Paediatrics (RCH) - Theses
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ItemInvestigating the influence of BCG and hepatitis B vaccine on neonatal immune responses( 2020)Neonatal infectious diseases result in an estimated 40% of neonatal deaths worldwide and contribute significantly to chronic morbidity. Childhood disease-specific immunisation is irrefutably linked to the decline in deaths from targeted infections over the last century. However, neonatal immunisation strategies are limited, in part, due to impairments in their adaptive immune function. Vaccine-induced protection from severe forms of tuberculosis (TB) with the Bacille Calmette-Guerin vaccine (BCG) and perinatally-acquired hepatitis B infection with the hepatitis B vaccine (HBV) are two exceptions, with these vaccines commonly administered to neonates worldwide. Evidence for heterologous (‘non-specific’) effects (NSEs) of various vaccines used in childhood, most notably for BCG, is increasing. This refers to the immunomodulatory capabilities of vaccines to influence immune outcomes beyond inducing protective immunity against the vaccine’s specific targeted disease. There is limited evidence for these effects in neonates, particularly for HBV. The research reported in this thesis aimed to explore the influence of BCG, HBV and concurrent administration of both vaccines at birth on the neonatal immune responses to unrelated antigens compared with unvaccinated babies in a randomised control trial (RCT): The Early Life Vaccine and Immunity Study (ELVIS). Neonatal blood samples from 128 participants, collected seven days after randomisation, were stimulated with various unrelated antigens for 20 hours. Cytokine responses, measured in the supernatants by an enzyme-linked immunosorbent assay (ELISA) method, were analysed using non-parametric statistical tests to determine differences in median responses between the four groups: BCG alone, HBV alone, concurrent BCG and HBV and the unvaccinated control group. I found minimal differences in the median in vitro cytokine responses to all stimulants between the four groups. However, both vaccines independently influenced cytokine-stimulant responses. Effects on responses were strongest for BCG-vaccinated babies, but only decreased interferon gamma (IFN-gamma) responses to the Toll-like receptor (TLR) ligand resiquimod (R848) and monocyte chemoattractant protein-1 (MCP-1) responses to heat-inactivated E. coli were significantly different from controls. Combined vaccines tended to induce similar cytokine-stimulant response patterns as BCG alone, although for some cytokine-stimulant pairs, the BCG-induced effects were mitigated by HBV and vice versa. This study adds to the evidence for NSEs of vaccines in neonates. It is the first study to investigate the influence of HBV immunisation on immune responses to unrelated antigens, finding no statistically significant differences in median cytokine responses compared with controls. The finding that concurrent vaccination with HBV and BCG at birth induced the same cytokine-stimulant response pattern as BCG alone, suggested that cytokine responses to unrelated stimulants are driven by BCG. This is also the first study to show that in neonates concurrent HBV and BCG vaccination at birth weakens the NSEs of BCG for certain cytokine-stimulant pairs. Further research into neonatal vaccine NSEs are warranted. Future studies should explore and further investigate the clinical relevance of certain cytokine-stimulant response signatures identified in my thesis and the mechanism for these observations in neonates. These results will direct research on how we could potentially exploit any beneficial vaccine NSEs to provide protection against infection in the very young.
