Background: Congenital heart disease (CHD) is the leading non-infectious cause of paediatric morbidity and mortality worldwide and a significant social and healthcare burden. The aetiology of CHD is poorly understood, though heritable genetic factors including copy number variants (CNVs) have been shown to contribute to the risk of CHD in individuals of European ancestry. However, the role of rare CNVs in the development of CHD in African populations including South Africa is unknown. This study aims to identify pathogenic and likely pathogenic CNVs in South African cases of CHD. To our knowledge, this is the first study to investigate the genetic basis of CHD in a South African cohort. Methods: The study cohort included 105 patients presenting to the cardiac clinics at Red Cross War Memorial Children's Hospital and Groote Schuur Hospital with non-syndromic isolated CHD (n = 76), nonsyndromic CHD with additional extra-cardiac anomalies (n = 17), and positive controls with syndromic CHD (n = 12). Genotyping was performed using the Affymetrix CytoScan HD platform. Rare CNVs were filtered using stringent criteria for their size and algorithm-specific quality score and were compared against a gene panel of known CHD-associated genes. Candidate genes were considered based on pLI scores and reported CHD phenotypes in mouse models. The identified CNVs were validated by quantifying the read-coverage of available whole-exome sequencing data of a similar overlapping cohort. Results: Chromosomal microarray analysis was successful for 101 participants (including 89 non-syndromic CHD cases and 12 control cases) and led to the identification of eight CNVs overlapping genes known to be causal for CHD (GATA4, TBX1, FLT4, CRKL, NSD1, and B3GAT3), and four CNVs encompassing candidate genes likely to play a role in the development of CHD (DGCR8, JARID2, KDM2A, and FSTL1). The CNVs were identified in nine unrelated individuals: five of the CNVs were classified as pathogenic or likely pathogenic (5.6% of the cohort) and four were classified as variants of unknown significance (4.6%). CNVs of interest were validated using the available whole-exome sequencing data. Conclusions: In this study, we show that chromosomal microarray analysis is an effective technique for identifying CNVs in patients diagnosed with CHD and that this approach can be performed locally in South Africa, producing results similar to those seen in international CHD studies. The findings of this thesis highlight the wide genetic heterogeneity of CHD and the growing importance of CHD genetic studies for both research and clinical purposes. Advancing our understanding of CHD aetiology will help define disease risk in South Africa and improve the way we care for and assess our cardiac patients.

Background Effective phototherapy reduces neonatal jaundice and its complications. Irradiance increases as the distance of the light source decreases from a single phototherapy light. There are limited studies of the effect of distance and positional changes on different LED light designs on achieving effective phototherapy. Objectives To describe and compare the effect of distance, angle and plastic barriers on three different LED lights of different design. Methods Comparisons were made using a Servolite LED light, a General Electric (GE) Lullaby and a Ningbo David LED phototherapy light. Measurements were done according to methods described by the International Electrotechnical Communission (IEC). The effective irradiated area was measured on a grid measuring 60 x 30 cm subdivided into 5 x 5 cm squares. Measurements were done for the following scenarios: light placed at the manufacturers' recommended distance, 20 cm closer, 20 cm further, at an angle, through clear plastic and through scuffed perspex. Results When the lights were placed closer to the irradiated surface than the manufacturers' recommendations, the maximum irradiance increased, but the median irradiance and uniformity ratio decreased. When the lights were angled at 45 the median irradiance was decreased. A decrease in the median irradiance was also seen when phototherapy lights passed through scuffed plastic and food grade plastic. Conclusion Our study demonstrated that placing LED lights closer than the manufacturers recommendations, the use of transparent barriers and the use of lights at an angle, compromised phototherapy irradiance and distribution. Only the GE light met IEC standards.

