Paediatrics (RCH) - Theses
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ItemThe development of feeding in infants with oesophageal atresia between birth and nine months of age( 2017)Feeding problems have been widely reported in babies with oesophageal atresia (OA) and are usually described in terms of growth or oesophageal functioning. The feeding problems may persist into adulthood. The aim of my study was to explore the nature of feeding development from a wider perspective, comprising biomedical, psychological and interactional factors relevant during the development of feeding in babies with OA. The RCH Neonatal Unit admitted forty babies with OA, eligible to participate in the research study over the recruitment period. Thirty families (75%) gave informed consent for participation in the research project. Twenty seven families finally participated in the study. My study used data from parent questionnaires, structured observation schedules, parent interviews and a medical record audit to describe infant, parent and interactional factors. I collected data at four times points (one month, three months, six months and nine months of age) and analysed data using descriptive statistics to investigate the pattern and extent of factors co-occurring with the development of feeding. I analysed semi-structured interviews qualitatively to reveal a range of parental perspectives related to feeding babies with OA. My study used mixed methodology to ensure inclusion of the parental experience of feeding their baby with oesophageal atresia. Comparison of quantitative and qualitative research findings revealed agreement between qualitative and quantitative results for the following: most babies were found to feed relatively well; the number of comorbid health conditions per baby did not significantly impact on feeding outcome; parents reported that feeding their baby with oesophageal atresia was stressful, supporting the results of validated measures of stress; mothers demonstrated relatively high levels of reflective functioning; and mother-infant interaction, in terms of maternal sensitivity and infant responsiveness was generally positive. Differences between quantitative findings and parental perception were apparent in the following: despite babies demonstrating poorer than expected growth, few parents reported concern regarding poor weight gain; potentially aversive procedures such as oesophageal dilatation did not impact on feeding outcomes even though some parental comment indicated otherwise; parents did not comment on the significantly reduced feeding efficiency detected in the quantitative data; and while a group of babies demonstrated resistance to feeding that matched resistance in babies with posttraumatic feeding disorder, parents overwhelmingly reported that their babies enjoyed mealtime. As hypothesised, I detected a statistically significant association between mothers’ acute stress symptoms when their babies were one month old and feeding success at nine months. In addition, mothers with higher levels of reflective functioning demonstrated higher levels of sensitivity during feeding and babies who resisted feeding at nine months of age had a poorer feeding outcome at the same age. While many questions remain unanswered, my study presents descriptions of a range of factors involved in the development of feeding disorder in babies with OA and a small number of significant associations between these factors and feeding outcomes. The development of feeding in infants with OA is multifactorial in nature and involves all three domains (psychological, interactional and biomedical). Findings from the study extend the body of evidence upon which to base further research. The study also presents findings that support the need for revision of clinical practice and the development of new, targeted interventions for feeding problems in babies with OA.
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ItemGAPTrap: a novel gene expression system for pluripotent stem cells and their differentiated derivatives( 2017)Human induced pluripotent stem cells (iPSCs or iPS cells) are derived by the in vitro reprogramming of somatic cells and resemble embryonic stem cells (hESCs) derived from the inner cell mass of the pre-implantation blastocyst stage embryo. In vitro, both iPSCs and hESCs can be directed to differentiate towards specific lineages and therefore represent a tractable platform to study important events during the specification of early human cell types. The ability to reliably express either fluorescent reporters or other genes of interest is critical for using pluripotent stem cells to investigate cellular differentiation pathways and gene function. We designed a simple expression system in which genes of interest can be inserted into the ubiquitously expressed GAPDH locus. In this system, transgenes are fused in frame with GAPDH via a T2A linker sequence that results in the generation of two separate proteins from a single GAPDH mRNA. Some versions of these vectors contain an additional element that enables targeted clones to be selected using antibiotic resistance. Independent PSC clones harbouring variations of this vector, which we have named GAPTrap, express remarkably consistent levels of the associated reporter genes. In vitro differentiation experiments, which generated cells representing the three embryonic germ layers, mesoderm, endoderm and ectoderm, showed that GAPTrap transgene expression remained consistent in a number of different cell types, including cardiomyocytes, blood cells and neurons. In addition, analysis of teratomas derived from iPSCs harbouring a GAPTrap-LacZ transgene showed that beta-galactosidase expression was maintained in a variety of differentiated cell types. Examination of cells containing a single allele insertion of the GAPTrap vector revealed that GAPDH activity was not reduced in undifferentiated transgenic PSCs. Nevertheless, western blot analysis showed that particular vector configurations adversely affected the level of GAPDH expression derived from the modified allele. Using this information, we redesigned the vectors to increase the level of transgene expression and of the upstream GAPDH protein. These new configurations subsequently enabled the isolation of doubly targeted cell lines: PSCs in which both alleles of GAPDH contained a different GAPTrap vector. The ability to independently target both GAPDH alleles provides further flexibility in designing more sophisticated experiments requiring multiple elements. Indeed, we used this information to create iPSCs harbouring two GAPTrap vectors encoding BCL-2 and MYC, each with independent regulatory elements to enable either protein to be activated in controllable manner. In summary, the GAPTrap vectors are a promising and straightforward system for expressing genes of interest in pluripotent stem cells and their differentiating derivatives.