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ItemExploring Efficient Generation of InsulinProducing Cells from iPSCs, to Enhance Cell Therapy for Type I Diabetes( 2020)Type I Diabetes Mellitus (TIDM) is one of the immune disorders and results from the loss of cells that make insulin (INS), a hormone that regulates the level and availability of glucose within the body. The most common treatment is exogenous insulin therapy which involves regular insulin injections or the use of an insulin pump. However, this treatment is imperfect and cumbersome, driving researchers to find more effective methods to control blood glucose levels. One potential treatment would involve making INS producing beta cells using pluripotent stem cells and then using these manufactured cells as a beta cell replacement therapy. Making insulin-producing cells in the laboratory involves several stages from stem cells to definitive endoderm, pancreatic endoderm, endocrine precursor, and the last step to mature beta cells. Any improvements in specific steps for the production of PSC derived pancreatic beta cells has the potential to lower the cost of an eventual treatment, making such therapy more widely available. In this project, we examined variations in the differentiation protocols with the ultimate aim of increasing the yield and functionality of the final cell product. One of the significant findings to emerge from this thesis is, in Chapter 4, we show that SCF addition has the potential to enhance cell proliferation during the stages of early differentiation. A surface receptor for Stem Cell Factor, cKIT, is widely applied to determine endoderm formation in the differentiation process of in vitro pluripotent stem cells. Despite its long-standing relationship with endoderm formation, a potential role for SCF itself during this process has not been systematically addressed. Using a suspension-based differentiation system, we show that the addition of SCF to differentiating cultures of PSCs enhanced the proliferation of early definitive endoderm, marked by the expression of CXCR4 and EPCAM. This effect of SCF was discernable within 2 days of differentiation initiation and coincided with the down-regulation of its receptor, cKIT. SCF treatment appeared not to affect the kinetics of differentiation, with key markers such as MIXL1, SOX17, CXCR4, and EPCAM showing identical expression patterns in untreated and treated cultures. Taken together, our results indicate that SCF addition at an early stage of differentiation will potentially reduce the number of input PSCs required to yield a given amount of endodermal products for the downstream application. This thesis is organized into six main chapters. Chapter 1 is a literature review concerning the introduction of pluripotent stem cells, Type I diabetes, pancreas development, normal molecules used in vivo differentiation protocol for beta cells generation. Chapter 2 describes the materials and methods of our experiments. Chapter 3 investigates the role of Activin A in definitive endoderm formation, which attaches great importance to adding Activin A from the beginning of differentiation. Chapter 4 shows that SCF promotes the proliferation during definitive endoderm differentiation. Chapter 5 presents images of the final cell product, which successfully show the signals of insulin. Chapter 6 is related to the limitations of previous research and the suggestions for future work.
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ItemNo Preview AvailableAutism Spectrum Disorder (ASD) and Anaesthesia( 2019)This thesis addresses the question of what is the best evidence-based management for children with ASD (autism spectrum disorder) coming under anaesthetic care in the hospital setting. The increasing prevalence of ASD (1) has meant that most anaesthetists need to become proficient in understanding and managing children with ASD. Children with ASD have higher hospital contact than their neurotypical peers.(2) Behavioural problems, sensory sensitivities, language deficit, and inflexibility with change contribute to the difficulties experienced by children with ASD in the hospital setting. (3)Hospitals may be inherently challenging to children with ASD: being inflexible places, with the sound of crying children, with invasive monitoring techniques and bright lights.(4) One unpleasant anaesthetic experience can lead to heightened anxiety and future refusal to attend hospital. In order to find the context for best anaesthetic care, we have reviewed the existing literature about ASD and its management in chapter one. The first part of chapter two is a review of anxiety and premedication in general terms. The evidence for current best practices in managing children with ASD in the perioperative period is outlined in the second part of chapter two. To further understand the family and staff perspective of optimal care, we conducted a qualitative study of 29 individuals including 15 parents of children with ASD who had had a recent anaesthetic and 14 staff members that had looked after them in different capacities at two hospitals in Melbourne, Australia in chapter three. Chapter four contains discussion and conclusion. It includes discussion about the discontinued preparation/premedication trial (CLOMID). The flaws in the design and obstacles in its execution are examined. Our data showed important organisational, educational and resource matters. Problems such as prolong waiting for an operation date, lack of training of staff including anaesthetists and nurses, lack of availability of simple equipment and private spaces in the recovery rooms- were to be addressed. Good communication, clear explanation, and friendly attitudes as well as flexibility and individualised care of patients were considered useful. The supplementary material includes a protocol for a preparation /premedication study that has not been concluded as well as two social stories that I have designed.
