Introduction
Background and rationale {6a}
Risk of morbidity and mortality (complete cohort)
Pathogenesis of NEC
Pathogenesis of late-onset sepsis
Antibiotic treatment
Probiotics as a nutritional intervention strategy in preterm infants
The preventative effect of B. infantis Bb-02, B. lactis Bb-12, and Str. Thermophilus TH4 on NEC and mortality
The preventative effect of B. infantis Bb-02, B. lactis Bb-12, and Str. Thermophilus TH4 on LOS
Study rationale
Feeding tolerance and postnatal growth (sub-cohort)
Postnatal growth failure
Feeding tolerance
Probiotics and feeding tolerance
Objectives {7}
Primary aim
Secondary aims
Tertiary aims
Trial design {8}
Methods: participants, interventions, and outcomes
Study setting {9}
Eligibility criteria {10}
Inclusion criteria
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Extremely preterm infants (born before 28 completed gestational weeks)
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Infants enrolled within 72 h after birth with informed consent
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Infants born at Karolinska University Hospital (sub-cohort)
Exclusion criteria
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Infants with severe complications with low chance of survival detected within 72 h after birth
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Infants with major congenital anomalies
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Infants participating in another interventional trial where main outcome includes incidence of NEC
Informed consent {26a}
Additional consent provisions for collection and use of participant data and biological specimens {26b}
Interventions
Choice of probiotic {6b}
Intervention description {11a}
Criteria for discontinuing or modifying the allocated interventions {11b}
Strategies to improve adherence to interventions {11c}
Relevant concomitant care permitted or prohibited during the trial {11d}
Provisions for post-trial care {30}
Outcomes {12}
Outcome variables complete cohort | |||
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Primary outcome variables | Analysis metrics | Method of aggregation | Time point |
Necrotizing enterocolitis | Incidence rate | Mean/Median | During study intervention (until 34 weeks of gestational age) |
Necrotizing enterocolitis Bell Stage >IIa Verified | Incidence rate | Mean/Median | During study intervention (until 34 weeks of gestational age) |
Necrotizing enterocolitis Bell Stage >IIIa Verified | Incidence rate | Mean/Median | During study intervention (until 34 weeks of gestational age) |
Necrotizing enterocolitis Operation | Date, time | Mean/Median | During study intervention (until 34 weeks of gestational age) |
Abdominal symptoms | Incidence of free gas, intermural intestinal gas, inflated abdomen discolored abdomen, stagnant intestinal loop >24 h | Sum | During study intervention (until 34 weeks of gestational age) |
Differential gastrointestinal diagnoses | Spontaneous intestinal perforation, Malrotation/volvulus, other | Sum | During study intervention (until 34 weeks of gestational age) |
X-ray of the abdomen | Incidence rate | Mean/Median | During study intervention (until 34 weeks of gestational age) |
Death | Incidence rate | Mean/Median | Third day postpartum until 40 weeks of gestational age |
Secondary outcome variables | |||
Late-onset sepsis | Incidence rate | Mean/Median | During study intervention (until 34 weeks of gestational age) |
Verified late-onset sepsisb | Incidence rate | Mean/Median | During study intervention (until 34 weeks of gestational age) |
Late-onset sepsis Culture-proven | Type of pathogen | Sum | During study intervention (until 34 weeks of gestational age) |
Late-onset sepsis date | Date of onset | Sum | During study intervention (until 34 weeks of gestational age) |
Late-onset sepsis Peripheral line at onset | Yes/No | Mean/Median | During study intervention (until 34 weeks of gestational age) |
Late-onset sepsis Central line at onset | Yes/No | Mean/Median | During study intervention (until 34 weeks of gestational age) |
Use of antibiotics | Rate of prescription (number) Duration of treatment (days) | Mean/Median | During study intervention and until 40 weeks of gestational age |
Length of stay | Number of days | Mean/Median | Length of stay at the neonatal intensive care unit Length of stay at homecare |
Effect on microbiome | Fecal samples | 1. Directly after inclusion ± 2 days 2. 14 ± 2 days gestational age 3. 34 weeks ± 2 days gestational age 4. 12 months corrected age ± 2 weeks | |
Serious adverse event (Necrotizing enterocolitis, death and late-onset sepsis included) | |||
