Effect of allopurinol in addition to hypothermia treatment in neonates for hypoxic-ischemic brain injury on neurocognitive outcome (ALBINO): study protocol of a blinded randomized placebo-controlled parallel group multicenter trial for superiority (phase III).

BACKGROUND: Perinatal asphyxia and resulting hypoxic-ischemic encephalopathy is a major cause of death and long-term disability in term born neonates. Up to 20,000 infants each year are affected by HIE in Europe and even more in regions with lower level of perinatal care. The only established therapy to improve outcome in these infants is therapeutic hypothermia. Allopurinol is a xanthine oxidase inhibitor that reduces the production of oxygen radicals as superoxide, which contributes to secondary energy failure and apoptosis in neurons and glial cells after reperfusion of hypoxic brain tissue and may further improve outcome if administered in addition to therapeutic hypothermia. METHODS: This study on the effects of ALlopurinol in addition to hypothermia treatment for hypoxic-ischemic Brain Injury on Neurocognitive Outcome (ALBINO), is a European double-blinded randomized placebo-controlled parallel group multicenter trial (Phase III) to evaluate the effect of postnatal allopurinol administered in addition to standard of care (including therapeutic hypothermia if indicated) on the incidence of death and severe neurodevelopmental impairment at 24 months of age in newborns with perinatal hypoxic-ischemic insult and signs of potentially evolving encephalopathy. Allopurinol or placebo will be given in addition to therapeutic hypothermia (where indicated) to infants with a gestational age ≥ 36 weeks and a birth weight ≥ 2500 g, with severe perinatal asphyxia and potentially evolving encephalopathy. The primary endpoint of this study will be death or severe neurodevelopmental impairment versus survival without severe neurodevelopmental impairment at the age of two years. Effects on brain injury by magnetic resonance imaging and cerebral ultrasound, electric brain activity, concentrations of peroxidation products and S100B, will also be studied along with effects on heart function and pharmacokinetics of allopurinol after iv-infusion. DISCUSSION: This trial will provide data to assess the efficacy and safety of early postnatal allopurinol in term infants with evolving hypoxic-ischemic encephalopathy. If proven efficacious and safe, allopurinol could become part of a neuroprotective pharmacological treatment strategy in addition to therapeutic hypothermia in children with perinatal asphyxia. TRIAL REGISTRATION: NCT03162653, www.ClinicalTrials.gov , May 22, 2017.

Neuroactive metabolites and bile acids are altered in extremely premature infants with brain injury.

The gut microbiome is associated with pathological neurophysiological evolvement in extremely premature infants suffering from brain injury. The exact underlying mechanism and its associated metabolic signatures in infants are not fully understood. To decipher metabolite profiles linked to neonatal brain injury, we investigate the fecal and plasma metabolome of samples obtained from a cohort of 51 extremely premature infants at several time points, using liquid chromatography (LC)-high-resolution mass spectrometry (MS)-based untargeted metabolomics and LC-MS/MS-based targeted analysis for investigating bile acids and amidated bile acid conjugates. The data are integrated with 16S rRNA gene amplicon gut microbiome profiles as well as patient cytokine, growth factor, and T cell profiles. We find an early onset of differentiation in neuroactive metabolites between infants with and without brain injury. We detect several bacterially derived bile acid amino acid conjugates in plasma and feces. These results provide insights into the early-life metabolome of extremely premature infants.

Detailed statistical analysis plan for ALBINO: effect of Allopurinol in addition to hypothermia for hypoxic-ischemic Brain Injury on Neurocognitive Outcome - a blinded randomized placebo-controlled parallel group multicenter trial for superiority (phase III).

