address

Department of Neonatology
King Edward Memorial Hospital for Women
374 Bagot road
Subiaco- 6008
WA

Country

Australia

Phone

+61452549299

Fax

chandra.rath@health.wa.gov.au

Contact person for public queries

Name

Chandra Prakash Rath

Address

Department of Neonatology
King Edward Memorial Hospital for Women
374 Bagot Road
Subiaco- 6008
WA

Country

Australia

Phone

+61 0864582222

Fax

chandra.rath@health.wa.gov.au

Contact person for scientific queries

Name

Chandra Prakash Rath

Address

Department of Neonatology
King Edward Memorial Hospital for Women
374 Bagot Road
Subiaco- 6008
WA

Country

Australia

Phone

+61 0864582222

Fax

chandra.rath@health.wa.gov.au

Data sharing statement

Will individual participant data (IPD) for this trial be available (including data dictionaries)?

Yes

What data in particular will be shared?

De-identified individual data shall be available for sharing.

When will data be available (start and end dates)?

Immediately following publication to 15 years after that.(After approval of institutional ethics committee)

Available to whom?

Researchers who will provide a methodologically sound proposal.

Available for what types of analyses?

For approved proposals and IPD meta-analysis.

How or where can data be obtained?

By contacting the principal investigator (chandra.rath@health.wa.gov.au)

What supporting documents are/will be available?

Doc. No.	Type	Citation	Link	Email	Other Details	Attachment
17141	Study protocol				Attachment	384650-(Uploaded-27-09-2022-00-58-31)-Study-related document.docx
17142	Informed consent form				Attachment	384650-(Uploaded-19-09-2022-15-11-21)-Study-related document.docx

Results publications and other study-related documents

Documents added manually

No documents have been uploaded by study researchers.

Documents added automatically

No additional documents have been identified.

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