Bronchopulmonary dysplasia | Yes/No | Mean/Median | During study intervention (until 34 weeks of gestational age) |
Respiratory distress syndrome | Yes/No | Mean/Median | From birth and during study intervention (until 34 weeks of gestational age) |
Patent ductus arteriosus (PDA) | Yes/No | Mean/Median | From birth and during study intervention (until 34 weeks of gestational age) |
Pulmonary hypertension | Yes/No | Mean/Median | During study intervention (until 34 weeks of gestational age) |
Intraventricular hemorrhage | Yes/No | Mean/Median | During study intervention (until 34 weeks of gestational age) |
Hydrocephalus | Yes/No | Mean/Median | During study intervention (until 34 weeks of gestational age) |
Retinopathy of prematurity | Yes/No | Mean/Median | During study intervention (until 34 weeks of gestational age) |
Characteristics/covariates | |||
Pregnancies | Number | Mean/Median | Baseline |
Preeclampsia | Yes/No | Mean/Median | Baseline |
Chorioamnionitis | Yes/No | Mean/Median | Baseline |
Infection pregnancy | Number of infections | Sum | Baseline |
Antibiotics during pregnancy | Period | Sum | Baseline |
Mode of delivery | Vaginal or Cesarean-section | Mean/Median | Baseline |
Apgar-score | At 1, 5 and 10 minutes | Mean/Median, Sum | Baseline |
Gender (x/y) | Boy/girl | Mean/Median | Baseline |
Multiple birth | Yes/No | Mean/Median | Baseline |
Gestational age | Number (gestation week) | Mean/Median | Baseline |
Birth weight | Kilograms | Mean/Median | Baseline |
Birth length | Centimeters | Mean/Median | Baseline |
Birth head circumference | Centimeters | Mean/Median | Baseline |
Z-scores from growth chart at birth | Standard deviation | Baseline | |
Respiratory support Intubation | Period of treatment | Mean/Median | From birth until 40 weeks of gestational age |
Respiratory support Continuous positive airway pressure (CPAP) | Period of treatment | Mean/Median | From birth until 40 weeks of gestational age |
Respiratory support High-flow nasal cannula | Period of treatment | Mean/Median | From birth until 40 weeks of gestational age |
Respiratory support Low-flow nasal cannula | Period of treatment | Mean/Median | From birth until 40 weeks of gestational age |
Patent ductus arteriosus closure strategies Paracetamol | Period of treatment | Mean/Median | From birth until 40 weeks of gestational age |
Patent ductus arteriosus closure strategies Operation | Date | Mean/Median, Sum | From birth until 40 weeks of gestational age |
Participant timeline {13}
Participant timeline for the sub cohort
Sample size {14}
Recruitment {15}
Assignment of interventions: allocation
Sequence generation {16a}
Concealment mechanism {16b}
Implementation {16c}
Assignment of interventions: blinding
Who will be blinded {17a}
Procedure for unblinding if needed {17b}
Data collection and management
Plans for assessment and collection of outcomes {18a}
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Date of randomization
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Group assignment
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Date of start of intervention
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Date of pause of intervention
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Infection during pregnancy
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Antenatal antibiotics
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If stool samples were taken (at baseline, 14 days post menstrual age (PMA), 34 weeks PMA, and 12 months corrected age)
Diagnostic/validation of NEC
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Abdominal symptoms in connection to NEC episode
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Radiological findings
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Possible differential diagnoses
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Abdominal surgery
Diagnostic/validation of LOS
Data collection and assessment for sub-cohort
Outcome variables sub cohort | |||
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Outcome variables - feeding tolerance | Analysis metrics | Method of aggregation | Time point |
Enteral nutrition | Number of days until full enteral feeds | Mean/Median | During study intervention (until 34 weeks of gestational age) |
Parental nutrition | Number of days with parental nutrition | Mean/Median | During study intervention (until 34 weeks of gestational age) |
Interruptions of enteral nutrition | Number of enteral nutrition interruptions >8 h | Mean/Median | During study intervention (until 34 weeks of gestational age) |
Enteral nutrition decreased due to clinical instability | Number of EN decrease >50% | Mean/Median | During study intervention (until 34 weeks of gestational age) |