BACKGROUND: Despite therapeutic hypothermia (TH) and neonatal intensive care, 45-50% of children affected by moderate-to-severe neonatal hypoxic-ischemic encephalopathy (HIE) die or suffer from long-term neurodevelopmental impairment. Additional neuroprotective therapies are sought, besides TH, to further improve the outcome of affected infants. Allopurinol - a xanthine oxidase inhibitor - reduced the production of oxygen radicals and subsequent brain damage in pre-clinical and preliminary human studies of cerebral ischemia and reperfusion, if administered before or early after the insult. This ALBINO trial aims to evaluate the efficacy and safety of allopurinol administered immediately after birth to (near-)term infants with early signs of HIE. METHODS/DESIGN: The ALBINO trial is an investigator-initiated, randomized, placebo-controlled, double-blinded, multi-national parallel group comparison for superiority investigating the effect of allopurinol in (near-)term infants with neonatal HIE. Primary endpoint is long-term outcome determined as survival with neurodevelopmental impairment versus death versus non-impaired survival at 2 years. RESULTS: The primary analysis with three mutually exclusive responses (healthy, death, composite outcome for impairment) will be on the intention-to-treat (ITT) population by a generalized logits model according to Bishop, Fienberg, Holland (Bishop YF, Discrete Multivariate Analysis: Therory and Practice, 1975) and ."will be stratified for the two treatment groups. DISCUSSION: The statistical analysis for the ALBINO study was defined in detail in the study protocol and implemented in this statistical analysis plan published prior to any data analysis. This is in accordance with the Declaration of Helsinki and the International Conference on Harmonization Good Clinical Practice guidelines. TRIAL REGISTRATION: ClinicalTrials.gov NCT03162653. Registered on 22 May 2017.

Inter-site generalizability of EEG based age prediction algorithms in the preterm infant.

Objective. To overcome the effects of site differences in EEG-based brain age prediction in preterm infants.Approach. We used a 'bag of features' with a combination function estimated using support vector regression (SVR) and feature selection (filter then wrapper) to predict post-menstrual age (PMA). The SVR was trained on a dataset containing 138 EEG recordings from 37 preterm infants (site 1). A separate set of 36 EEG recordings from 36 preterm infants was used to validate the age predictor (site 2). The feature distributions were compared between sites and a restricted feature set was constructed using only features that were not significantly different between sites. The mean absolute error between predicted age and PMA was used to define the accuracy of prediction and successful validation was defined as no significant differences in error between site 1 (cross-validation) and site 2.Main results. The age predictor based on all features and trained on site 1 was not validated on site 2 (p< 0.001; MAE site 1 = 1.0 weeks,n= 59 versus MAE site 2 = 2.1 weeks,n= 36). The MAE was improved by training on a restricted features set (MAE site 1 = 1.0 weeks,n= 59 versus MAE site 2 = 1.1 weeks,n= 36), resulting in a validated age predictor when applied to site 2 (p= 0.68). The features selected from the restricted feature set when training on site 1 closely aligned with features selected when trained on a combination of data from site 1 and site 2.Significance. The ability of EEG classifiers, such as brain age prediction, to maintain accuracy on data collected at other sites may be challenged by unexpected, site-dependent differences in EEG signals. Permitting a small amount of data leakage between sites improves generalization, leading towards universal methods of EEG interpretation in preterm infants.

Amplitude-integrated EEG pattern predicts further outcome in preterm infants.

Changes in EEG background activity are powerful but nonspecific markers of brain dysfunction. Early EEG and amplitude-integrated EEG (aEEG) pattern predict further neurodevelopmental outcome in term infants; however, sufficient data for prognostic value of aEEG in preterm infants are not available so far. The aim of the study was to evaluate whether aEEG predicts further outcome and to compare it to cerebral ultrasound assessment. In 143 preterm infants, aEEG within the first 2 wk of life and outcome data at 3 y of age (Bayley Scales) could be obtained.aEEG was classified into a graded score according to background activity, appearance of sleep-wake cycling, and occurrence of seizure activity. In preterm infants, aEEG was significantly associated with further outcome. Specificity was 73% for assessment within the first and increased to 95% in the second week of life, whereas sensitivity stayed nearly the same 87% (first week) to 83% (second week). Cerebral ultrasound showed a specificity of 86% within the first and second week, sensitivity also stayed nearly the same (74 and 75%). aEEG has a predictive value for later outcome in preterm infants and can be used as an early prognostic tool.

