Paediatrics (RCH) - Theses

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We could do more, but should we? The approach to decisions about life-prolonging treatments for children with life-limiting conditions

Vemuri, Sidharth ( 2023-05)

Children with life-limiting conditions are anticipated to have episodes of clinical deterioration in their health. These episodes of deterioration can be significant and may result in the child’s death. During these episodes, decisions about whether to embark on treatments designed to prolong life arise. These treatments involve intensive care therapies, such as intubation and mechanical ventilation, and resuscitative measures. Decision making about these treatments for children with life-limiting conditions commonly occurs between the child’s paediatrician and their parents. This is because the child often lacks developmental capacity to participate in decision making for themselves. Making decisions about life-prolonging treatments, otherwise known as end-of-life decision making, is often viewed with significant trepidation by paediatricians because of the gravity and emotional nature of these decisions. Little is known about how these decisions are made in practice. Despite this gap in knowledge, consensus guidelines in middle- to high-income countries recommend the practice of shared decision making and advance care planning. Shared decision making and advance care planning are concepts that emerge in the care of a decisionally competent adult patient, to preserve and respect their autonomy in decision making. These concepts are poorly understood in the paediatric context where decisions are being made for the child rather than with the child. This thesis explores how paediatricians approach end-of-life decision making for children with life-limiting conditions. This exploration adopts an interpretivist approach which that assumes that a paediatrician’s experience of end-of-life decision making will shape their practice. To achieve this, this thesis draws on two qualitative studies involving semi-structured interviews. The first involves paediatricians primed by a paper-based vignette; the second involves paediatricians and parent-actors primed by a clinical simulation. Development of the latter study was supported by a scoping review of methodological literature. The overall findings were that paediatricians identified and valued a preparatory process of discussions with parents for end-of-life decision making for children with life-limiting conditions. Most paediatricians did not regard this preparation as advance care planning but considered it the practice of shared decision making. Despite reporting this practice as shared decision making, what paediatricians often described were physician-led decision-making approaches. Detail of how paediatricians work within and lead this preparatory process was revealed, with their overarching aim being to facilitate alignment of parental views with the paediatrician’s goals of care for the child. The mechanism by which paediatricians intend to achieve this by leading parents through this process of discussion in preparation for end-of-life decision making was discovered; this mechanism has been termed ‘shepherding’. By developing understanding of paediatricians’ perspectives of the process of preparing for end-of-life decision making for children with life-limiting conditions, there is an increased awareness of the broader ethical landscape in which end-of-life decision making occurs. This thesis makes the end-of-life decision making process for children with life-limiting conditions visible, providing a shared language that can facilitate consistency of practice and training, and future targeted research. Finally, this thesis contributes to knowledge about the role of simulation as a reliable and feasible option in qualitative research inquiry, which should encourage its use in future research of highly sensitive phenomena.
Quantifying oedema in children in intensive care

Gelbart, Ben ( 2023-07)

Oedema is the accumulation of fluid in tissues and an important clinical sign in the management of critically ill children. It indicates that fluid extravasation from the intravascular to the interstitial space has exceeded physiological clearance mechanisms. The initial mechanistic descriptions in the 19th and 20th centuries still hold true, but there is now greater understanding of the pathophysiology of diseases and interventions that cause oedema, particularly regarding the role of endothelial injury and dysfunction. Oedema is clinically recognisable by clinicians; however, its clinical assessment is inexact and therefore its relevance poorly understood. Positive fluid balance (or fluid accumulation) on the other hand, as measured by fluid balance charts, is a widely reported metric, measurable, and over the past two decades, numerous studies have reported its associated harm in critically ill children. Virtually no data exist regarding the outcomes associated with oedema. This is, at least in part, because standardised, validated methods for quantifying oedema do not exist. The overarching aims of this thesis focused on the development of methods for quantifying oedema and investigating their precision and validity. A prospective observational study in infants with congenital heart disease in intensive care was conducted in this regard. There are virtually no data on the clinical measurement of oedema and therefore the nature of this study is exploratory. The study investigated simple and novel techniques and investigated their validity as well as their relationships to important clinical outcomes such as vital organ oedema and organ dysfunction. This study was supported by preliminary pilot work to establish feasibility and safety of such methods and a retrospective study which investigated the associated outcomes of fluid accumulation measured by fluid balance charts. This thesis addresses an important component of critical illness where a knowledge gap exists and the commonly utilised and reported metric, fluid balance, insufficiently defines fluid status during critical illness.
Investigation of approaches for handling missing data in complex epidemiological studies