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Item"What about me?": The impact of cystic fibrosis on parental differential treatment, sibling relationships and adjustment( 2017)Cystic fibrosis (CF) is an inherited, multisystem disorder that primarily affects the respiratory and digestive systems. Currently, approximately half the individuals with CF in Australia, the United States and Canada are adults, which represents a remarkable change in the prognosis of this disease over the past 20 years. However, despite recent advances in diagnosis and treatment, CF shortens the life span, and the daily treatment regimen is complex, burdensome and time-consuming, taking 2-4 hours per day. The treatment regimen requires a high level of parental supervision in infancy, childhood and adolescence. The demands CF care places on parents raise the question of how time and attention are allocated when there are other, healthy siblings in the family. The overall objective of this study was to assess the extent of parental differential treatment in two types of families - those with a well child and a child with CF and those with two healthy children. In addition, the impact of differential treatment on the quality of the sibling relationship and the social and emotional adjustment of well siblings in families caring for a child with CF were examined. This study evaluated a cohort of 39 Australian children growing up with a younger sibling with CF and 29 comparison families with similarly aged, healthy children. Information on the amount of time parents spent in daily activities with the younger and older siblings was collected from both mothers and fathers using daily phone diaries. Data were collected from older siblings on a range of social and emotional variables and both younger and older siblings rated the quality of the sibling relationship. This study found evidence of parental differential treatment for fathers but not for mothers. Fathers spent more time with the younger child with CF than the older healthy sibling. High levels of fathers’ differential treatment were consistently associated with older healthy siblings indicating a perception of parental bias towards the child with CF. Despite the limitations of cross-sectional research, a major strength of this study was the recruitment of fathers, whose contribution to family life is often neglected in studies of families caring for children with chronic health conditions. Clinical implications and recommendations for future research are discussed.
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ItemUnveiling the biology of collecting duct epithelium repair and regeneration( 2017)The healthy functioning of the kidney requires the orchestrated action of its two functional units, nephrons and the collecting duct system (CD). Therefore malfunction of either of these two essential compartments can lead to kidney failure. Diseases affecting the collecting duct system (CD), congenital abnormalities of the kidney and urinary tract (CAKUTs), are the most frequent cause of End-Stage Renal Disease (ESRD) in children. At this stage, the only available therapeutic options for treating kidney failure are dialysis or organ transplantation. Given the fact that only one in three patients will receive a transplant, and that dialysis comes with a high risk of mortality, the study of the mechanisms underlying the repair and regeneration of the collecting duct system is vital because it will facilitate new therapeutic strategies for treating kidney disease which are available to more people. The CD system originates from one of the two progenitor populations which give rise to the kidney, the ureteric bud (UB). The formation of the UB is a substantial part of the nephrogenesis process, and develops into a branched tree-like structure which will ultimately form the ducts of the urinary collecting system. This PhD thesis investigates recent concepts in normal kidney organogenesis, repair, and regeneration, and focuses on the CD system. In the first part of this research, we investigated the capacity of an endogenous kidney mesenchymal stem cell population (k-MSC), previously reported by our laboratory, which both arose from the collecting duct epithelium and also then integrated into the same compartment. To this end, we generated and examined the functional capacity of Pkd1 defective k-MSCs to trigger autosomal dominant polycystic kidney disease (ADPKD) into wild type mice. Given that micro-injection of double transgenic k-MSCsPkd1del2-4/TMTO+ did not produce significant evidence for cyst formation in the recipient mice, we therefore ruled out the possibility that k-MSCs represent a preferable population for effecting repair. Nevertheless, we showed the capacity to generate k-MSCs with mutant genes, which may prove useful as cellular models of human diseases.