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ItemImproving the diagnosis of scabies in low-resource settings( 2020)Scabies is a parasitic disease and a global health problem that predominantly affects disadvantaged communities in low-resource settings. Scabies significantly impacts the health and quality of life of those with the disease. To accurately assess the global burden of disease and to compare data across regions, standardised diagnosis with consistent disease definitions is necessary. In low-resource settings, diagnosis by clinical assessment is the principal diagnostic method. In the absence of available experts, non-expert health workers are likely to play critical roles in diagnosis, including for prevalence mapping. Currently, standardised processes for clinical diagnosis for scabies do not exist. This thesis explores the diagnosis of scabies in low-resource settings and the role and utility of non-expert health workers in the diagnosis of scabies. Chapter 3 describes the evaluation of non-expert health workers in the diagnosis of scabies and impetigo using clinical criteria. The diagnosis of four briefly-trained nurses was compared to the consensus diagnosis of two experienced doctors. The sensitivity of the nurses’ diagnosis compared to the reference standard was 55.3% for scabies with a specificity of 89.9% Sensitivity for moderate to severe scabies was 93.5% with a specificity of 74%. The accuracy of diagnosis by non-expert health workers is promising and may be acceptable for scabies and impetigo disease mapping in low-resource settings. Chapter 4 describes the development and evaluation of a training protocol for the diagnosis of scabies and impetigo for non-expert health workers. The aim of this study was to measure the change in knowledge and confidence of the participants and explore their experience and perceptions of the training. Training was evaluated using a case-based test, a questionnaire and semi-structured interviews. The overall results of the case-based test were 90% for scabies and 75.5% for impetigo. The mean score for both self-reported knowledge and confidence increased from 2.5 to 4.5 following training and scores increased for all nurses (mean difference 2, 95%CI 1.1-2.9, P=0.005). The study showed that training local health staff in scabies diagnosis was enjoyable for participants and led to improvements in self-reported knowledge and confidence. Chapter 5 investigates the prevalence of scabies and impetigo using a cross-sectional study in a primary school in Gizo in the Solomon Islands. Using the International Alliance for the Control of Scabies (IACS) diagnostic criteria the classified the diagnosis of scabies. The prevalence of scabies was 54.3% and prevalence of impetigo was 32.1%. 63.5% of those with impetigo had scabies, corresponding to a population attributable risk of 11.8%. The study highlighted the extremely high burden of these diseases supporting the need for interventions for scabies in this community. Chapter 6 evaluates the methods of data collection, analysis and display for describing in detail the distribution of scabies lesions in a pilot study. The study used a novel technique of representation of dermatological lesions in the form of a choropleth map. The study found that the methods used were feasible for a larger population and would describe valuable detailed information on specific lesion location in scabies. The study will provide information on lesions at specific body sites to determine if simplified examinations are appropriate for prevalence surveys. The public health control of scabies requires identification of high-prevalence communities to target interventions, as well as methods to monitor the effectiveness of interventions. Such programs would be dependent on accurate and standardised diagnosis for population mapping. This thesis suggests methods to improve the diagnosis of scabies in low-resource settings. Modifications to training and diagnostic methods are likely to improve diagnostic accuracy. Improvements to scabies diagnosis will contribute to efficient collection methods and reliability of prevalence data.
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ItemInvestigating the biology of paediatric T cell acute lymphoblastic leukaemia to facilitate more effective individualised therapy( 2019)Acute lymphoblastic leukaemia (ALL) takes up the highest percentage of paediatric cancer. The treatment requires intensive chemotherapy for two to three years, as well as haematopoietic stem cell transplantation for poor-prognosis cases. Compared to B cell lineage ALL (B-ALL), T cell lineage ALL (T-ALL) has a lower 5-year event free rate, higher rate of relapse, and a worse outcome for relapsed cases. Individualised therapy, targeting at oncogenic changes in each patient, can make treatment more effective and less harmful. This requires understanding of the oncogenic biology of each individual leukaemia. We have attempted to develop a T-ALL model based on hiPSC-derived T cells, which will be in human origin, maintain normal genetic pattern, mimic in vivo T cell development, and can be massively produced for high throughput lab work. This model may make up for the shortcomings of conventional leukaemia cell lines and mouse models. This project investigates the biology of T-ALL by focusing on two novel fusion genes – TCF7-CSF1R and ETV6-CRX – identified by RNA sequencing of paediatric T-ALL patient samples. We have shown that TCF7-CSF1R is sufficient to immortalise mIL-3 dependent Ba/F3 cells. The ETV6-CRX fusion gene is anticipated to block differentiation. Establishing consistent expression of this fusion will require further optimization. The feasibility of setting up a hiPSC-derived T-ALL model was also assessed, with respect to protein expression in human T-ALL/lymphoma cell lines, hiPSC differentiation efficiency, hiPSC-derived T cell lentiviral infection rate, and cytokine withdrawal during differentiation. This project provides potential directions for improvement of methods for exogenous gene expression, such as the usage of CRISPR-Cas9 based techniques to introduce gene modifications for fusion genes such as ETV6-CRX that are difficult to express, particularly in hiPSC-derived T cells that have a low viral infection level. The T cell differentiation protocols also need to be optimised to make the T cell production easier and efficient. Detailed functional assay during T cell differentiation needs to be conducted in the future. In this thesis, Chapter 1 presents the background of this project; a literature review introducing human haematopoietic system, in vivo thymocyte development, paediatric TALL, novel oncogenic fusion-related genes, and in vitro T cell generation; the aims and hypothesis. Chapter 2 introduces the methods and materials used in this project. Chapter 3 presents the identification, cloning, and expression of novel fusion genes. Chapter 4 investigates the ability of these novel fusion genes to support cell survival and proliferation in conventional Ba/F3 cell line. Chapter 5 assesses the feasibility of setting up the hiPSC-derived T-ALL model. Chapter 6 makes a discussion on the results and concludes the whole project.