Use of breastmilk fortifiers | Number of days until breastmilk fortifiers is induced | Mean/Median | During study intervention (until 34 weeks of gestational age) |
Feeding tube requirement | Number of days requiring a feeding tube | Mean/Median | During study intervention and until 40 weeks of gestational age |
Macronutrient intake | Amount of macronutrients given (kcal/kg/day) | Mean/Median | During study intervention (until 34 weeks of gestational age) |
Micronutrient intake | Amount of micronutrients given (μg or mg/kg/day) | Mean/Median | During study intervention (until 34 weeks of gestational age) |
Outcome variables - postnatal growth | |||
Attaining birth weight | Number of days | Mean/Median | After attaining birth weight after normal initial weight loss postpartum |
Body weight | Kilograms | Mean/Median | 1. Once every week during study intervention 2. 40 weeks of gestational age 3. 3 months of corrected age 4. 12 months of corrected age 5. 2.5 years of corrected age 6. 5.5 years of age |
Body length | Centimeters | Mean/Median | 1. Once every week during study intervention 2. 40 weeks of gestational age 3. 3 months of corrected age 4. 12 months of corrected age 5. 2.5 years of corrected age 6. 5.5 years of age |
Head circumference | Centimeters | Mean/Median | 1. Once every week during study intervention 2. 40 weeks of gestational age 3. 3 months of corrected age 4. 12 months of corrected age 5. 2.5 years of corrected age 6. 5.5 years of age |
Growth chart Z-score | Standard deviation | 1. Once every week during study intervention 2. 40 weeks of gestational age 3. 3 months of corrected age 4. 12 months of corrected age 5. 2.5 years of corrected age 6. 5.5 years of age | |
Body composition | Fat mass, fat-free mass (%) | Average | 3 months corrected age |
Characteristics/covariates | |||
Use of medical steroids | Period of treatment | Mean/Median | From birth until 5.5 years of age |
Use of tetracyclines | Period of treatment | Mean/Median | From birth until 5.5 years of age |
Type of enteral nutrition mothers’ own milk, donated breastmilk, formula | Percentages (%) | Proportion | During study intervention (until 34 weeks of gestational age) |
Breastmilk fortification | Type | Proportion | During study intervention (until 34 weeks of gestational age) |
Breastfeeding at discharge | Full, partly, none | Proportion | Discharge from neonatal intensive care unit/homecare |
Plans to promote participant retention and complete follow-up {18b}
Data management {19}
Confidentiality {27}
Plans for collection, laboratory evaluation, and storage of biological specimens for genetic or molecular analysis in this trial/future use {33}
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Directly after inclusion or at the timepoint when infant first produces stool
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14 ± 2 days gestational age
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34 weeks ± 2 days gestational age
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12 months corrected age ± 2 weeks
Statistical methods
Statistical methods for primary and secondary outcomes {20a}
Primary aim 1 and secondary aims 1–3
Secondary aims 4 and 5
Secondary aim 6
Tertiary aims 1–2
Interim analyses {21b}
Methods for additional analyses (e.g., subgroup analyses) {20b}
Methods in analysis to handle protocol non-adherence and any statistical methods to handle missing data {20c}
Plans to give access to the full protocol, participant-level data, and statistical code {31c}
Oversight and monitoring
Composition of the coordinating center and trial steering committee {5d}
Composition of the data monitoring committee, its role and reporting structure {21a}
Adverse event reporting and harms {22}
Adverse events
Serious adverse events
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Infections; LOS, meningitis, cytomegalovirus (CMV) infection
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Gastrointestinal; NEC, spontaneous intestinal perforations, volvulus, ileus, other malrotation
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Lung function; bronchopulmonary dysplasia (BPD), respiratory distress syndrome
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Circulation; patent ductus arteriosus (PDA), pulmonary hypertension
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Brain function; intraventricular hemorrhage (IVH), periventricular leukomalacia (PVL), hydrocephalus
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Retinopathy of prematurity (ROP)
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Death