Probiotic Supplementation in Extremely Preterm Infants in Scandinavia

Study plan

PURPOSE AND AIMS Studies have shown conclusive results on reduced incidence of necrotizing enterocolitis (NEC) in preterm infants born after 28 gestational weeks (1, 2). However, previous research has been underpowered for extremely preterm (EPT) infants and thus, the efficacy in this group of infants is not fully eluted. Therefore, the Swedish guidelines for probiotic supplementation in neonatal care currently excludes EPT infants from being eligible to receive probiotic supplementation (3). Notably, EPT infants is the patient group with highest mortality and NEC rate, and thus most at need for strategies that can reduce this incidence (4). We believe our study design will enable to answer if probiotic supplementation can be expanded to also include EPT infants.

Overall aim The overall aim is to determine whether early probiotic supplementation can be used as a preventable nutritional intervention to reduce the risk of NEC and neonatal mortality among EPT infants (born less than 28 gestational weeks) using a double-blinded randomized control design. Probiotic supplement effect on other common neonatal morbidities will also be studied. The study will be carried out at the neonatal intensive care unit (NICU) at Karolinska University Hospital and collaborating tertiary neonatal centres in Sweden and in Denmark.

Primary aim

1. To determine whether the usage of probiotic supplementation will reduce the incidence of a composite endpoint of NEC and neonatal mortality among EPT infants.

Secondary aims 2. To determine whether probiotic supplementation will reduce the incidence of NEC. 3. To determine whether probiotic supplementation will reduce the incidence of neonatal mortality.

3\. To determine whether probiotic supplementation will reduce the incidence of neonatal sepsis 4. To determine whether probiotic supplementation will reduce the rate of prescription and duration of antibiotic use during hospital stay.

5\. To determine whether probiotic supplementation will reduce duration of hospital stay.

6\. To determine whether probiotic supplementation affect development of microbiome.

Tertiary aims

1. To assess whether probiotic supplementation will reduce the incidence of enteral feeding intolerance. 2. To assess postnatal growth and body composition by following participants post-NICU hospitalization at Karolinska University Hospital.

SURVEY OF THE FIELD Care of preterm infants has improved considerably during the last decade. Despite this, about 20% of EPT infants (born \<28 gestational weeks) in Sweden still dies (4). In EPT infants, NEC is a common cause of death (5). NEC is a gastrointestinal disease that causes an inflammatory response and is the most severe gastrointestinal-related morbidity in preterm infants (5-7). The pathogenesis of NEC is multifactorial and not fully understood, but the principle underlying cause believes to be immature gastrointestinal system. The current conservative treatment for NEC is exclusive parental nutrition and antibiotics. In up to 50% of cases, gastrointestinal surgery is necessary.

In a systematic review and meta-analysis from 2021 summarizing 45 trials including 12 320 infants, it was observed that a combination of Bifidobacterium and Lactobacillus was associated with a 54% lower rate of NEC and a 44% lower rate of total mortality (1). The ProPrems trial, conducted in Australia and New Zealand randomized 1099 infants born \<32 gestational weeks. In the ProPrems trial, a probiotic combination B. infantis Bb-02, B. lactis Bb-12, and Str. thermophilus TH4 was tested (2). However, this trial was underpowered for EPT infants and thus, the efficacy and safety cannot be generalized to this vulnerably patient group. The recent position paper from year 2020 on "Probiotics and Preterm Infants" by the European Society for Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) has investigated the results from existing randomized controlled trials, meta-analysis, and systematic reviews (8). They concluded that two probiotic supplements can be used on infants with birth weight \<1500 grams. The committee recommends providing either L. rhamnosus GG ATCC53103 or the combination of B. infantis Bb-02, B. lactis Bb-12, and Str. thermophilus TH4 which will be used in our study. Because of uncertainty regarding efficacy for EPT infants, the Swedish recommendations only include infants born above 28 gestational weeks (4). There is, to our knowledge, no ongoing randomized controlled trials investigating probiotic supplements to EPT infants. We believe that our research will provide high-quality evidence and that study results will contribute to the knowledge on whether current recommendations on probiotics supplementation can be expanded to also include EPT infants in Sweden, Denmark and that the results can be generalized globally.

STUDY DESIGN We will conduct a double-blinded randomized trial testing the effect of probiotic supplementation on the incidence of NEC and neonatal mortality. The study will be performed at Karolinska University Hospital in Solna and Huddinge and 10 other tertiary neonatal units in Sweden and Denmark. The trial will be designed in accordance with recommendations for interventional trials by the SPIRIT Statement (9) and Consolidated Standards for Reporting of Trials CONSORT guidelines (10).

POPULATION For all studies in the current proposal, we will adapt a double-blinded multicenter randomized controlled and registry-based trial examining the effect of probiotic supplementation on EPT infants. We will recruit EPT infants, born between 22 0/7 and 27 6/7 weeks of gestation. The recruitment period will be between August 2022 and June 2026 or until enough patients have been included. Infants will be recruited within 72 hours from birth, legal guardians will be informed about the study and asked to participate. Informed written consent will be retrieved prior to inclusion. Patients with severe complications and low chance of survival or major congenital abnormalities or patients participating in other trial with the same or overlapping outcome measures will be excluded.

The inclusion of patients in the trial will be performed in Sweden and Denmark at ten different hospitals, the patient may however be relocated to other hospitals during the trial. In case of relocation prior to completion (34w GA) continuation of the intervention or control will be granted under the supervision of the referring hospital. Included hospitals in Sweden are Karolinska University Hospital in Solna and Huddinge, Skåne University Hospital, Sahlgrenska University Hospital, Linköping University Hospital, Umeå University Hospital and Uppsala University Hospital. Included hospitals in Denmark are Rigshospitalet Copenhagen, Rigshospitalet Glostrup, Aarhus University Hospital and Odense University Hospital. The inclusion of patient will start in August of 2022 and prolong approximately until December of 2026 or until sufficient number of patients has been included.