Middleton, Melissa ( 2023-04)

Missing data are ubiquitous in epidemiological studies and multiple imputation (MI) and inverse probability weighting (IPW) are popular statistical tools to address this. Both approaches can reduce bias in the target effect estimate compared to the standard complete-case analysis, with MI also often improving precision. However, MI may result in biased target effect estimates if the imputation model is misspecified, particularly when imputing large amounts of missing information. Conversely, IPW may not be effective to handle missing data in a disorganised pattern across multiple variables. An alternative approach is to combine MI and IPW, where MI is used to handle small amounts of data missing across multiple variables, while IPW is used to address larger proportions of missingness. The aim of this thesis is to explore the use of MI in combination with IPW, including how best to implement a combined approach, and how it compares to either MI or IPW alone. Simulation and case studies are used to evaluate the approaches, motivated by two case studies considered within the Barwon Infant Study: a case-cohort study with a binary endpoint, and a longitudinal study investigating the effect of an exposure on an outcome measured at a given timepoint. In case-cohort studies, a large proportion of exposure data is missing ‘by design’ and is typically handled with IPW, while unintended missing confounder data are handled using MI. One key consideration of MI is that the imputation model should be compatible with the target analysis model, that is all features and variables of the analysis model should be considered in the imputation model. In the context of using a combined MI/IPW approach, this requires the weighting to be incorporated into the imputation model. There is currently a lack of guidance on how best to do this. It is also unclear whether MI or IPW alone may provide any benefit over a combined approach. In longitudinal studies, missing data is typically handled using MI, however, when there are large proportions of incomplete outcome data due to drop-out, it is unclear whether a combined MI/IPW approach may be preferrable. Within a combined approach, incomplete outcome data could be handled using IPW, while MI is used to handle incomplete covariates. It is unclear whether a combined MI/IPW approach offers any benefit over MI or IPW alone in this setting. The findings from this thesis suggest that in case-cohort studies with a binary endpoint, inclusion of the weights as a predictor in the imputation model when using a combined approach performed well suggesting that it may be sufficient for imputation model compatibility. Additionally, a combined MI/IPW approach provides relatively unbiased target parameter estimates and was preferable to an MI-only or IPW-only approach. In the longitudinal setting, there was no gain in terms of bias or precision when using a combined MI/IPW approach over MI-only, with both performing well. Given that MI-only is simpler to implement, this may be the preferrable approach in this context. The findings from this thesis provide guidance on the use of a combined MI/IPW approach in the context of case-cohort studies with binary outcomes and longitudinal studies estimating the effect of an exposure on an outcome measured at a given timepoint. They address a much-needed gap in the literature for handling missing data in complex epidemiological studies.
Safety of Bacille Calmette-Guérin vaccination and revaccination

Villanueva, Paola ( 2022-12)