Background: Long term survival of children living with HIV due to improved early access to antiretroviral therapy (ART) is contributing to a growing population of adolescents living with perinatally acquired HIV (PHIV+) at risk of developing chronic multisystem comorbidity. There is limited knowledge on the overall burden, progression and causes of morbidity in PHIV+ adolescents, especially in resource limited settings. Much of what is known about morbidity in PHIV+ adolescents relates to single organ system pathology and there is a lack of a holistic approach to PHIV+ adolescents and their overall health. The aim of this PhD project was therefore to investigate the spectrum and determinants of chronic morbidity, the progression of disease and intercurrent illness in PHIV+ adolescents on ART over a 4- year period. Methods: This was a prospective study of participants enrolled in the Cape Town Adolescent Antiretroviral Cohort (CTAAC), a longitudinal cohort study, that recruited 515 PHIV+ adolescents and 110 HIV negative (HIV-) adolescents matched by age from 7 health care sites in Cape Town, South Africa. Eligibility criteria included PHIV+ adolescents who were aged 9-14 years, who had been on ART for at least 6 months and were aware of their HIV status. All adolescents and caregivers gave informed consent/assent. Participants were followed 6-monthly with questionnaires, clinical examination with detailed pulmonary (lung function), neurocognitive (magnetic resonance imaging and a battery of neurocognitive tests), cardiovascular (echocardiogram and ECG) and laboratory investigations. Analyses for each specific objective of the PhD were developed. Three analyses used data from the enrolment visit and were primarily descriptive and two were longitudinal and examined the incidence of hospitalizations, QuantiFERON conversion (an interferon gamma release assay used to measure Mycobacterium tuberculosis infection) and Tuberculosis (TB) disease. Results: Five hundred fifteen PHIV+ and 109 HIV- participants had a median follow-up of 4.1 years (IQR: 3.7–4.6). At enrollment, PHIV+ adolescents had a median duration of ART of 7.6 years (IQR: 4.6–9.2), median CD4 count of 713 cells/mm3 (IQR: 561.0–957.5) and 387 (75%) had a viral load of <50 copies/mL. Neurocognitive impairment was present in more than half of the PHIV+ cohort (56.3% vs. 45.3% in HIV-, p=0.05) but renal impairment was rare (2.3% in PHIV+ vs. 2.1% in HIV-, p=0.89). Microalbuminuria was also rare (8.0 in PHIV+ vs. 9.0% in HIV-, p=0.80). Respiratory or cardiac impairment were more common in PHIV+ adolescents than in HIV- participants (27.1% vs. 14.7%, p=0.01 and 46.1% vs. 33.7%, p=0.03, respectively). Multisystem impairment (defined as impairment of ≥ 3 systems) was uncommon, with only 10% of PHIV+ adolescents having 4-system impairment. Metabolic abnormalities, such as insulin resistance (IR), were relatively common but IR rates did not differ compared to HIV- adolescents (18 vs. 20%, p= 0.17). Incidence rates for hospitalization were 6.6 per 100-person-years (PY) in PHIV+ adolescents, three times that of HIV- adolescents. Sixty percent of hospitalization episodes were due to non-infectious causes and 24% due to infectious causes, of which pneumonia and TB were the predominant causes. PHIV+ adolescents had a substantially higher incidence of TB disease than HIV- adolescents (2.2/100 PY, 95% CI 1.6-3.1 vs. 0.3/100 PY, 95% CI 0.04-2.2), despite a similar rate of TB infection, as measured by QuantiFERON positivity. TB disease was associated with low CD4 counts and high viral loads in PHIV+ adolescents. Conclusion: Chronic single system morbidity experienced by PHIV+ adolescents on ART was common and merits further study, as this population begins to engage in adult lifestyle factors, such as smoking and alcohol use, that may compound these abnormalities. However, multisystem morbidity was relatively rare. In addition, in a relatively small percentage of adolescents there were subclinical metabolic abnormalities (IR and microalbuminuria) that may result in increased morbidity especially with regards to diabetes and cardiovascular disease in later life. The high burden of hospitalization and intercurrent disease, mainly due to TB, could be prevented by proven strategies, such as TB preventive therapy and ensuring adherence to optimal ART regimens.

Background Many critically ill children in South Africa are cared for in regional hospitals by general Paediatricians. Critically ill adults and children are usually cared for in the same units. There is limited data on the numbers of children admitted and the outcomes of these children. Objective To describe the patient profile and outcomes of children admitted to a general high care unit (HCU) in a regional hospital in the Western Cape, South Africa. Methods This was a retrospective descriptive study of all children admitted to the HCU of George Regional Hospital during a one year period (2016). Demographic data, HIV, anthropometric data, immunisation status, diagnoses, medical interventions, length of stay, death or survival, and referral data to the tertiary paediatric intensive care unit (PICU) were collected. The PIM3 score and Standardized Mortality Ratio (SMR) was calculated. Results Thirty percent (144/468) of the HCU admissions were children. Most (70%) were admitted after hours. Half were under 9 months (range 3 days to 149 months). Sixty-five percent of the children required respiratory support and 45% needed inotropic support. Twenty percent of the children were transferred to the PICU. Twelve children (8,5%) died with most deaths (75%) occurring at regional level. Half of the deaths were due to sepsis with pneumonia (25%) and diarrhoea with shock (25%) accounting for the rest. The cumulative PIM3 score was 9.049 (95%CI 6.430-11.668) with an SMR of 1.326 (95%CI 1.028-1.866) observed. Conclusion Critically ill children accounted for a third of HCU admissions. Most children needed medical interventions. These require specific training and equipment that are often lacking. After hours admissions also put strain on limited staff. Most children were successfully discharged demonstrating a good outcome. This was achievable with good channels of communication and transport to a tertiary PICU.