A Novel Approach for More Effective Emergency Equipment Storage: The Task-Based Package-Organized Neonatal Emergency Backpack.

Objectives: To evaluate a new task-based package-organized (TPO) neonatal emergency backpack and to compare it to the classical (ABC- and material-based) backpack. Methods: Simulation-based assessment of time to retrieve equipment for three different tasks [intraosseous access (IO), intubation and adrenaline administration] using the TPO and the classical emergency backpack was compared. Results: Equipment retrieval times for the three tasks were assessed for 24 nurses (12 intermediate care, 12 intensive care) and were significantly faster in the TPO than in the classical backpack (IO 33 vs. 75 s, p < 0.001; intubation 53 vs. 70 s, p = 0,001; adrenaline 22 vs. 45 s, p < 0.001). The number of missing items was significantly lower using the TPO backpack for IO and adrenaline retrieval (IO 0,9 vs. 2,3 items, p < 00001, adrenaline 0.04 vs. 1, p < 0.001) but not for intubation equipment (0.9 vs. 1, not significant). The subjective rating of overall clearness was significantly higher for the TPO compared with the classical backpack (5,9 vs. 3,5, p < 0.001). Conclusion: Task-based package organization of neonatal emergency backpacks is feasible and might be superior to ABC-/material-oriented storage.

Aberrant gut-microbiota-immune-brain axis development in premature neonates with brain damage.

Premature infants are at substantial risk for suffering from perinatal white matter injury. Though the gut microbiota has been implicated in early-life development, a detailed understanding of the gut-microbiota-immune-brain axis in premature neonates is lacking. Here, we profiled the gut microbiota, immunological, and neurophysiological development of 60 extremely premature infants, which received standard hospital care including antibiotics and probiotics. We found that maturation of electrocortical activity is suppressed in infants with severe brain damage. This is accompanied by elevated γδ T cell levels and increased T cell secretion of vascular endothelial growth factor and reduced secretion of neuroprotectants. Notably, Klebsiella overgrowth in the gut is highly predictive for brain damage and is associated with a pro-inflammatory immunological tone. These results suggest that aberrant development of the gut-microbiota-immune-brain axis may drive or exacerbate brain injury in extremely premature neonates and represents a promising target for novel intervention strategies.

Cerebrospinal fluid drainage in posthaemorrhagic ventricular dilatation leads to improvement in amplitude-integrated electroencephalographic activity.

AIM: Progressive posthaemorrhagic ventricular dilatation (PHVD) may induce abnormal amplitude-integrated electroencephalographic (aEEG) activity prior to clinical deterioration or significant cerebral ultrasound changes. These abnormalities might be ameliorated with cerebrospinal fluid (CSF) drainage. The aims of this study were to investigate the occurrence of aEEG-abnormalities with progressive PHVD in relation to clinical and cerebral ultrasound changes and to evaluate whether CSF drainage results in aEEG improvement. METHODS: aEEG and cerebral ultrasound scans were performed in 12 infants with PHVD, before and after CSF drainage, until normalization of aEEG occurred. RESULTS: aEEG was abnormal with progressive PHVD in all patients. Concurrently, 60% of the patients were clinically stable without deterioration in ultrasonographic cerebral abnormalities. Post drainage, continuous pattern was restored in all but one patient, whereas the frequency of discontinuous pattern decreased in nine patients and burst-suppression pattern decreased in all but one patient. Low-voltage pattern was only observed in one patient who suffered severe grade IV IVH and died one week after EVD placement. Sleep-wake cycling matured in 75%. CONCLUSION: These findings demonstrate the impact of CSF drainage on compromised aEEG-activity associated with PHVD. aEEG changes indicative of impaired cerebral function were apparent before clinical deterioration or major ultrasound changes. These changes were reversible with CSF drainage. aEEG should therefore be used in addition to clinical observation and ultrasound when monitoring PHVD.