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ItemWnt signalling pathway in fetal and neonatal cardiopulmonary development( 2017)Cardiopulmonary development and maturation involves numerous intracellular protein signalling pathways. Any alteration to these pathways may lead to the development of congenital heart and lung defects. One such important pathway is the canonical and the non-canonical Wnt (wingless-type MMTV integration site member) signalling pathway. As such, the involvement of Wnt protein intermediates in the heart and lung of late gestation fetuses and neonates have not been previously studied in large animal models. Therefore, my PhD aims to characterise the role of Wnt signalling proteins during normal heart and lung development in late gestation ovine model, the response of the Wnt proteins under in utero stress (IUS) and after short-term betamethasone (BM)-administration in preterm fetuses and in newborn lambs.
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ItemDevelopment of working memory in very preterm children( 2017)Children born very preterm (VP; <32 weeks’ gestation) show wide-ranging long-term neuropsychological difficulties compared with their term-born peers, including reduced language, attention, memory and learning. One area of concern in VP children is working memory (WM), referring to the capacity to maintain and to manipulate information for a brief period of time. WM is a core capacity for higher-order human behaviours, such as reading comprehension, abstract reasoning and arithmetic. In healthy children, the development of WM has been reported to be associated with the protracted neurobiological maturation of the frontal-parietal neuronal network, continuing into late adolescence. However, the development of WM in VP children is largely unknown. It is not clear whether VP children continue showing WM difficulties compared with term-born peers across child development, or whether the neuronal basis of WM in VP children differs from that of their term-born peers. The objective of this dissertation was to address these gaps in the research literature by conducting three research studies using a longitudinal cohort of VP children and term-born controls.
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ItemDeveloping cell and gene therapy for Friedreich ataxia( 2017)Friedreich ataxia (FRDA) is a debilitating neurodegenerative disorder which affects 1 in 29 000 individuals of Caucasian descent. Disease onset is typically between 10-15 years of age and it is primarily characterised by progressive gait and limb ataxia. Individuals with FRDA experience an ongoing loss of motor coordination and become wheelchair-dependent within 15 years after disease onset – decreasing their quality of life. Other disease symptoms include scoliosis, muscle weakness and spasticity and hypertrophic cardiomyopathy. Complications of the heart are the most common causes of death in FRDA with life expectancy decreased to an average of 37 years old. As there is no current treatment which can cure or slow the neurodegeneration inherent to FRDA, individuals with FRDA manage disease symptoms with various symptomatic treatments. The development of new and more effective treatments capable of slowing or halting disease progression is therefore essential to improve the quality of life for individuals with FRDA. In most cases, FRDA is caused by homozygous GAA trinucleotide repeat expansions within intron 1 of FXN which encodes for frataxin, a nuclear-encoded mitochondrial protein. The expansion results in reduced levels of frataxin, but does not alter the protein sequence and structure. Accumulating evidence indicates that increasing frataxin will be therapeutically beneficial to individuals with FRDA. With bone marrow (BM) cells being the ultimate target for gene correction through cell and gene therapy for FRDA, this study first examined the therapeutic potential of bone marrow transplantation (BMT). To investigate the therapeutic potential of BMT as a cell-based therapy for FRDA, we transplanted the mouse model of FRDA (YG8 mice) with either GFP-positive wild-type BM cells (referred as corrected YG8 mice) or FRDA BM cells (referred as non-corrected YG8 mice). GFP-positive donor cells allowed tracking of donor cell engraftment in the body of corrected YG8 mice post-BMT. Donor BM-derived cells successfully reconstituted the haematopoietic system of BMT recipient mice at eight weeks post-BMT. Long term engraftment of donor BM-derived cells in the haematopoietic system was also identified as late as 13.5 months post-BMT. Neurobehavioural assays, however, showed no significant difference in the motor coordination, locomotor activity and limb flexion response between corrected and non-corrected YG8 mice at all time points tested. To evaluate if the transplantation of BM cells itself was beneficial, YG8 non-transplanted mice were also tested on the various neurobehavioural assays and showed a significant decrease in motor coordination, locomotor activity and limb flexion response over time. Comparison between corrected/non-corrected YG8 mice and non-transplanted YG8 mice showed no significant difference in the motor coordination of corrected/non-corrected YG8 mice compared to non-transplanted YG8 mice. However, since there was a significant decrease in motor coordination of YG8 non-transplanted mice over time while BMT mice demonstrated consistent