Introduction: Vaccination is one of the most efficient ways to prevent infectious diseases, however due to the naivety and regulation of immunity found in infants, induction of vaccine-mediated immunity is challenging. Respiratory and diarrheal diseases are major contributors to infant mortality. Additionally, Human Immunodeficiency Virus-1 (HIV) infections increase the risk of mortality. Current advances in Prevention of Mother-to-Child Transmission (PMTCT) have prevented HIV infections in almost 97% of infants being born to HIV-infected mothers. As a result there is an increasing number of HIV exposed, uninfected infants (HEU). HEU infants have a higher rate of infectious disease related mortality and morbidity compared to unexposed infants, the underling causes of these differences are still not understood. In this dissertation, responses to two childhood vaccines, live, attenuated rotaviral vaccine (Rotarix) and acellular Pertussis (aP), were analyzed in HEU infants, with specific focus on T-cell responses to Rotarix and aP, due to the current lack of published data on T-cell responses. Additionally, the influence of feeding mode, that is breast or formula feeding, was also assessed as it is well established that breast fed infants contract fewer infections compared to formula fed infants. Methods: This dissertation included infants from a larger cohort which includes three groups of infants; HIV unexposed breast fed (UBF), HIV exposed breast-fed (EBF) and HIV exposed formula fed (EFF) infants. Infants were recruited at birth and followed up until 36 weeks of age. As no Rotavirus vaccine T-cell assay was previously published, multiple techniques were utilized to attempt to optimize an assay capable of detecting Rotavirus (RV) vaccine-specific T-cell responses. To determine T-cell responses to Bordertella pertussis (BP), blood was collected from infants at each time-point and 200ul was stimulated with BP antigen in a 12-hour whole blood assay. Cells from all assays were fixed and stained for flow cytometric analysis of CD4 and CD8 T-cell responses. The markers used included live/ dead, CD3, CD4 and CD8 for identification of T-cell populations, IFNγ, IL-2 and TNFα cytokines, HLA-DR and Ki67 for activation and proliferation, and CD45RA and CD27 memory differentiation. Data analysis was then completed using Microsoft Excel, Flow.Jo V9, GraphPad prism V6, Pestle 1.7 and Spice V5.33 software packages. Results: Despite multiple attemps it was not possible to optimise an assay capable of consistently detecting Rotavirus vaccine specific responses. This was partly due to interferance from contaminating agents in the protein antigens used, and difficulty in culturing and purification of whole virus. Assessment of aP spcific CD4+ T-cell memory demonstrated an overall increase in terminally differentiated (TD) memory cells accross time. This mirrored the ontogeny of the total T cell pool which showed an overall decrease in naïve T-cell frequencies with a consequent increase in late and terminally differentiated CD4 and CD8 T-cell populations over time through the first months of life. Both total and aP specific CD4+ early differentiated (ED) memory T-cells remained unchanged over time. ED CD8+ memory T-cells peaked at week 15 in EBF infants. A similar observation was found in UBF infants but at a non-significant level. EFF infants had no significant changes in CD8+ naive, ED and late differentiated (LD) memory populations over time. Additionally all infants demonstrated high levels of Ki67 expression at D4-7, which is prior to vaccination and maintained this level of proliferation after vaccination. HEU infants had higher levels of activation compared to HU infants in the first week of life but this normalised to HU infant levels by week 7. Furthermore EFF infants had peak T-cell activation at week 7 as compared to week 15 in EBF infants. In addition HU infants had better cytokine responses than HEU infants at week 7 but similar responses at week 15 and 36. In Addition, EFF infants also had increased vaccine specific CD4+ responses at week 7 and week 36 compared to EBF infants. This was true for overall cytokine expressing CD4 T-cells and single TNFα expressing CD4+ T-cells. Disscussion: Given the important role T-cells play in the clearance of Rotavirus, it is important that an assay capable of detecting RV vaccine specific T-cell responses be developed. Furthermore, T cells play a role in providing help for antibody responses to BP and for killing of intracellular bacteria. Our findings regarding immunity to aP suggest that all infants, regardless of HIV exposure status and feeding mode, are able to mount a T cell response to aP vaccination. However the differing ontogeny of responses seen in all three groups of infants lends some insight on the complex determinants of vaccine T -cell immunogenicity. In this case, age since vaccination, HIV exposure, and feeding mode resulted in apparent changes in vaccine responses as well as T cell differetiation and activation.