Bibliography: leaves 129-163. / There is little information on the aetiology and outcome of HIV-associated pneumonia in African children and no comprehensive data from South Africa. Studies of HIV-infected adult in Africa reported that the spectrum of pulmonary disease differs from that of developed countries with tuberculosis and pyogenic pneumonia predominating and Pneumocystis carinii pneumonia (PCP) occurring uncommonly. Knowledge of the aetiology and outcome of pneumonia is important for the development of paediatric management guidelines and of policies for allocation of resources especially in South Africa, where the HIV pandemic has resulted in increasing numbers of HIV-positive children requiring admission to hospital or intensive care units for pneumonia. Furthermore in countries with limited resources, development of cost effective diagnostic procedures to investigate the aetiology of pneumonia is necessary.

Includes bibliographical references / In this study we sought to more comprehensively analyse antigen-specific CD4 T cell responses induced by vaccination and to examine the effects of latent M. tb infection on these responses. We had two broad objectives: Firstly, to determine the effects of latent M. tb infection on epitope recognition by mycobacteria-specific CD4 T cells and to design HLA class II tetramers for detection of these cells. Secondly, to characterise antigen-specific CD4 T cells following vaccination with the novel vaccine candidate, H1:IC31, by measuring transcriptomic, phenotypic and functional attributes, and to determine the effects of latent infection on these responses. Firstly, we found that acquisition of M.tb infection did not alter the breadth and/or pattern of Ag85A/ B CD4 T cell epitopes recognised. We determined the HLA allele restriction of identified epitopes, and designed HLA class II tetramers for detection of Ag85-specific CD4 T cells. These results suggest that latent infection does not alter CD4 T cell epitope breadth within Ag85A/ B elicited by BCG vaccination and/or exposure to environmental mycobacteria. The second finding of this work is that underlying infection drives a more effector-like H1-specific CD4 T response after vaccination. Following vaccination M. tb-infected adolescents had higher frequencies of H1-specific CD4 T cells compared with uninfected adolescents. Additionally, H1-specific CD4 T cells from infected adolescents predominantly displayed a CCR7 - CD45RA - effector memory phenotype, had higher proportions of IFN-γ + TNF-α + IL-2 + cells, and expressed higher levels mRNA transcripts encoding effector molecules such as granzyme K and perforin, compared with uninfected adolescents. By contrast, H1-specific CD4 T cells in uninfected adolescents displayed a less differentiated memory phenotype, and had increased expression of central memory genes, compared to cells from infected adolescents. Thirdly, we found that Ag85B and ESAT-6-specific CD4 T cells exhibited markedly distinct transcriptomic profiles, memory phenotypes and cytokine expression patterns in M.tb infected adolescents. The data suggested that ESAT-6-specific cells preferentially drove the effector-like H1-specific response in M.tb infected adolescents. We conclude that while underlying M.tb infection does not affect the epitopes recognized by mycobacteria-specific CD4 T cells, but may promote and maintain effector memory antigen-specific CD4 T cells endowed with immediate effector function and tissue homing.