motor coordination over time, this indicated there was a greater decline in the motor coordination of YG8 non-transplanted mice over time. In addition, YG8 non-transplanted mice performed better on the accelerating rotarod at first testing compared to BMT mice. There was a significant increase in the locomotor activity of corrected mice compared to YG8 non-transplanted mice but there was no significant difference in the locomotor activity between non-corrected mice and YG8 non-transplanted mice. Overall, transplantation of either wild-type or FRDA BM cells slowed or halted the decline in motor functions and was beneficial to motor behavioural outcomes in FRDA, with wild-type BM cells conferring a greater benefit than YG8 BM cells. This could mean that BMT may have the potential to slow or halt motor declination in individuals with FRDA, therefore delaying or preventing their dependence on a wheelchair. Thus, BMT could be the difference between needing a walker instead of being wheelchair-bound for life. GFP-positive cells were also identified in the dorsal root ganglia (DRG) and spinal cord of corrected mice, indicating successful engraftment of donor BM-derived stem cells in major sites of neuropathology post-BMT and demonstrating low-level chimerism. To investigate if BMT had an effect at the molecular and cellular level, western blotting analyses showed no significant difference in the level of mature frataxin protein between corrected and non-corrected mice in all tissues analysed in the short term and long term post-BMT. However, immunofluorescence studies showed increased neuronal marking in the DRG of corrected mice compared to non-corrected mice, in particular the proprioceptive neurons which are highly affected in individuals with FRDA. In conclusion, delayed or halted motor dysfunction and increased number of proprioceptive neurons with improved morphology post-BMT together illustrate the corrective potential of BMT to treat FRDA and provide an avenue for delivering lentiviral gene modified BM cells for autologous cell and gene therapy.
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ItemMotor outcomes and their impact on activities of daily living, following paediatric arterial ischemic stroke( 2017)Paediatric arterial ischaemic stroke (AIS) affects between 1.6-2.7 per 100,000 children per year. Motor impairments are the most commonly reported adverse consequence following paediatric arterial ischaemic stroke, yet the long-term impact of stroke remains poorly understood. As childhood is a time of rapid brain development and growth, stroke during this developmental stage has the potential to affect not only established skills, but also the development on new and emerging skills with resultant difficulties spanning a lifetime. The aim of this study was to measure and describe motor and functional difficulties of children following AIS and to explore factors associated with outcomes including lesion characteristics (size, location and laterality), early neurological impairment and age at stroke onset. This thesis addresses these aims through three studies with supplementary data presented in a fourth results chapter. Study one explored motor and functional outcomes across the first year following AIS. Early predictors of 12-month outcomes were also explored, as was the relationship between fine motor and gross motor function and adaptive behaviour. A larger lesion size was associated with poorer gross motor outcome. Trajectories of recovery differed depending on age at stroke onset and poorer fine and gross motor outcomes were associated with poorer adaptive behaviour skills. Study two evaluated the relationship between neurological outcome at 1 month following diagnosis of paediatric AIS and motor and adaptive behaviour outcomes at 12 months. Presence of impairment on the paediatric stroke outcome measure (PSOM) at 1 month was associated with poorer 12-month fine motor (FM) skills, gross motor (GM) skills and adaptive behaviour. One month PSOM impairment score was more predictive than age group or lesion size of 12-month motor and adaptive behaviour outcome. Study three investigated motor impairment and functional motor outcomes 4-6 years after AIS as well as factors associated with these outcomes. At 4-6 years post stroke, motor function, quality of life, fatigue, adaptive behaviour, activities of daily living and speed of handwriting were significantly poorer than age expectations. The pre- school group had the highest percentage of fine and gross motor impairment. Poorer fine motor skills were associated with subcortical only strokes and large lesion size. Poorer gross motor outcomes were correlated with pre-school age, bilateral lesions and PSOM impairment at 1 month. The findings from these studies highlight the fact that children are at elevated risk for motor and functional impairments following AIS, with the pre-school age group most vulnerable. Early predictors of poor outcomes were identified, including age at stroke onset, lesion size and early neurological impairment, which helps to facilitate targeted early intervention and long-term rehabilitation. These results support the need for long term surveillance following paediatric stroke with children demonstrating changes in function beyond the first year following diagnosis. These findings indicate the need for functional measures to be used in routine follow up screening.