Enrollment, randomization and blinding Clinicians will approach the parents for enrollment during the first 48 hours postpartum or antenatally if appropriate. After the parents have given written consent and patient given a Study-ID, the legal guardian will be randomly assigned to intervention- or control group in a 1:1 fashion. Randomization will be done within 24 hours after parents have given written consent. The study will be double-blinded. The preparation for intervention- and control group will be made by assistant nurses working in the nutrition kitchen where the enteral nutrition is being prepared for all patients at NICUs in Sweden. Thus, the medical and nursing teams caring for the infants, as well as participating researchers will be unaware of the type of supplementation and will not have knowledge on whether group A or B is receiving the probiotic supplement. See detailed description about enrollment, randomization- and blinding process under heading "Material: Patient selection - population, sample".

INTERVENTION The intervention product, commercially known as ProPrems®, is a combination of (one billion freeze-dried bacteria per 0.5 g in a maltodextrin base powder: Bifidobacterium infantis Bb-02 (DSM 33361) 300 million, Bifidobacterium lactis (BB-12®) 350 million, and Streptococcus thermophilus (TH-4®) 350 million. The supplementation of probiotics or pure breastmilk will be administered by gastric tube and starts when the infant tolerates at least 3 mL breastmilk per meal. For the intervention group, the standard dose of 0.5 grams will be mixed with 3 mL breastmilk. If the mother's breastmilk is available this will be used as first choice, if not, donated breastmilk will be used. The dose will be administered once daily until 34 weeks of gestational age and will not change with increasing age and weight. If enteral feeds and probiotic supplementation feeds are not tolerated after initiation and have to be discontinued or reduced below 3ml/meal, supplementation will be paused and only started again when the infant is tolerating 3 mL per meal again.

CONTROL The control group will receive 3 mL of mothers' own breastmilk or donated breastmilk without the probiotic supplement and administered by gastric tube. This may however be administered as bolus or continues feeds according to local feeding strategy.

OUTCOME Primary outcome variables are NEC rate and overall mortality. Secondary outcomes are incidence of sepsis, duration of hospital stay, use of antibiotics. Tertiary outcomes include feeding tolerance, and growth and body composition after hospital discharge.

RESEARCH QUESTIONS Primary aim and secondary aim 1 and 2: Do probiotic supplementation reduce the incidence of a composite endpoint of NEC and neonatal mortality among EPT infants? Participants: EPT infants, 22 0/7 to 26 6/7 gestational weeks, at all sites. Data collection: Incidence of NEC and mortality will be registered and retrieved from the Swedish Neonatal Quality (SNQ) Register.

Incidence of NEC will be defined according to Bell's stage II and above. Bell stage II includes clinical signs with abdominal symptoms and radiological findings. Stage III includes findings in stage II in addition to severe clinical condition such as need of intubation and/or need for surgery. Registered incidence of NEC in SNQ will be assessed by a neonatologist before inclusion in the analyses and must include the following:

1. Abdominal symptoms in connection to NEC episode 2. Radiological findings 3. Possible differential diagnoses 4. Abdominal surgery Data analysis: Relative risk (RR) and 95% confidence intervals (CI) using standardized logistic regression (see "STATISTICAL METHODS" for more information.

Secondary aim 3: Does probiotic supplementation reduce the incidence of neonatal sepsis? Participants: EPT infants, 22 0/7 to 26 6/7 gestational weeks, at all sites. Data collection: Incidence of neonatal sepsis will be registered and retrieved from SNQ.

Incidence of sepsis will be defined as blood culture proven sepsis and/or laboratory inflammatory response were at least one of the following must be confirmed:

1. LPK \< 5 or \> 20 (x109 cells/L) 2. TPK \< 100 (x109 cells/L) 3. CRP \> 15 mg/L Data analysis: RR and 95% CI using standardized logistic regression.

Secondary aim 4: Does probiotic supplementation reduce the rate of prescription and duration of antibiotic use during hospital stay? Participants: EPT infants, 22 0/7 to 26 6/7 gestational weeks, at all sites. Data collection: Prescription and duration of antibiotics will be registered and retrieved from SNQ.

Data analysis: RR and 95% CI using standardized logistic regression.

Secondary aim 5: Does probiotic supplementation reduce duration of hospital stay? Participants: EPT infants, 22 0/7 to 26 6/7 gestational weeks, at all sites. Data collection: Duration (number of days) of hospital stay will be registered and retrieved from SNQ.

D

• Sepsis [Срок оценки: Depending on gestational age at birth (22+0 wGA - 27+6 wGA) up to the end of intervention at 34 w GA + 2 weeks. This gives a time frame of 8-14 weeks]
• Anibiotic use [Срок оценки: Depending on gestational age at birth (22+0 wGA - 27+6 wGA) up to the end of intervention at 34 w GA + 2 weeks. This gives a time frame of 8-14 weeks]
• Anibiotic use [Срок оценки: Depending on gestational age at birth (22+0 wGA - 27+6 wGA) up to the end of intervention at 34 w GA + 2 weeks. This gives a time frame of 8-14 weeks]
• Feeding intolerance [Срок оценки: Depending on gestational age at birth (22+0 wGA - 27+6 wGA) up to the end of intervention at 34 w GA + 2 weeks. This gives a time frame of 8-14 weeks]
• Gut microbiome analysis [Срок оценки: immediately after inclusion, 14 days of life, 34 week of gestational age and 12 months corrected age.]