Paediatrics (RCH) - Theses
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ItemNo Preview AvailableA Blood Based Study of COVID-19( 2022)Background: Coronavirus Disease 2019 (COVID-19), caused by the virus SARS-CoV-2, has had substantial impact on global health. In addition to the severe respiratory symptoms of COVID-19, approximately 20% of adult COVID-19 patients experience a clinically significant thrombosis. The relationship between COVID-19 and thrombosis is poorly understood, however the presence of thrombosis in even mild COVID-19 suggests that the haemostatic system is fundamental to COVID-19 pathogenesis. Children, while somewhat protected from COVID-19 coagulopathy, experience a severe, post-COVID-19 inflammatory condition known as multisystem inflammatory syndrome in children (MIS-C). Little is known about the underlying pathophysiology of MIS-C, and what differentiates it from typical presentation of severe COVID-19. Objectives: This research program explores the blood-based impacts of COVID-19 in adults and children, with a particular attention to understanding the differential thrombotic risk between adults and children, and MIS-C which seems to occur uniquely in children. This project investigated platelet phenotype and function in COVID-19 in vivo. SARS-CoV-2 infection induced fibrin-clot structure and characteristics were investigated in vitro. Additionally, the biological mechanisms underpinning MISC and severe COVID-19 were investigated in children. Methods: Platelet phenotype and function was investigated in 67 children and adults with mild COVID-19, by performing whole-blood platelet flow cytometry. These were compared to platelet flow cytometry samples collected prior to the COVID-19 pandemic. Fibrin clots were investigated in a novel in vitro model of endothelial involvement in Haemostasis. Human umbilical vein endothelial cells (HUVECs) were cultured and infected with SARS-CoV-2 and incubated with plasma from healthy children, healthy adults and adult vasculopaths. Experiments were repeated with the addition of anticoagulants bivalirudin, defibrotide, unfractionated heparin (UFH), and low molecular weight heparin (LMWH). Fibrin clot structure was determined using scanning electron microscopy. The plasma proteome of children who developed MIS-C or COVID-19 acute respiratory distress syndrome (ARDS) were compared to healthy children, by mass spectrometry. Abundance of proteins was compared to establish a protein signature of MIS-C and ARDS, and to determine underlying biological pathways in either syndrome. Results: Platelet activation increased in adults and children with mild COVID-19, which persisted at least four weeks after their initial infection. Additionally, close household contacts of the index case who did not test positive to COVID-19, also showed increase in platelet activation by PAC-1 and CD62P. Fibrin clots were induced in SARS-CoV- 2 infected cells. The fibrin network was denser and more composed of thinner fibrin fibres in the vasculopathic adults compared to healthy adults, and in healthy adults compared to healthy children. UFH and LMWH were effective at preventing fibrin clot formation, even at a half (LMWH) and quarter (UFH) prophylactic doses. 85 proteins were differentially expressed in MIS-C patients while 52 were differentially expressed in COVID-19 ARDS patients. Complement and coagulation pathways were impacted in both MIS-C and ARDS, however MIS-C showed specific FcGR and BCR activation while COVID-19 showed changes in haem scavenging and retinoid metabolism. Conclusion: COVID-19 induced changes to the coagulation system across three major aspects of haemostasis: platelets, a SARS-CoV-2 infected endothelium, and the plasma proteins. We show that there are prothrombotic platelet changes in the setting of mild COVID-19, and that vasculopathy patients may have increased risk of thrombosis. Our evidence that UFH is effective in reducing SARS CoV-2 induced coagulation in vitro sets the scene for future clinical research in establishing treatment of COVID-19 coagulopathy. We show that severe COVID-19 phenotypes (MIS-C and ARDS) are driven by coagulation and complement, showing the crucial interplay between immunity and coagulation in COVID-19.