Bacille Calmette-Guerin (BCG) vaccine, derived from live-attenuated Mycobacterium bovis, is commonly given as a single dose in infancy to protect against tuberculosis (TB) in high TB-prevalence settings. There is growing interest in BCG revaccination for the protection of adolescents and adults against TB. Increasing recognition of potential novel applications for BCG to protect against diseases other than TB also mean that BCG vaccination (and revaccination) may become increasingly used in broader age groups and settings. For these reasons, a more comprehensive and detailed understanding of the safety of BCG vaccination and revaccination, particularly in adults, is necessary to help inform public health policy. My PhD research aimed to evaluate the incidence and predictors of reactions (normal, accelerated and adverse) to BCG vaccination, the impact of revaccination on BCG reactions, and the clinical management of adverse reactions to the vaccine. These aims were investigated using active safety surveillance of BCG vaccination reactions within an international randomised controlled trial of nearly 7000 adult participants – the BRACE Trial (BCG vaccination to reduce the impact of COVID-19 in healthcare workers), as well as through two systematic reviews and a meta-analysis of BCG vaccination safety in children. My thesis comprises three sections characterising reactions following BCG vaccination and revaccination: (i) the normal reaction; (ii) accelerated reactions; and (iii) adverse events. The first section reports a cohort study to assess the prevalence, factors influencing formation and vaccinee perception of BCG scarring (normal BCG reaction) following vaccination and revaccination in adults. The second section includes a systematic review and meta-analysis to investigate the clinical significance of an accelerated BCG reaction in children, reporting an association with prior mycobacterial exposure. A cohort study is then presented that evaluated the incidence and clinical implications of an accelerated BCG reaction in asymptomatic adults in low and high TB-prevalence settings. The third section includes a systematic review of the management strategies for BCG-associated lymphadenitis and injection site abscess in children. This is followed by a cohort study to assess the incidence and risk factors (host immune- and vaccination-related) for BCG-associated lymphadenitis and injection site abscess in adults. An additional cohort study in adults assesses the safety of co-administration of BCG with the influenza vaccine. The overall incidence of local adverse events and serious adverse events in the BRACE trial is then described, as well as the impact of previous BCG vaccination on local injection site reactions. Overall, BCG revaccination in adults was associated with increased frequency of normal (scar), accelerated and local adverse reactions (injection site abscess and lymphadenitis), compared to receiving BCG for the first time. Younger age was also associated with increased frequency of normal and local adverse reactions. Similarly, female sex was associated with an increased frequency of normal reactions and lymphadenitis following BCG vaccination, as well as increased frequency of accelerated BCG reactions. Latent TB infection was found to predispose to an accelerated BCG reaction, but was not associated with injection site abscess. Nonetheless, regardless of prior BCG vaccination status, age or sex, the majority of reactions were mild in nature, localised to the injection site and resolved without intervention. In summary, the studies in my thesis indicate that BCG vaccination and revaccination have an acceptable safety profile in children and adults.
Exploring the investigation, diagnosis and clinical management of tree nut and peanut allergy in children

Brettig, Timothy William ( 2023-03)

By the age of one, 1 in 10 Australian children have developed a food allergy. Most people are aware of common allergens such as cow’s milk, egg, and peanut, but tree nut allergy is less recognised and researched despite having a prevalence similar to peanut allergy in older children. Peanut and tree nuts are responsible for the majority of fatal or near-fatal allergic reactions to food and with natural allergy resolution for peanut reported as 30%, and tree nuts collectively reported as 10%, a diagnosis of peanut or tree nut allergy carries a poor prognosis. Diagnosis of food allergy is not simple. Clinical history determines whether a clinician may perform a diagnostic test such as a skin prick test or blood test to determine the likelihood of allergy. If the result is not conclusive, then an oral food challenge (OFC), or medically supervised food introduction is required to achieve an accurate diagnosis. Whilst OFCs are the gold standard for diagnosis, they are time and labour intensive, expensive and put the individual at risk of severe allergic reaction, or anaphylaxis. They are also the ‘bottle-neck’ in the diagnostic process with limited access relative to the burden of food allergy assessments. With limited understanding of the accuracy of current diagnostic tools there is a real risk for over-diagnosis of tree nut and peanut allergy. This thesis aims to improve the diagnostic approach to tree nut and peanut allergy. It explores several areas involved in the diagnosis of tree nut and peanut allergy including describing the prevalence of cashew allergy and the diagnostic accuracy of currently used tests for specific tree nuts. It also explores the use of diagnostic algorithms as a method to improve diagnostic accuracy and reduce the need for OFCs in both modelled and ‘real world’ datasets for cashew and peanut allergy. The cost of these interventions is also assessed in the first economic evaluation for use of diagnostic algorithms in tree nut allergy diagnosis. Using the population-based EarlyNuts cohort (n=1933), I found the prevalence of cashew allergy in 1 year old infants to be 1.49%, with sensitisation (SPT result >/= 3mm) seen in 1.96%. Infants with eczema in the first year of life were more likely to be cashew allergic (adjusted odds ratio=5.75) and there was also an association between peanut allergy and cashew allergy (adjusted odds ratio=19.30). Cashew was introduced before 12 months of age in 25% of participants, which was low compared to other foods such as peanut. I have undertaken a systematic review of the accuracy of the current tests used in diagnosing tree nut allergy. This review highlighted that for some tree nuts, such as cashew or walnut, the accuracy of tests was reasonable, but many nuts lacked evidence about a conclusive diagnostic cut-off, increasing the likelihood of requiring an OFC for definitive diagnosis. Tests for cashew allergy were most accurate compared to other tree nuts, and newer blood tests, specifically IgE to the cashew component ana o 3, were superior to the commonly used method of a cashew skin prick test for diagnosis. To investigate this further, I combined and analysed global data from studies of patients with possible cashew allergy that had data on both test results and food challenge outcomes, demonstrating that using ana o 3 or a diagnostic algorithm incorporating sIgE to cashew followed by ana o 3 resulted in an 80% reduction in the need for an OFC to get a definitive diagnosis compared to skin prick testing alone. I performed a cost-comparison analysis comparing these approaches to skin prick testing alone (the current clinical practice standard in Australia) again using modelled data. This demonstrated a 46% cost reduction to the healthcare system. The final step of my PhD was to investigate the clinical and economic impact of a similar 2-step algorithm used in a large tertiary hospital allergy department for diagnosis of peanut allergy. This retrospective clinical audit (n=8826) demonstrated an uptake of the diagnostic algorithm of 41.95% of eligible presentations, which resulted in a 27.8% reduction in OFCs performed compared to using peanut SPT alone. I also demonstrated a 32% cost reduction as a result of implementing this pathway. The outcomes of my thesis can be directly translated into modifications of clinical practice resulting in significant clinical impact by achieving more accurate and timely clinical outcomes with fewer OFCs, improved safety for individuals and substantial cost reductions to the health system.
Towards timely screening and diagnosis of neonatal jaundice