This work is concerned with the application of scintigraphy in studies of foregut motility in paediatric patients; notably oesophageal transit during swallowing, gastro oesophageal reflux (GOR) and stomach emptying as measured by the gastric contents 30 and 120 minutes after deglutition of liquid. Relevant anatomy, physiology, pathophysiology and non-radioisotopic methods of gastro oesophageal examination are outlined. The extremely important question of dose deposited by ionising radiation in paediatric patients is also considered. Because currently available estimations of absorbed dose after swallowing non- absorbable radionuclides are unsatisfactory and often contradictory, a new model was derived. It is based on more physiological principles than previous models, with a continuously varying exponential passage between four compartments in the gastrointestinal tract. From a review of previous work and our experience, a standard method of examination was developed. There is emphasis on normal physiological manoeuvres. The broad extent of useful information available in the study stems from the positions in which the patient is investigated, the quantity of labelled fluid swallowed, its physiological nature and observations of oesophageal transit, GOR, gastric emptying and pulmonary aspiration after two hours. The wide range of paediatric problems amenable to investigation by this means is described, including those of a life-threatening nature. Also, the importance of using this examination to determine the likelihood of successful surgical intervention in the gastro oesophageal region is explained. By using the observations made during swallows in neonates, the closure of the lower oesophageal sphincter (LOS) was observed during a short series of rapid deglutitions. This extends previous knowledge of the function of the neonatal LOS. These findings were very clearly demonstrated by the condensed image technique. A comparison between the abilities of radiology and scintigraphy to detect GOR in paediatric patients, with and without oesophagitis, gives unequivocal results in favour of scintigraphy. Two independent clear correlations between oesophageal transit time and the severity of GOR and age are demonstrated by variance and correlation analysis. The mean duration of GOR, maximum height of a GOR, gastric contents 30 and 120 min after deglutition, the effect of the nature of liquid swallowed and age are also examined and their relations are described. The application of the method to assess drug response is illustrated by the paediatric use of cisapride, an experimental drug which increases acetylcholinesterase release. The deductions from this work include values of the radiation dose deposited in paediatric patients of various ages, after swallowing non- absorbed Tc-99m compounds. Other contributions are a scintigraphic means to allow recognition of the importance of oesophageal dysmotility in paediatrics, especially in the absence of GOR and its relevance to the efficacy of surgery in cases of apparent GOR. Also, this gastro oesophageal scintigraphic study provided information on the paediatric use of cisapride, the relevance of GOR to repeated respiratory problems and the recognition of a new paediatric syndrome analogous to the diffuse oesophageal spasm syndrome of adulthood. It has been demonstrated that a routine, uncomplicated, paediatric scintigraphic examination, useful for investigating a wide range of problems in both a first and third world setting is possible for a nuclear medicine practitioner with normally available radiopharmaceuticals, equipment and radiographical expertise.

Introduction: There are limited data on the chest X-ray (CXR) abnormalities in human immunodeficiency virus (HIV)-infected children in low- and middle-income countries (LMIC's). Aim: To investigate the evolution of CXR abnormalities in HIV-infected children in LMIC's, to correlate this with the severity of HIV-disease, and to assess the impact of anti-retroviral therapy (ART). Method: A prospective longitudinal study evaluating clinical, immunological and radiographic parameters at regular intervals over a minimum of 24 months. CXR abnormalities were stratified by severity and deemed persistent if present for 6 consecutive months or longer. Univariate and multiple logistic regression analyses assessed associations between radiological and clinical/immunological parameters at enrolment. An ordinal multiple logistic regression model assessed the association of enrolment and time-dependent variables with CXR findings over time.

Introduction/Purpose: Lennox-Gastaut Syndrome (LGS) is one of the most common refractory epilepsies of childhood with significant morbidity and mortality. However there is paucity of data of this syndrome in resource limited settings (RLS). We sought to delineate the phenomenology, diagnosis, aetiologies, management and outcomes of children referred with LGS in the Western Cape Province of South Africa. To further identify early clinical markers differentiating LGS from other types of epilepsy. Methods. This retrospective observational cohort study included all children between 1 to < 18 years of age in the neurology database with a referral label of LGS between 2000-2018. The group were critiqued for those who met the diagnostic criteria of LGS. Then were categorized into those with confirmed LGS and remainder were not- LGS. Data of the social demographics, age of seizure onset, etiology, preceding epileptic spasms, and semiology of epilepsy types, management interventions were reviewed to identify key diagnostic indicators to permit early and targeted interventions for children with this epilepsy syndrome. Results; Of 2551 children managed with epilepsy, 110 were suspected at presentation to have LGS of these 66 records were available for assessment. The median (IQR) age in months at presentation was 35(16.0-54.5) with a slight male (37/66) predominance. 43(65%) met the criteria LGS and 23(35%) were not-LGS. 34(52%) had no identifiable cause for their epilepsy whilst a structural and metabolic cause were identified in 25(38%) and 3(5%) respectively. Moderate or severe cognitive impairment was associated with LGS (OR 2.59, p = 0.02 and OR =3.15, p = 0.01) and so were tonic seizures (OR=4.03, p=0.04). The most common diagnoses in the not-LGS group were other types of DEE not meeting the LGS criteria (15%) and uncategorised epileptic syndromes 11%. Conclusion; Over third of the children in this cohort were erroneously referred with LGS early in their course. This has implications for their management and prognostic counselling. Identification of indicators such as tonic seizures and moderate or severe intellectual/cognitive impairment are useful early markers which support a diagnosis of LGS and could be viable for use in our setting.