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ItemEarly life oral exposures as risk factors for food allergy and the impact of infant feeding guidelines( 2021)Food allergy is a severe immune reaction affecting around 10% of infants. The recent rise in food allergy is a growing public health concern. Susceptibility to food allergy is affected by environmental exposures that affect infant gut immunity, including oral food and microbial exposures. Changes in infant feeding guidelines recommending introduction of allergenic foods in the first year were the first public health measure introduced specifically to prevent food allergy. I aimed to evaluate their impact on timing of allergenic food introduction and prevalence of food allergy, as well as investigating oral microbial exposure as a potential food allergy risk factor. The effect of 2016 updates to infant feeding guidelines on food allergy was evaluated using two large population-based cross-sectional studies (n=5,300 and n=1,933) that recruited 12-month-old infants in Melbourne, Australia 10-years apart (2007-2011 and 2018-2019) to study allergenic food introduction and peanut allergy. Microbial exposure was investigated in relation to food allergy using data from a cohort study (n=1,072) based in south-east Australia which measured pacifier cleaning methods in the first year of life. Data were analyzed using multivariable logistic regression models and direct standardization. Regression models were adjusted for known food allergy risk factors, depending on the exposure. An overview of systematic reviews searched systematic reviews in four databases to examine age of complementary feeding in relation to other health outcomes. These studies are the first report that peanut introduction by 12 months of age (inclusive) in the population increased (88% in 2018-2019 vs 28% in 2007-2011) following the implementation of 2016 infant feeding guidelines. Despite this massive shift, peanut allergy prevalence did not decrease as much as expected (3.1% to 2.6%). However, the increase in peanut introduction seems to have halted the rise of peanut allergy in one-year-old infants. The systematic review found 27 reviews on age of complementary or allergenic food introduction on multiple outcomes. There was no evidence of a detrimental effect on other health outcomes of the recommended introduction of solids and allergenic foods starting around 6 months of age, though solids introduced earlier than 4 months could increase the risk of overweight. Furthermore, sanitization of infant pacifier with antiseptics at 6 months of age increased the risk of food allergy at age one, possibly an effect of the chemicals on oral or gut microbiome. An increase in early introduction following 2016 guideline changes has halted the overall rise in peanut allergy. Nevertheless, peanut allergy is still high in Melbourne, thus additional prevention strategies are required. Reassuringly, other health outcomes are not risked by the recommended age of allergenic food introduction. The findings on cleaning pacifiers with antiseptics suggests that suggests microbial exposure can protect against food allergy. This warrants further interventional studies to determine if microbial exposures could prevent food allergy. Moreover, early life risk factors, prior to food introduction, need to be explored further.
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ItemEpigenetic Markers As A Predictor Of Lung Disease Severity In Cystic Fibrosis( 2020)Cystic fibrosis (CF) is a multisystem disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Lung disease which is the major source of morbidity and mortality in CF, begins in infancy and persists and progresses in the first years of life. The most sensitive measure of CF lung disease is computed tomography (CT) and the majority of children show evidence of bronchiectasis, which represents irreversible structural lung disease, by five years of age. The rate with which lung disease develops and progresses varies greatly between people with CF. Accurate prediction of future lung disease severity in people with CF would facilitate significant improvements in clinical care, as well as provide helpful information to people with CF and their families, health authorities and CF researchers. It has been demonstrated that CF lung disease severity is determined by a combination of non-CFTR genetic and environmental factors. However, no robust predictive biomarkers have been developed. Epigenetic mechanisms are those which regulate gene expression but do not alter the DNA sequence itself. DNA methylation is the most widely studied epigenetic mechanism, and an individual’s DNA methylation profile is determined by their underlying genotype and environmental exposures (including in utero). DNA methylation has been studied as a predictive biomarker in other childhood diseases, and given it is determined by the same factors which determine CF lung disease severity, is well positioned to act as a biomarker in CF. Despite this, DNA methylation based biomarkers of future lung disease severity have not been explored in CF. The thesis presents a series of projects which aimed to identify DNA methylation based biomarkers of future CF lung disease severity. The projects in this thesis utilised biospecimens and clinical data from the world leading Australian Respiratory Early Surveillance Team for Cystic Fibrosis (AREST CF) cohort. In order to better understand previous attempts at identifying predictive biomarkers, Chapter Two and Three describe reviews of the evidence regarding non-CFTR genetic modifiers of CF lung disease and DNA methylation based biomarkers of future health outcomes in children. The reviews highlight the importance of appropriate study design when trying to identify predictive biomarkers, which then informed design of the subsequent epigenome wide association study (EWAS). Chapters Four and Five describe the development of methods that were necessary for subsequent EWAS. In Chapter Four protocols for cryopreservation, and flow cytometry based phenotyping and sorting of specific cell populations were developed and validated for use with paediatric bronchoalveolar lavage (BAL). These methods were then used in Chapter Five to obtain purified populations of the common cell types in BAL, which were then used to develop reference epigenomes for these cell types. These reference epigenomes allowed adjustment for cell composition in the subsequent EWAS, which is a crucial step in DNA methylation studies involving samples with multiple cell types. Chapters Six and Seven describe the EWAS studies that attempted to identify predictive biomarkers. Chapter Six involved genome wide DNA methylation profiling of BAL collected at six years of age, and assessed the ability to predict the presence or absence of bronchiectasis at nine years of age. No predictive biomarkers were identified. Chapter Seven involved genome wide DNA methylation profiling of BAL collected at one year of age, and assessed the ability to predict the presence or absence of bronchiectasis at five years of age. Seven predictive biomarkers were identified, which when assessed using area under the receiver operator curve analysis had extremely good performance as predictive biomarkers. Several of the predictive biomarkers were related to genes that are relevant to CF lung disease pathophysiology and hence may represent therapeutic targets. By following best practice for EWAS, and in particular using tissue specific samples, adjusting for cell composition and assessing outcome using the gold standard measure, this thesis succeeded in identifying DNA methylation based biomarkers of future lung disease severity in CF. If these biomarkers are validated in external cohorts they could dramatically improve the care of children with CF around the world. N.B. There are two files that have been submitted separate to this thesis document as supplementary files. They have been highlighted as appropriate in the thesis. In addition, the code used for statistical analysis is available at: https://atlassian.petermac.org.au/bitbucket/pages/OS/paed-cf- methylation/master/browse/docs/index.html
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ItemAssessment of health needs of children entering out-of-home care in Victoria: a mixed methods study( 2020)In Australia, state-based statutory Child Protection (CP) systems intervene when children are deemed to be at risk of harm from exposure to neglect and/or abuse. On 30th June 2019, 44,906 (8.0 per 1,000) Australian children were subject to court orders placing them with a relative (kinship) or foster carer in an out-of-home care (OOHC) placement. These children have often experienced socio-economic disadvantage in addition to adverse experiences such as neglect or abuse, with resultant high health needs. Australian and international child health experts uniformly recommend that health is routinely assessed upon entry to OOHC. Australia has national standards that stipulate an initial assessment within 30 days and a comprehensive assessment (paediatrician-led, with dental, audiology, and optometry assessments) within three months of entering OOHC. Despite this, Victorian state policy is less clear. Some routine assessments (medical, dental, optical, and auditory) are advised within one month of entry to care. While some areas have access to dedicated multi-disciplinary assessment clinics, there is no state-wide recommendation for routine comprehensive assessment. The majority of Victoria has no model of adequate service provision for health assessments. There are no data that monitor health assessments at either a state or national level, and little is known in Australia about the barriers to healthcare for children in OOHC. This research therefore aimed to determine the extent to which Victorian children entering OOHC were having their health needs assessed and addressed in a timely way, and to identify barriers and enablers to timely health assessment. This was a convergent parallel mixed-methods study with 3 sub-studies: Study 1, an audit of children seen at a multidisciplinary OOHC comprehensive assessment clinic, confirmed high rates of need and identified that only 25% of children attended within timeliness recommendations. Study 2 included a survey of 290 foster and kinship carers and interviews with 19 survey participants. Barriers encountered when accessing health services for children in OOHC included time-consuming consent processes, insufficient publicly funded health services, long wait times, poor communication of health information and delays in receiving key identifying information such as the child’s Medicare number. Increased health navigation support, greater prioritisation of health within CP, greater prioritisation of children in OOHC by health services, and flexible delivery of healthcare were identified as potential solutions. Study 3, a retrospective data linkage cohort study, examined the health service use of 6,201 children in the first year after entry to OOHC between 2010 and 2015, through analysis of administrative health data from one federal (Medicare) and four Victorian datasets. Only 41 children had attended all recommended services (GP, paediatrician, dentist, audiologist, and optometrist). While 90% attended a GP, fewer than 40% attended other health services, and a minority attended within recommended timeframes. This research has demonstrated that health assessment for Victorian children upon entry to OOHC is neither routine nor timely, and potentially modifiable barriers exist within the health and child protection systems. The findings have implications for cross-sector service and policy development, and for monitoring of compliance with national standards.
