The VISNAT trial is a PILOT STUDY due to absence of previous evidences in literature on using probiotic in newborns with perinatal asphyxia. It has been developed according to SPIRIT checklist (Supplementary 1). It is designed as a randomized, placebo-controlled, blinded, multicentre superiority trial with two parallel groups and a primary outcome of mortality and/or disability at 18 months of age. After informed consent (Supplementary 2) is obtained from both parents, randomization will be performed as block randomization with a 1:1 allocation using a computer-generated allocation sequence, while the allocation concealment will be performed using locked bags. Randomization data and allocation list will be stored in a secure place and will not be available to any of the components of the study apart of data collectors.
Participant files will be stored for a period of 10 years after completion of the study. All the components of the study will be blinded including: participants and their parents, healthcare providers, outcome assessors, data collectors, data analysts.
Patient and Public Involvement
Patient and Public Involvement was not sought in the design of this study.
Participants: inclusion/exclusion criteria
- Newborns with gestational age >/= 35 weeks and birth weight >/= 1800 grams
- Intrapartum asphyxia defined according at least one of the following:
- APGAR index at 10 minutes </=5;
- Resuscitation with endotracheal tube or mask IPPV for more than 10 minutes;
- pH </= 7 or excess of base >/= 12 mmol/l on arterial blood gas (ABG) analysis within the first 60 minutes of life
- Moderate/severe Hypoxic-ischemic encephalopathy assessed between 30 and 60 minutes of life according to Sarnat & Sarnat definition
- Hypothermic treatment. According to the Italian national guidelines on Perinatal Asphyxia and Therapeutic Hypothermia, the hypothermic therapy will be started in the first six hours of life and will last 72 hours.
- Inability to obtain the informed consent from both parents
- Congenital major malformations or syndromes
- Surgical diseases
No concomitant care and interventions are prohibited during the trial.
Setting and recruitment
In order to reach the prespecified sample size (see below 13. Statistical Issue: hypothesis, sample size and power) the VISNAT trial will involve 9 centres:
- Azienda Ospedaliera Universitaria Consorziale Policlinico di Bari ([email protected]) (coordinator centre)
- Azienda Ospedaliera Regionale San Carlo di Potenza (Camilla Gizzi; [email protected])
- Azienda Ospedaliera “V. Fazzi” di Lecce (Giuseppe Presta; [email protected])
- Azienda Ospedaliera “A. Perrino” di Brindisi (Enrico Rosati)
- Azienda Ospedaliera Universitaria di Foggia – Ospedali Riuniti (Giuseppe Popolo; [email protected])
- Azienda Ospedaliera Universitaria “Federico II” di Napoli (Maria Vendemmia)
- Azienda Ospedaliera Universitaria di Modena (Licia Lugli)
- Azienda Ospedaliera Universitaria “Sant’Anna” di Ferrara (Elisa Ballardini)
- Azienda Ospedaliera “Bolognini” di Seriate (Felicia Varsalone)
Each clinical centre involved in the VISNAT was chosen based on documentation for patient availability.
The coordinator centre will be the Neonatal Intensive Care Unit “A.Mautone” of Policlinico di Bari. It is an academic department specialized in the care of newborns at risk due to several conditions such as extreme prematurity, surgical and cardiac diseases, metabolic diseases, neurological diseases and perinatal asphyxia treated with systemic hypothermia. Our research team is made of neonatologists with considerable experience in intensive care, neonatal gastroenterology and neurology, supported by a group of residents and young paediatricians constantly involved in research and clinical trials. Our team also actively collaborates with other academic units, as well as, Neuroradiology unit for brain MRI in asphyxiated infants treated with systemic hypothermia and Laboratory unit for all blood chemistry tests. During time we have performed many research protocols with scientific papers published in peer-reviewed scientific journal.
Each of the others centres has a Neonatal Intensive Care Unit and has been chosen based on patient recruitment capability.
After obtaining the informed consent, the healthcare providers will enrol the patients that meet the inclusion criteria and proceed to randomization. All the interventions will take place in the Neonatal Intensive Care Units.
The recruitment period will extend over 2 years, while the follow-up will last up to 2 years.
The experimental intervention will consist in administrating a high-dose multi-strain probiotic (SIVOMIXXTM) for 30 days in addition to hypothermic therapy. The administration of probiotics will begin during the first 24 hours of hypothermia, at the dose of 1 capsule open in 5 millilitre of water. It will be administered daily for 30 days, either via nasogastric tube or orally.
The comparison intervention will consist in administrating placebo for 30 days in addition to hypothermic therapy. The administration of placebo will begin during the first 24 hours of hypothermia, at the dose of 1 capsule open in 5 millilitre of water. It will be administered daily for 30 days, either via nasogastric tube or orally.