Small volume enemas do not accelerate meconium evacuation in very low birth weight infants.

We hypothesized that small volume enemas accelerate meconium evacuation in very low birth weight (VLBW) infants. In a randomized controlled trial, VLBW infants (n = 81) received either repeated daily small volume enemas if complete spontaneous meconium passage failed within 24 h or no intervention. Small volume enemas did not accelerate complete meconium evacuation, which occurred after 6.0 to 9.6 (95% CI) d in the intervention group and after 7.7 to 11.0 (95% CI) d in the control group. No adverse events were observed. Daily administration of small volume enemas had no effect on total meconium evacuation defined by the time of last meconium passage.

3-D ultrasonographic imaging of the cerebral ventricular system in very low birth weight infants.

The purpose of the study was to assess reference ranges for lateral ventricular volume of very low birth weight (VLBW) infants using 3-D ultrasound (US). A total of 108 patients with birth weights < or =1500 g or mother's postmenstrual age < or =32 weeks were examined prospectively in a longitudinal study. Infants in conditions considered being potential confounders such as intraventricular haemorrhage (IVH) and periventricular leukomalacia (PVL) were not included in the calculations. Hence, 77 subjects remained for final statistical analysis. Mean postmenstrual age at birth was 194.5 (27 weeks and 5.5 days) +/- 14 SD days, mean birth weight was 972.5 +/- 236.3 SD g. Reference ranges for lateral ventricle volume were established from serial images. The exponential regression analyses revealed a weekly increase in volume of 6.3% (95% CI 4.4%-8.3%) and 6.6% (95% CI 4.7%-8.6%) in respect to the left and the right ventricle (p < 0.001). Postmenstrual age correlated significantly (p < or = 0.015) with ventricle volume. No significant association to head circumference could be determined. Establishment of reference values for the lateral ventricle volume of VLBW infants should facilitate application of 3-D US in routine diagnostics in neonatal intensive care units and detection of ventricular enlargement as a prediction of risk for poor neurodevelopmental outcome in high-risk cohorts.

Mortality and morbidity in extremely preterm infants (22 to 26 weeks of gestation): Austria 1999-2001.

OBJECTIVE: The aim of this retrospective study was to analyze the mortality and morbidity for extremely preterm infants with a gestational age from 22 to 26 weeks. All infants were born in Austria during the years 1999-2001. METHODS: Data were collected from 16 neonatal intensive care units in Austria. Main outcome criteria were mortality, the rates of chronic lung disease (CLD) and severe retinopathy of prematurity (ROP, stage > or =3) to determine the short-term outcome; the rate of cerebral palsy (CP) at the corrected age of twelve months to assess the long-term outcome. RESULTS: Overall, 796 preterm infants with a gestational age less than 27 weeks were born in Austria and 581 (73%) were registered as live-born infants. Of those live born, 508 (87%) were analyzed. The mortality rates were 83%, 76%, 43%, 26% and 13% for 22, 23, 24, 25 and 26 weeks' gestation, respectively. The rates of CLD were 33% (22 weeks), 36% (23 weeks), 42% (24 weeks), 31% (25 weeks) and 22% (26 weeks). The rates of ROP of stage > or =3 were 0% (22 weeks), 29% (23 weeks), 23% (24 weeks), 18% (25 weeks) and 10% (26 weeks). The rates of CP at the corrected age of 12 months were 33%, 50%, 33%, 26% and 25% for 22, 23, 24, 25 and 26 weeks' gestation, respectively. CONCLUSIONS: The results of this national study are in accordance with the international literature: mortality and morbidity increased with decreasing gestational age.