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ItemProximal femoral osteotomy in children and adolescents with cerebral palsy( 2018)Background Cerebral Palsy (CP) is the most common cause of physical disability affecting children in developed countries. Approximately one third of children with CP may develop hip displacement. Non-ambulant children at Gross Motor Function Classification System (GMFCS) levels IV and V are at highest risk. Without early detection through surveillance programs, hip displacement can progress to hip dislocation, which is frequently painful and negatively impacts health-related quality of life (HRQoL). Injections of Botulinum Neurotoxin A (BoNT-A) have no role, and soft tissue surgery has a limited role in preventing hip displacement in non-ambulant children with CP. Bony hip reconstruction surgery such as a proximal femoral osteotomy (PFO) is effective in stabilising the hip and HRQoL. PFOs include Femoral Derotation Osteotomies (FDO) which aim to improve the gait of an ambulant child (GMFCS I-III), and Varus Derotation Osteotomies (VDRO) which aim to contain the hips in non-ambulant children (GMFCS IV-V). However, PFOs can carry high risks, especially in children with medical co-morbidities such as respiratory disease, nutritional deficiencies, hypertonia and osteopenia. Aim This thesis involved a series of three studies, which aimed to expand our knowledge of the trainee learning curve, outcomes and adverse events relating to PFO in children and adolescents with CP. Method and Results A new implant combining locking and cannulated technology (Locking Cannulated Blade Plate, LCBP) was recently developed for use in PFO. A pilot study was performed on the first 25 patients who had surgery with the LCBP, at the Royal Children’s Hospital (RCH), Melbourne. This study established safety for use in children as young as three, with weights as low as eleven kilograms. A further prospective, parallel cohort study of 90 consecutive children with CP was conducted to compare the LCBP against with existing non-cannulated, non-locking implant (Angled Blade Plate, ABP). Technical and radiological outcomes of surgery were similar between implants. However, the surgical technique was reported by trainees to be easier when using the LCBP, with less technical errors. Approximately 60 percent of the children experienced minor adverse events including: constipation, inadequate pain control, and respiratory compromise. However, a CP specific tool was not available to classify the severity of events. Study three was performed to clarify the Modified Clavien-Dindo (MCD) system for lower limb surgery in children with CP and test its’ reliability for classifying adverse events. Very good reliability was demonstrated amongst members within a multidisciplinary team. Conclusion Novel findings from these studies may help improve the safety and efficacy of the management of hip displacement in children with CP. Further research should address the long-term outcomes of PFO in children with CP, evaluate the validity of the MCD for children in CP and determine if the MCD can be embedded in the electronic medical records (EMR) as a routine tool for audit and clinical research.
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ItemCirculating microRNAs in Rett syndrome( 2019)Rett Syndrome (RTT) is a severe neurological disorder with diagnostic challenges and undetermined patholophysiology. Small regulatory RNAs known as miRNAs are essential in a multiple biological processes, including brain development. Using next generation sequencing in a mouse model of RTT, I identified circulating, plasma miRNAs that are differentially expressed in RTT mice. Many of these miRNAs were previously shown to have neurological functions or be involved in RTT. The biomarker potential of these miRNAs was further explored using quantitative PCR.
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ItemDaytime predictors of sleep disordered breathing in Duchenne muscular dystrophy( 2017)Sleep disordered breathing (SDB) in Duchenne muscular dystrophy (DMD) progresses to respiratory failure. The gold standard for diagnosis is polysomnography (PSG) but the optimal timing is unknown. Twenty boys with DMD, aged 10 – 16 previously undiagnosed with nocturnal hypoventilation (NH), had repeat PSG. Concurrent clinical data was correlated to outcomes of PSG. 90% had OSA, O% NH (PtcCO2 > 50 mmHg > 25% TST) after 2 years. Boys younger than 10 years of age with DMD need PSG to elicit OSA.