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ItemNo Preview AvailableImproving diagnostic tests for tuberculosis: mycobacterial antigen-stimulated cytokine biomarkers for diagnosis and monitoring therapy( 2017)Tuberculosis (TB) is the leading cause of infectious disease mortality worldwide. Accurate and timely diagnosis of both active TB and latent TB infection (LTBI) is essential to controlling the TB pandemic. Current diagnostic tests for TB have several limitations, including poor performance in children, in the immunocompromised and in patients with extra-pulmonary TB. At present, commercial immunodiagnostic tests for TB do not distinguish between active TB and LTBI. In addition, there is no established biomarker that functions as a ‘test of cure’ for TB. Such a biomarker would allow treatment to be tailored to individual response, potentially decreasing treatment duration and reducing drug toxicity. My PhD research project focussed on evaluating candidate mycobacterial antigen-specific cytokine biomarkers to improve the performance of immunodiagnostic tests for TB in children and adults. The first section of this thesis focuses on cytokines to diagnose and distinguish between active TB and LTBI. It includes a systematic review of cytokine biomarkers that have been previously evaluated for their ability to distinguish between active TB and LTBI. Two separate research studies, one in a cohort of children, and one in adults, are then described. The first was a follow-up study in a new paediatric cohort to validate the role of several candidate biomarkers previously identified by our research group that distinguish between active TB and LTBI. Following on from this study in children, I led a larger study to evaluate the role of these cytokine biomarkers in the diagnosis of active TB and LTBI in adults, and to determine their specificity for TB infection. This study confirmed the findings in children that mycobacterial antigen-stimulated IL 1ra, IL-10 and TNF- responses are significantly higher in individuals with active TB than LTBI, although there was considerable overlap in responses between the two groups. The second section of this thesis focuses on cytokines for monitoring anti-tuberculous therapy. It includes a published systematic review of cytokines that have previously been evaluated for this purpose. This is followed by a published study in an adult patient cohort which investigated whether mycobacterial antigen-specific cytokine responses change during treatment for TB. It was found that mycobacterial antigen-specific IL-1ra responses decreased during therapy in both active TB and LTBI. An additional ex vivo research study, also published, confirmed that changes in cytokine response observed during therapy were unlikely to be due to any immunomodulatory effects of anti-tuberculous treatment. IL-1ra was therefore identified as a candidate ‘test of cure’ biomarker that should be investigated in future longitudinal studies.
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ItemNeurological risk factors in infants having noncardiac surgery and their relationship to growth and neurodevelopmental outcome( 2017)INTRODUCTION: With advancements and improvements in surgical techniques, neonatal and antenatal care over the last sixty years, the mortality following neonatal non-cardiac surgery (NCS) has fallen to less than five percent. Consequently, the focus has now shifted to morbidity and long-term outcomes with increasing concern that these children are at risk of neurodevelopmental (ND) impairment. Other high risk neonatal groups, including those born prematurely and those who require early cardiac surgery, are known to have increased risk of ND impairment and frequently have abnormalities detected on brain imaging in the newborn period. The neurobiological substrates for ND impairment following NCS are unclear and likely to be multi-factorial but may include brain injury. The effect of neonatal NCS on brain structure, growth and development has not been described. AIMS: The aim of the study was to investigate neurological risk factors, in particular brain injury, in infants requiring neonatal NCS, and their impact on growth and ND outcomes over the first two years of life. METHODS: Infants undergoing major NCS in the first two weeks of life at The Royal Children’s Hospital, Melbourne for a diagnosis of oesophageal atresia (OA), congenital diaphragmatic hernia (CDH) or abdominal wall defect (AWD), were enrolled in this case-controlled prospective study. Infants were excluded if they were born before 32 weeks’ gestation or if they had a known genetic anomaly associated with ND impairment. Magnetic resonance imaging (MRI) of the brain was performed, time and clinical status allowing, pre-operatively and repeated prior to hospital discharge along with an assessment of neurobehaviour