Background patterns and sleep-wake cycles on amplitude-integrated electroencephalography in preterms younger than 30 weeks gestational age with peri-/intraventricular haemorrhage.

AIM: The objective of this prospective study was to evaluate the influence of peri-/intraventricular haemorrhage (PIVH) grades I-IV on amplitude-integrated electroencephalographic (aEEG) activity in preterm infants<30 weeks gestational age (GA). METHODS: The aEEG tracings of the first 2 weeks of life of 56 preterm infants younger than 30 weeks GA (2 groups: group A=23-26 weeks GA, group B=27-29 weeks GA) born during a 4-year period with PIVH grades I-IV were assessed for the relative duration of four background aEEG activity patterns (continuous pattern, discontinuous high-voltage pattern, discontinuous low-voltage pattern and nearly isoelectric pattern), the presence of seizure activity and the appearance of sleep-wake cycles and compared to the tracings of 75 neurologically healthy preterms without PIVH. RESULTS: Analysis of aEEG background activity showed a decrease of continuous activity whereas discontinuous activity increased in both groups with larger haemorrhages (grades III and IV) and when compared to controls. Suspected seizure activity was more common with increasing degree of bleeding in group A (50% with PIVH I or II, 75% with PIVH III or IV) and when compared to controls and was the same with increasing degree of bleeding in group B (47% with PIVH I or II, 45% with PIVH III or IV). Sleep-wake cycles were less common with larger haemorrhages in both groups (group A: 41% with PIVH I or II, 25% with PIVH III or IV; group B: 52% with PIVH I or II, 9% with PIVH III or IV) and when compared to controls. CONCLUSIONS: The aEEG characteristics of severe PIVH consist in a combination of a more discontinuous background pattern, a lack of sleep-wake cycles and a higher likelihood of seizure activity when compared to age-matched controls.

Intra- and extrauterine maturation of amplitude-integrated electroencephalographic activity in preterm infants younger than 30 weeks of gestation.

OBJECTIVE: To prospectively investigate the longitudinal changes of amplitude-integrated electroencephalographic (aEEG) activity in preterm infants <30 weeks gestational age (GA). METHODS: Infants (GA <30 weeks) without evidence of neurological abnormalities had weekly aEEG recordings performed. The relative duration of the three aEEG patterns (discontinuous low voltage, discontinuous high voltage and continuous) was determined and the influence of GA and postnatal age (PNA) on the occurrence of each pattern was assessed. RESULTS: Ninety-eight infants (median GA 26 weeks; range 23-29 weeks) were studied. With higher GA (OR 1.68, 95% CI 1.33-2.13) and PNA (OR 1.91, 95% CI 1.53-2.38), the likelihood for the occurrence of continuous activity increased. The discontinuous low-voltage pattern was less likely to occur with increasing GA (OR 0.68, 95% CI 0.55-0.83) and PNA (OR 0.70, 95% CI 0.61-0.81). CONCLUSION: Maturation of aEEG activity in preterm infants is influenced by both GA and PNA.

Effects of a combined therapy of erythropoietin, iron, folate, and vitamin B12 on the transfusion requirements of extremely low birth weight infants.

OBJECTIVES: Erythropoietin is frequently administered to premature infants to stimulate erythropoiesis. The primary goal of erythropoietin therapy is to reduce transfusions, but the efficacy of erythropoietin has not been convincingly demonstrated in this regard. The aim of this trial was to investigate whether combined administration of vitamin B12, folic acid, iron, and erythropoietin could decrease transfusion requirements in extremely low birth weight infants. PATIENTS AND METHODS: In a randomized, controlled trial, extremely low birth weight infants with a birth weight < or = 800 g and a gestational age < or = 32 weeks were randomly assigned to a group receiving combination treatment or a control arm. RESULTS: The treatment increased levels of folate in red blood cells, vitamin B12, ferritin, transferrin receptor levels in plasma, and reticulocyte counts. The proportion of infants requiring no transfusions was lower in the treatment group (38%) as compared with controls (5%). The treatment group and the need for mechanical ventilation were independent predictors of the number of transfusions in multiple regression analysis. Cox regression analysis indicated that combined therapy resulted in a 79% risk reduction for any transfusion. CONCLUSION: Combined treatment with erythropoietin, intravenous iron, folate, and vitamin B12 during the first weeks reduces the need for transfusion in extremely low birth weight infants.