Thomas, Mercy ( 2023-04)

ABSTRACT Neonatal jaundice remains one of the primary reasons for newborn hospital readmissions, despite the efforts to recognise at risk newborns before birth hospital discharge. Due to the increasing prevalence of early hospital discharge of neonates, community based screening, including appropriate point of care technology, is essential for the timely detection of jaundice. This thesis aimed to generate and compile evidence to advance the timely screening of neonatal jaundice in community settings. The thesis contains one review paper, a discussion paper and three research studies, each investigating aspects of the evidence required for advancing neonatal jaundice screening policy. The first study determined the rate and severity of jaundice readmissions to tertiary level neonatal services in Victoria. Medical records of newborns from four major metropolitan health services were retrospectively reviewed for April to September of three consecutive years 2018, 2019 and 2020. The overall incident rate of jaundice readmissions was high at 3.3%. While the rate of readmissions differed between hospitals, the percentage of newborns readmitted remained the same across the study period at most sites. A substantial number of newborns (11%) were readmitted with severe jaundice, and this presentation was similar across both the gestational age groups of 35 to 37+6 weeks and greater than or equal to 38 weeks newborns. Moreover, a significant proportion (37%) of newborns were readmitted earlier than usual (within three days of age) following discharge from the birth hospital. The second study assessed current screening practices, including the enablers and barriers to screening and opportunities to optimise neonatal jaundice screening in community settings. An anonymous, purpose-designed web-based (REDCap) survey was sent to maternal and child health (MCH) nurses who worked across the local government areas from metropolitan and regional Victoria, and to domiciliary midwives from two selected hospitals in Metropolitan Melbourne. The most common enablers were physical examination of newborns and discharge summaries from the birth hospital for MCH nurses, compared to years of experience, and jaundice screening documentation tools for domiciliary midwives. Common barriers included screening newborns with darker skin tones, communication difficulties among culturally and linguistically diverse families, and lack of point of care screening tools. Recommendations from this study include the need for point of care screening tools, optimising the screening process by providing parent and staff education, a standardised, evidence based practice guideline, and a timely streamlined handover process between birth services and MCH services. The third study investigated the possibility of developing a low cost point of care screening tool to aid in the onsite, real-time diagnosis of neonatal jaundice in community settings. New insights in transporters of bilirubin metabolism hypothesise that a proportion of bilirubin is excreted via the renal system. This study aimed to determine if unconjugated bilirubin, the key biomarker responsible for neonatal jaundice, could be measured in the urine of newborns. Urine samples of jaundiced (SBR greater than150 umol per Litre) newborns (n=22) were collected within the first 72 hours of life. Urine samples were analysed using two methods 1) High-performance liquid chromatography and 2) Liquid chromatography triple quadrupole mass spectrometry. The preliminary findings indicated the presence of unconjugated bilirubin in the urine of newborns but at low levels. While urine bilirubin could facilitate a less invasive jaundice screening method, the correction for urine concentration and correlation to serum bilirubin needs would need to be established before validating the diagnostic utility of urine bilirubin. The results of this project highlight the existing gaps in practice for the timely detection of neonatal jaundice in community settings, which can lead to avoidable hospital readmissions and severe cases of jaundice. The study emphasises the opportunity to enhance preventative strategies, including improving infant feeding practices, enhanced communication pathways between birth services and MCH services, implementing community-based screening protocols, and utilising point of care screening tools to improve newborn outcomes. These findings have important implications for public health policy, practice, and research. To ensure the successful implementation of these recommendations, an implementation framework is proposed involving collaboration among healthcare providers, policymakers, and researchers to develop guidelines, education resources and a communication pathway between birth services and MCH services to ensure optimal screening practices and timely diagnosis of neonatal jaundice.
The Epilepsies of Cerebral Palsy