Neurodevelopmental and behavioural tools, used to assess children's cognitive and psychological development, play important supportive roles in clinical decision-making processes. It is therefore important that inferences made based on the information generated by these tools are valid and reliable. Because validity and reliability are inextricably tied to the context in which the tool is administered, it is critical that tools, including established “gold standard” tools, undergo ongoing psychometric evaluation. This is particularly important when tools are used in contexts different to that in which the original tool was developed. The use of poorly performing measures in clinical settings may result in a higher risk of misinterpretation of results or misdiagnosis, carrying serious implications for provision of interventions. This dissertation explored the psychometric properties of two frequently used behavioural tools in a South African context. The first aim of the dissertation was to investigate the use, cultural appropriateness, and psychometric soundness of Achenbach System of Empirically Based Assessment (ASEBA) forms in sub-Saharan Africa. The ASEBA forms are used worldwide to screen children and adolescents for behavioural and emotional problems. To achieve this aim, I conducted a systematic review of the psychometric properties of the ASEBA forms in sub-Saharan Africa. The second aim was to evaluate the structural and construct validity of a commonly used measure of AttentionDeficit/Hyperactivity Disorder (ADHD), the Swanson, Nolan, and Pelham ADHD Rating Scale (SNAP-IV), in a sample of South African children with neurodevelopmental disorders (NDDs), including Autism Spectrum Disorder and Intellectual Disability. The systematic review identified 58 studies with sub-Saharan African participants that reported measurement properties of the ASEBA forms. Most studies came from Southern (n = 29, 50%) or East African (n = 25, 43%) countries. Forty-nine studies (84%) used translated versions of the tool, but details regarding the translation process, if available, were often sparse. Most studies (n = 47, 81%) only reported internal consistency (using coefficient alpha) for one or more subscales. The methodological quality of the psychometric evaluations varied considerably across all measurement properties, except for internal consistency. There is limited good quality psychometric evidence available for the ASEBA forms in sub-Saharan Africa. Recommendations include implementing a standardised procedure for conducting and reporting translation processes and conducting more comprehensive psychometric evaluations of the translated versions of the tools. Parents of 109 children with one or more diagnosed NDDs completed the SNAP-IV. A subset of parents (n = 79) also completed the ASEBA Child Behaviour Checklist (CBCL/6-18). We conducted a confirmatory factor analysis to inspect the two-factor structure of the SNAP-IV (Inattention + Hyperactivity-Impulsivity). We also calculated ordinal coefficient alpha (α) to estimate internal consistency. Finally, we correlated scores of SNAP-IV and CBCL/6-18 subscales to estimate concurrent, convergent (ADHD + externalizing behaviour), and discriminant (ADHD + internalizing behaviour) validity respectively. The two-factor model performed acceptably (χ2 (134) = 249.82, p < 0.001, TLI = 0.868, RMSEA = 0.089, p < 0.001). The model fit improved after removing three problematic items, two of which were dependent on the child's level of spoken language (χ2 (89) = 135.17, p < 0.01, TLI = 0.942, RMSEA = 0.069, p = 0.096). The revised SNAP-IV subscales had acceptable internal consistencies (α = 0.85-0.86). Correlation coefficients between the SNAP-IV and ADHD-related CBCL/6-18 subscales were significant (r = 0.53- 0.62, p < 0.001). Correlations between ADHD and externalizing behaviours (r = 0.45, p < 0.001) and internalizing behaviours (r = 0.38, p < 0.001) respectively were not significantly different (z = 0.97, p = 0.165). The findings tentatively support the use of the SNAP-IV in this group of children. However, there are limitations to its performance in this population likely related to the presence of NDDs. Taken together, the findings of these two studies highlight the need for clinicians and researchers to conduct ongoing psychometric testing of behavioural tools for use with linguistically and culturally diverse sub-Saharan African populations. The data also reveal important insights regarding problems associated with using standard behavioural tools in children with complex clinical presentations.