A randomized, controlled trial of the effects of adding vitamin B12 and folate to erythropoietin for the treatment of anemia of prematurity.

BACKGROUND: Premature infants, especially those with birth weights of <1500 g, often suffer from anemia of prematurity and associated problems. Erythropoietin therapy is a safe effective way to prevent and to treat anemia of prematurity. We hypothesized that combined administration of vitamin B12 and folate with erythropoietin and iron would enhance erythropoietin-induced erythropoiesis. METHODS: In a randomized, controlled trial, 64 premature infants (birth weight: 801-1300 g) receiving erythropoietin and iron supplementation were assigned randomly to receive either vitamin B12 (3 microg/kg per day) and folate (100 microg/kg per day) (treatment group) or a lower dose of folate (60 microg/kg per day) (control group). RESULTS: During the 4-week observation period, vitamin B12 and folate enhanced erythropoietin-induced erythropoiesis significantly, as indicated by a 10% increase in red blood cell counts, compared with folate alone. Hemoglobin and hematocrit levels remained stable in the treatment group, whereas they decreased in the control group. Vitamin B12 levels in the treatment group increased over baseline and control values, whereas red blood cell folate levels were comparable between the groups. Subsequent analysis showed slight nonsignificant differences in baseline red blood cell count, hemoglobin level, hematocrit level, and mean corpuscular volume values, which must be addressed as a limitation. CONCLUSIONS: With the limitation of a slight imbalance in baseline data between the study groups, combined therapy with vitamin B12, folate, erythropoietin, and orally and intravenously administered iron seemed more effective in stimulating erythropoiesis among premature infants, compared with erythropoietin, iron, and low-dose folate alone. Additional trials are necessary to confirm these data.

Reference values for amplitude-integrated electroencephalographic activity in preterm infants younger than 30 weeks' gestational age.

OBJECTIVE: To prospectively investigate the development of amplitude-integrated electroencephalographic (aEEG) activity during the first 2 weeks of life in neurologically normal and clinically stable preterm infants <30 weeks' gestational age (GA). PATIENTS AND METHODS: Infants with a GA of <30 weeks admitted to the neonatal intensive care unit of the Vienna University Children's Hospital (Vienna, Austria) were studied prospectively by using aEEG and cranial ultrasound. Clinically stable infants without clinical or sonographic evidence of neurologic abnormalities were eligible for inclusion in the reference group. The distribution of 3 background aEEG activity patterns (discontinuous low-voltage, discontinuous high-voltage, and continuous), presence of sleep-wake cycles, and number of bursts per hour in the reference group were determined by visual analysis. RESULTS: Seventy-five infants (median GA: 27 weeks; range: 23-29 weeks) were eligible for inclusion in the reference group and had aEEG recordings during the first 2 weeks of life available. Analysis of aEEG background activity showed that with higher GA the relative amount of continuous activity increased while discontinuous patterns decreased. The number of bursts per hour decreased with increasing GA. Cyclical changes in aEEG background activity resembling early sleep-wake cycles were observed in all infants. CONCLUSIONS: Normal values for aEEG background activity were determined in preterm infants <30 weeks' GA. Clinically stable and neurologically normal preterm infants exhibit at least 2 different patterns of aEEG activity. There is a correlation between the GA and the relative duration of continuous aEEG activity.