using Prechtl’s general movements. Surviving infants were followed up at six, 12 and 24 months when growth and neurodevelopment were assessed using the Alberta Infant Motor Scale (AIMS) and/or the Bayley Scales of Infant and Toddler Development, 3rd Edition (BSID-III). Thirty-nine infants were matched for gestation at birth, gestation at time of post-operative MRI and gender with controls. Control infants were recruited from a neighbouring perinatal hospital as part of another research trial. RESULTS: Over 27 months, 54 infants were recruited with slight male preponderance of 61%. The mean (standard deviation [SD]) gestation at birth was 37.9 (2.3) weeks with a mean birth weight of 2.93 (0.67) kilos. Twenty-one of the participants had AWD, 17 had OA and 16 had CDH. Surgery was performed at a median (interquartile range [IQR]) age of 24 (10 – 53) hours with a mean (SD) duration of 133 (56) minutes. Four infants died prior to 24 month follow-up (mortality rate 7%), with three of the four dying before hospital discharge. Infants requiring neonatal NCS had poor growth in the first six months, in particular prior to hospital discharge. There was evidence of catch-up growth for weight and height by 24 months. However, head growth remained poor, in particular for those children with CDH. Compared to control infants, there was poor head growth in the first month of life in infants following NCS. At 24 months of age, infants who had NCS were lighter and tended to be shorter than matched controls. Pre-operative MR imaging of the brain, obtained in a small number of participants (n=7), revealed delay in brain maturation in six of the seven participants. Post-operative imaging, obtained in 51 participants, also revealed high rates of delay in brain maturation (47%), with delay in cortical folding more common than delay in myelination. Abnormalities on MR imaging were detected in 51% of cases with white matter signal abnormalities (WMSA) and ventricular dilatation seen most frequently at 18% and 27% respectively. WMSA were associated with delay in brain maturation and with intracranial haemorrhage. Compared to matched controls, infants undergoing NCS had smaller biparietal diameters, smaller transcerebellar diameters and larger ventricles. Infants requiring NCS were more likely to have delay in cortical folding [odds ratio (OR) 5.92 (95% confidence interval (CI) 1.91, 19.47), p < 0.001] and WMSA [OR 9.81 (95%CI 1.18, 446.25) p = 0.01]. Following NCS, infants had significantly lower scores than test norms on AIMS assessment at six and 12 months (p = 0.003 and < 0.001 respectively). BSID-III assessment was performed at mean (SD) age of 12.4 (0.8) months in 48 survivors and at a mean (SD) age of 27.0 (2.4) months in 45 survivors. Language and motor composite scores were lower than test norms at both 12 and 24 months, with p < 0.001 and 0.04 for language at 12 and 24 months, and p < 0.001 and 0.01 for motor, at 12 and 24 months. Participants were performing in the low average range, with mean (SD) scores of 91.5 (15.2) and 94.2 (14.4) at 12 and 24 months for language and 90.4 (11.4) and 94.4 (13.6) for motor. Mild developmental impairment at 24 months was found in one third of the group with composite scores greater than 1 SD below the mean in at least one domain, with 9% having delay in all three domains. ND impairment at 12 months was predictive of ND impairment at 24 months. Compared to matched controls, infants requiring neonatal surgery had lower composite scores for cognition, language and motor at 24 months. Similar to previous reports, the control group performed significantly better across all domains than test norms. Perinatal and peri-operative risk factors associated with ND impairment at 24 months were compromise at birth with an Agpar score of less than seven at one minute, markers of illness severity, feeding dysfunction and failure to thrive, in particular poor weight gain in the first six months. Markers of illness severity including a rising base excess in the first 24 hours post-operatively, longer duration of mechanical ventilation and hospital stay. Increasing left lateral ventricular size was the only MRI parameter that remained significant for ND impairment on multivariate analysis. Gestational age at birth had little impact on ND outcome at 24 months, for infants requiring NCS born at 32 weeks’ or more of gestation. CONCLUSIONS: Infants requiring early NCS are at increased risk of ND impairment at 24 months. Post-operative MR brain imaging revealed high rates of brain immaturity and abnormalities, including white matter signal abnormalities and ventricular dilatation. A clear-cut link between early brain injury and maturational delay and subsequent ND impairment was not found. Increasing illness severity, feeding dysfunction and failure to thrive are associated with ND impairment at 24 months.