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ItemNovel technology for the measurement of newborn and infant heart rate( 2017)Background: Monitoring heart rate in newborns and infants is crucially important in guiding resuscitation and medical care. Established methods for heart rate assessment of these children have inherent drawbacks. In recent years, novel methods for assessing neonatal and infant heart rate have been developed, with varying levels of evaluation conducted. Digital stethoscopes may provide a better means of heart rate assessment for newborns and infants. Aim: The aim of this thesis was to comprehensively review existing established and novel technologies used to monitor newborn and infant heart rate, and compare new digital stethoscope technology with the gold standard, electrocardiogram (ECG). Methods: This thesis (a) outlines the definition and importance of heart rate in medicine, presented in the context of a review of cardiac anatomy and physiology relevant to understanding this vital sign and aspects of its measurement in neonates and infants; (b) presents a narrative review of established methods for monitoring heart rate; (c) expands the scope of this review from established to emerging methods for monitoring heart rate with a systematic literature review of novel methods for newborn and infant heart rate assessment; (d) describes original research using a prototype digital stethoscope attached to a smart device containing software for detecting and displaying heart rate in real-time that was conducted on infants in the neonatal intensive and special care setting, as well in the delivery room setting using an improved version of the device and software. Results: A review of the literature analysing methods of assessing neonatal and infant heart rate found strengths as well as significant weaknesses in the various methods in clinical use or in development. In the neonatal unit, a prototype digital stethoscope and smartphone device for assessing heart rate had a mean difference (±2 standard deviations) of 7.4 (48.5) beats per minute (bpm) when compared to the gold standard of electrocardiography. The mean (interquartile range) time to first digital stethoscope heart rate display was 4.8 (1 to 7) seconds, and the device failed in 12.3% of use attempts. Repeating the comparison in the delivery room setting using an updated algorithm and new hardware, Bland-Altman analysis revealed a smaller mean difference (±2 standard deviations) between the digital stethoscope and electrocardiography of 0.2 (-18 to +18) bpm including crying periods (Figure 23), and 1.0 (-11 to +12) bpm excluding crying periods. The improved digital stethoscope took a median (interquartile range) of 7 (5 to 11.5) seconds after application to display a heart rate. It failed to detect heart rate in 37% of cases, all of which were in crying infants. Conclusion: A digital stethoscope and smart device with software can rapidly detect neonatal and infant heart rate. In the delivery room, device failure primarily occurred during infant crying, with improved accuracy during non-crying periods.
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ItemA tool for risk profiling and accurate prognostication in paediatric glioma integrating clinical features with epigenetics: it is time to move on from the binary classification( 2016)Paediatric glioma, the most common group of brain tumours in children, encompasses a wide range of entities with highly variable prognoses. Gliomas are grouped by histopathological features into high and low grade glioma but this classification does not take into account many established and emerging risk factors in this disease. Research into the molecular features of these lesions has shown that histology does not always correlate with biology and, where they differ, molecular features are usually superior at predicting outcome. Risk classifications have been developed for other paediatric malignancies which combine clinical, radiological, pathological and molecular factors to predict disease risk and prognosis. A comprehensive risk classification has not been published for paediatric glioma despite many risk factors being established in this disease. Using a clinical cohort of all paediatric glioma treated at a single institution (Royal Children’s Hospital, Melbourne, Australia) over an 18 year period a database was developed incorporating clinical, radiological, pathological and treatment factors. Where sufficient tumour tissue was available genome-wide methylation analysis was performed. The results of this were processed and evaluated by an established cluster analysis algorithm. Breaking the cohort into clinically appropriate subgroups, risk factors for disease progression and death were determined and prognosis estimated for distinct groups. A tool for robust risk profiling and prognostication was established with 5 main risk groups and 10 subgroups. Patients in the very low risk group have a predicted 100% overall survival and the majority require no treatment. In contrast there was a group of patients with 100% mortality within a short space of time where attempts at curative therapy are futile and may be deleterious to quality of life. The intermediate risk group contained a number of entities with a prognosis genuinely falling between that of low grade glioma and that of high grade glioma. Histopathological diagnosis retained prognostic importance for some, but not all, groups and methylation analysis was shown to have a significant role to play, particularly in high grade lesions or where diagnosis was unclear. This is likely to become part of routine care in the future and its place is strongly supported by the analysis presented here. Risk profiling is possible in paediatric glioma with far more accuracy than the current binary classification provides. A combination of established clinical factors and emerging molecular features provides an accurate and nuanced assessment of risk and prognosis.