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ItemReflecting on Babies in the NICU: An exploration of Parental Reflective Functioning in a quaternary neonatal intensive care unit( 2020)When an infant is admitted to the neonatal intensive care unit (NICU), parents are confronted with the challenge of developing relationships with their very sick infant while trying to comprehend the medical situation and their own emotional reactions. Although Parental Reflective Functioning (PRF) is important for early parent-infant relationships, its role in mental health and attachment relationship outcomes of infants admitted to NICU and their parents has not been evaluated. The prospective “Reflecting on Babies in NICU” (ROBIN) study aimed to determine whether PRF was measurable in NICU and predicted subsequent mental health problems. Parents of infants who were at least 3 weeks of age and admitted to a quaternary NICU at the Royal Children’s Hospital Melbourne without ever having been home were prospectively enrolled. PRF was evaluated using the Parental Reflective Functioning Questionnaire (PRFQ) and, in a nested substudy, a specifically developed NICU version of the Parent Development Interview (PDI:NICU). Acute stress disorder (ASD) and post-traumatic stress disorder (PTSD) were classified according to the Acute Stress Disorder Scale (ASDS) and Posttraumatic Stress Disorder Checklist (PCL-5) respectively. The association between baseline PRF and parent self-reported feelings of attachment, measured by the Maternal, and Paternal, Postnatal Attachment Scales (MPAS/PPAS), and parental emotional functioning, was evaluated both while the infant remained in NICU and at 10 months of infant-age. Evaluable data were available for 69 infants (67 mothers and 38 fathers) enrolled between July 2014 and April 2016. Baseline PRFQ subscale scores were similar to previously published studies. Baseline PRF scores from the PDI:NICU were available for 19 mothers. The mean overall score was 5.4 (SD 1.1, range 4-7), and the pattern of individual demand question scores was similar to previously published profiles. This indicates that PRF is measurable in the NICU-setting. Paired PRFQ results at baseline and at 10 months were available for 57 parents. For mothers, the PRFQ subscales correlated closely over time, whereas for fathers only the Pre-Mentalizing Mode subscale was correlated. This suggests that the PRF being measured in NICU may be an enduring trait, rather than a state influenced by the acute condition, more for mothers than fathers. PRF at baseline was highly correlated with self-reported attachment-quality at 10 months. Specifically, high PRFQ Pre-Mentalizing Mode (PM) subscale scores in NICU predicted dyads with subsequent “at risk” attachment, implying that parents with poorer PRF in NICU are more likely have parent-infant relationship difficulties as their child grows. Acute stress disorder affected 38% of mothers and 41% of fathers at baseline. Higher baseline PRF, especially the PRFQ Interest and Curiosity about Mental States (IC) subscale, was associated with risk of concurrent ASD. Higher baseline IC scores were also associated with risk of PTSD at 10 months in mothers. These findings present a dilemma. Lower capacity for PRF in NICU may jeopardise the developing parent-infant relationship, but higher PRF may create emotional trauma for parents. Infant Mental Health resources and a “nested mentalization” approach to care are vital for infants and their at-risk parents in NICU, so they can be held “in mind” in supported ways.