Type of milk
Newborns will not be randomized for type of milk. Mothers will be encouraged to give their own breast milk. In case of unavailability of breast milk, all enrolled newborns will be fed with the same artificial milk.
The type of milk (breast/artificial) will be registered in clinical notes.
Clinical assessment, laboratory tests and brain imaging (Supplementary 3)
- Weekly auxological data (weight, height, head circumference) will be recorded during hospitalisation.
Upon discharge, enrolled patients will be followed up in the outpatient service with periodical clinical evaluation.
Bayley Mental Development Index (MDI) will be performed at 18 months to assess the degree of disability (no disability, mild, moderate or severe disability).
- Blood sample (3 ml) for inflammatory cytokines levels (IL-1β, IL-6, IL-8 and TNF-α) will be collected within 24 hours of life and then daily throughout the 72-hour hypothermic treatment and at the end of probiotic supplementation (day 30). The analysis of the samples will be performed by ELISA assay at the coordinator centre (Dr. Thea Magrone - Department of Basic Medical Sciences, Neurosciences and Sensory Organs, A.O.U. Policlinico of Bari). Blood sample (3 ml) for Tau and neurofilament light protein levels will be collected within 24 hours of life, daily during hypothermia and at the end of the intervention (day 30) and analysed by ELISA assay at the coordinator centre (Prof. Salvatore Scacco - Department of Basic Medical Sciences, Neurosciences and Sensory Organs, A.O.U. Policlinico of Bari).
A stool sample will be collected for microbiome analysis in every enrolled newborn at the end of hypothermic treatment (i.e. before the beginning of oral feeding) and at the end of probiotic treatment. The analysis of the samples will be performed by Real-time PCR at the Department of Microbiology – University “La Sapienza”, Rome (Prof. Paola Mastromarino).
- According to the Italian national guidelines on neonatal asphyxia and therapeutic hypothermia, brain MRI will be scheduled at the end of the first week of life and during the second month of life.
Primary outcome will be to compare mortality and/or disability at 18 month of age between the two groups.
Disability will be assessed by means of the Bayley MDI.
Severe disability will be defined by any of the following: Bayley MDI < 70; Gross Motor Function (GMF) level of 3-5; blindness or profound hearing loss requiring amplification.
Moderate disability will be defined as Bayley MDI between 70-84 and any of the following: GMF level = 2, a seizure disorder or a hearing deficit.
Mild disability will be defined by either Bayley MDI of 70-84 alone or a MDI > 85 and any of the following: GMF level = 2, seizure disorder or hearing loss not requiring amplification.
Infants with Bayley MDI > 85, GMF level = 1, absence of any neurosensory deficits will be defined as “no disability”.
Secondary outcomes will be to compare inflammatory cytokines level and brain injury biomarkers (i.e. Tau and neurofilament light protein) between groups.
A subgroup analysis will be performed stratifying patients according to both probiotic/placebo treatment and breast/artificial milk. The aim will be to assess changes in gut microbiota and compare oral tolerance, the latter being defined as the number of days to reach full enteral feeding (140 ml/kg), in each subgroup.
Statistical Issue: hypothesis, sample size and power
This is prospective superiority study. The sample size, due to absence of previous evidences in literature on using probiotic in newborns with perinatal asphyxia, was established on the bases of TOBY’s results in which 45% of mortality and/or disability in the group with hypothermic therapy was observed.19 Hypotizing a 30% (with a superiority margin of 10% ) of reduction in the proportion of mortality and/or disability in the group with a high-dose multi-strain probiotic in addition to hypothermic therapy respect to the hypothermic treatment group, a power 1-b=0.80 and a significant level a=0.05, a sample of 55 newborns for each group are needed. Considering a 10% of dropout we decided to enrol 120 newborns over a two-year period (60 probiotics, 60 placebo).20
Statistical analysis will be performed by Prof. Margherita Fanelli.
The primary outcome will be evaluated comparing proportions of mortality and/or disability by means of z test. The effect of probiotics on the primary outcome will be evaluated adjusting for the centre and some clinically relevant variables (gestational age, birth weight) by means of logistic regression model. A survival analysis will be performed by Kaplan Meyer Overall Survival and Free Survival curves that will be compared by Log Rank test. Cox regression models will be built to evaluate the effects of probiotic and other clinically variables on the survival time.
The secondary outcomes will be analysed as follows: binary outcomes will be compared between the two arms using Chi-square test, inflammatory cytokines level and brain injury biomarkers will be compared between the two arms using Student t test or Mann-Whitney test, as appropriate while number of days to reach full feeds will be evaluated by Kaplan Meyer curves compared between the two arms by log rank test. Logistic regression models will be built to evaluate the effect of probiotics on the binary outcomes, adjusting for the centre and some clinically relevant variables (gestational age, birth weight, Sarnat).
A p-value less than 0.05 will be considered statistically significant. Statistical analysis will be performed using SPSS vers. 21.