Cooper, Monica Sophie ( 2023-05)

Epilepsy in children with cerebral palsy (CP) places a significant burden on individuals, society and the health care system. There is limited data on the association between aetiology, neuroimaging, EEG findings and epilepsy syndrome classification for children with CP. I have conducted a population-based study to characterise the epilepsy according to single pathological substrates: white matter injury (WMI) and grey matter injury (GMI), which can be further subclassified to deep GMI, watershed injury or an arterial ischaemic stroke (AIS). I also analysed the prevalence, type and outcomes of epilepsy in children with CP. This will help guide counselling and treatment.
The innate immune mechanisms underlying the off-target effects of Bacillus Calmette-Guérin vaccine in infants

Bannister, Samantha Ann ( 2022-12)

Bacillus Calmette Guerin vaccine (BCG) is administered to over 100 million newborn infants globally each year with the aim of protecting against tuberculosis. In addition to its specific anti tuberculous effects, BCG vaccine has off target effects that protect against a broad range of non mycobacterial infections in both children and adults. Immunological studies have implicated trained immunity in the mechanism underlying the off target effects of BCG vaccine in adults. Trained immunity is a de facto innate immune memory underpinned by the functional epigenetic and metabolic reprogramming of innate immune cells, such as monocytes. While much research has focused on elucidating the immunological mechanisms underlying the off target effects of BCG in adults, few studies to date have investigated the immunological mechanisms in children. Using samples from a well characterised sub cohort of 130 infants from MIS BAIR, a randomised trial of neonatal BCG vaccination in Australia, the projects in this thesis aim to characterise the phenotypic, functional, and epigenetic effects of neonatal BCG vaccination on the infant immune system in the first year of life. Chapter 2 of this thesis sought to better understand the role of vaccination and natural infection in shaping the human epigenome by a systematic review of the literature. This review identified a paucity of paediatric studies and highlighted a need for further research investigating the epigenetic mechanisms underlying the off target effects of BCG vaccine in children. This knowledge gap is addressed by the subsequent projects of this thesis. Immunological studies in infants are hampered by the ethical and logistical challenges of obtaining blood samples from this age group. Optimisation experiments detailed in Chapter 3 led to the refinement of laboratory methods that generate high dimensional immunological data from small volume infant blood samples containing as few as 3 million peripheral blood mononuclear cells (PBMC). Multi parameter flow cytometry was used to comprehensively profile and sort cell populations of interest for downstream analysis. The monocyte fraction was then used for parallel DNA methylation analysis and ex vivo heterologous stimulation assays. This analysis pipeline produced rich normative data on baseline immune cell profiles and monocyte cytokine responses in BCG naive infants. Subsequent studies in Chapters 4 and 5 found that BCG vaccinated infants have increased proportions of circulating classical and non classical monocytes, and activated regulatory CD4 T cells, compared to BCG naive infants at 13 months of age. Monocytes from BCG vaccinated infants also have an attenuated inflammatory response following heterologous stimulation and retain a DNA methylation signature of early life BCG exposure. Genes associated with this signature are involved in interferon signalling pathways and viral immunity. In summary, the studies in this thesis demonstrate that neonatal BCG vaccination induces durable changes in circulating immune cell profiles, monocyte cytokine responses and DNA methylation remodelling that persist over the first year of life. These changes are different to those observed in adults and strengthen evidence that immune responses to BCG vaccination are age dependent. Collectively, these findings contribute to a better understanding of the immunological mechanisms underlying the off target effects of BCG vaccine in infants that will inform the development of new immunomodulatory therapies and guide global vaccine policy.
Investigation of the short- and long-term complications of anorexia nervosa/atypical anorexia nervosa and the impact of the coronavirus pandemic

Springall, Gabriella Anne Cornell ( 2022-10)

Anorexia nervosa (AN) is an increasingly prevalent psychiatric disorder in which restricted energy intake leads to significantly low weight, a fear of weight gain, and a distorted perception of one’s body image. Individuals with this condition experience complications which affect the heart, muscles and bones, hormones, and mental health. The long-term prognosis for AN is difficult to predict; whilst many complications resolve with refeeding and weight gain, some damage may be permanent and disordered cognitions may persist. Furthermore, the impact of the coronavirus (COVID-19) pandemic on mental health and eating disorders is increasingly evident. However, reasons for declining mental health and the onset of disordered behaviours, as well as how the treatment of individuals is affected, remains unclear. The aim of this project was to (1) assess long-term cardiovascular and mental health following recovery from AN, and (2) determine the impact of the COVID-19 pandemic on both current and former AN patients. To achieve this, four separate but interrelated studies were conducted. The first study involved a four-year retrospective chart review of all patients presenting to the Royal Children’s Hospital Eating Disorder Service between 2017-2020. To assess the impact of the COVID-19 pandemic, the number and severity of presentations was compared between years via analyses of variance. The second study investigated long-term psychological outcomes and predictors of recovery and prognosis by administering online questionnaires to former AN patients from the Royal Children’s Hospital and Monash Children’s Hospital. The questionnaires assessed eating and exercising behaviours, mood, and COVID-19 impact. The third study involved non-invasive physical testing of the former patients to assess long-term cardiovascular health post AN recovery. Finally, further interrogation of carotid ultrasound images using wave analysis techniques provided valuable insight into the biomechanical interactions between the heart and vascular network. All outcome measures were compared to healthy controls and/or normal thresholds. Results revealed there was a surge in new AN cases under COVID-19 restrictions in 2020. Changes to routines, and feelings of isolation and loneliness were recognised as triggers for disordered behaviours. Mixed responses to telehealth and online learning were also reported. Former patient questionnaire responses indicated that psychological recovery from AN can be sustained long-term when individuals receive early and intense treatment. However, elevated levels of stress were exhibited by the former AN patients. This may reflect inherent personality features, such as perfectionism, which have been closely correlated with the condition. Despite increased stress and a history of an eating disorder, the former AN patients did not appear more susceptible to the impacts of the COVID-19 pandemic and faired comparably to controls. Cardiovascular assessments indicated that an increased propensity for cardiovascular disease may be associated with AN even after recovery. Regional changes in arterial stiffness, endothelial dysfunction, and abnormal autonomic regulation were identified in early adulthood. Overall, as the first research study to report on long-term cardiovascular and mental health following AN recovery, and the impact of the COVID-19 pandemic on an adolescent population within Melbourne (the most locked down city in the world), this thesis fills a critical gap in eating disorders literature. It identifies factors that contribute to patient course and outcome, reveals persisting cardiovascular adaptations, and informs of specific issues faced by both current and former AN patients during the pandemic. These findings have the potential to guide disease surveillance strategies and optimise clinical interventions.
A Blood Based Study of COVID-19

Mccafferty, Conor James Francis ( 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.

Paediatrics (RCH) - Theses

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