Tenofovir (TFV) and emtricitabine (FTC) are nucleoside reverse transcriptase inhibitors, often used in preexposure prophylaxis (PrEP) trials: where antiretroviral drugs are administered to high-risk, HIV-negative individuals to prevent HIV infection. Both drugs are safe when taken either daily or intermittently, which is ideal for PrEP regimens where adherence may not be high. The minimum number of doses estimated to confer high PrEP efficacy for a TFV/FTC regimen is four or more doses per week, resulting in a 95% lower risk of HIV acquisition. However, this is highly dependent on various host factors, of which adherence plays the largest role. The aim of the project was to develop a novel sensitive, specific, and robust direct method for the measurement of adherence, utilising tenofovir-diphosphate (TFV-DP) in dry blood spots (DBS) through LC-MS/MS analysis, to replace the current costly and laborious indirect method currently used to elucidate adherence of patients. This indirect method faces challenges, due to the polar nature of TFV and its metabolites, leading to separation and retention issues. The existing method applied a technique which separated the parent drug from the metabolite and then back-converted all metabolites to the parent drug before analysing the samples on LC-MS/MS. The developed alternative method aimed to reduce the time taken for each assay and the associated cost of consumables. TFV-DP is a highly polar compound and traditional reverse-phase chromatography has poor retention and separation capabilities when used to retain polar compounds, therefore alternative strategies were implemented. In this developed direct method, an anion exchange column was used along with a pH gradient, with the aim of improving separation and chromatography of TFV, TFV-DP, and tenofovir-monophosphate (TFV-MP). The method was optimised and validated using current U.S. Food and Drug Administration (FDA) and European Medical Agency (EMA) guidelines. The use of the anion exchange column resulted in a marked increase in retention time and allowed baseline separation of TFV, TFV-DP, and TFV-MP. Determination of TFV-DP from DBS was performed using three 3 mm DBS punches per sample, which underwent an extraction procedure followed by high-performance liquid chromatography with tandem mass spectrometry detection on an AB Sciex Qtrap 5500 mass spectrometer. The transitions of the protonated precursor ions were monitored at m/z 448.0 and 452.9 to the product ions m/z 350.0 and 354.9 for TFV-DP and the deuterated TFVDP internal standard, respectively. The method was validated over a range of 50–6400 fmol/punch for TFV-DP. The developed direct method had a lower limit of quantification (LLOQ) of 50 fmol/punch, which was higher than that of the indirect method; therefore, it had less sensitivity. The reduced sensitivity was acceptable, since the methods were meant for the measurement of adherence. The direct method had an ULOQ of 6400 fmol/punch, which was similar to that of the indirect method. The direct method also required significantly less on-bench sample processing and, therefore, was less time consuming and costly. To determine the suitability and accuracy of the direct method in comparison to the indirect method a comparative analysis was completed by analysing the same samples using both the indirect and direct method. The developed method met all the validation requirements and a strong correlation was observed between the results of the indirect and direct methods during the comparative analysis.

Includes bibliographical references.

Includes bibliographical references. / Short-term treatment outcomes of children starting ART in the ICU, general medical wards and outpatient HIV clinics at Red Cross War Memorial Children’s Hospital (RCWMCH): A Retrospective Cohort Study. Background: Antiretroviral therapy (ART) has proven to decrease morbidity and mortality in HIV-infected children and improve immunologic, virologic and clinical outcomes. As clinical management policies evolved, an emphasis on early infant testing was adopted resulting in an increasing number of children being diagnosed and commenced on therapy before the onset of severe disease progression. However, a fair proportion still remain untested and subsequently present to hospital with advanced immunosuppression and severe disease. Since the advent of the 2013 national Standard Treatment Guidelines which encourage expedited initiation of ART within 7 days of HIV diagnosis in all children under the age of 12 months and in those with advanced immunosuppression, it is likely that many HIV-infected children are being initiated on ART during hospitalisation in South Africa. No local published data on these outcomes exist. We assessed the short-term outcomes of children initiated on ART in the intensive care unit (ICU), general medical wards (GMWs) and outpatient HIV clinics (OHCs) at RCWMCH. Methods: Structured Literature Review A Pubmed search looking at outcomes of treatment naïve HIV-infected children and adolescents up to 19 years of age living in South Africa commenced on 1st line ART regimens in accordance to the national guidelines presiding at the time, over a 10 year period was performed. This served to identify gaps in knowledge around paediatric ART in a South African context warranting further research. Retrospective Cohort Study We conducted a retrospective cohort study of HIV-infected children <13 years of age, commenced on first line ART between January 2008 and December 2011 at RCWMCH. Outcome measures included death, virologic suppression and changes in CD4 count and percentage. Kaplan-Meier estimates, multivariate Cox proportional hazard ratios and logistic regression were used to estimate outcomes 6 months after ART initiation. Results: Structured Literature Review This review identified several knowledge gaps. One of these gaps, the treatment outcomes of children started on ART at different service levels within tertiary health care settings was addressed in our retrospective cohort study and described in section C of this dissertation Retrospective Cohort Study Seven hundred and forty-nine children were included: 106 were commenced on ART in the ICU, 509 in the GMWs and 127 in the OHCs. Four hundred and ninety-two (65.7%) children were <12 months old. Children in the ICU and GMW cohorts were significantly younger than the OHC cohort (median ages: 3 and 5 months respectively vs. 22 months) and had lower WAZ scores (-2.48 and -2.33 respectively vs -1.14). Three hundred and eighty-five (51.4%) children qualified for rapid ART initiation within 7 days of HIV diagnosis or hospitalisation, based on CD4 criteria in the 2013 national Standard Treatment Guidelines. Overall mortality was 6.4% (CI: 4.9 - 8.4). Mortality was significantly higher in the ICU cohort i.e. 14 (13.2%) deaths compared to 28 (5.5%) and 5 (3.9%) deaths in the GMWs and OHCs cohorts, logrank p=0.004. Predictors of mortality included being moderately underweight HR 2.4 (CI: 1.1 – 5.2; p=0.02), severely underweight HR 3.2 (CI: 1.6 – 6.5; p=0.001), absence of caregiver counselling sessions HR 2.9 (CI: 1.4 – 6.0; p=0.005) and ART initiation in ICU HR 2.6 (CI: 1.4 – 4.9; p=0.003). Conclusion: The findings of our retrospective cohort study serve as a basis for understanding the implications of ART initiation in children during hospitalisation.

Includes bibliographical references. / This study explores the illness experiences of twenty one patients who presented for treatment at primary health care settings in Mamre, a small "coloured" community approximately 50km from Cape Town, with the aim of investigating the possible contribution of psychological factors to presentation for primary health care. It also attempts to investigate how, psychologically, we may understand the process of illness identification and help-seeking. Selected literature on psychological issues in primary health care is reviewed. A hermeneutically-oriented medical anthropological approach to the study of illness and health care is outlined. The participants for the study were selected from all patients presenting at general practitioners in Mamre and for O.P.D treatment at Wesfleur hospital over a period of one week. A semi-structured interview, consisting mainly of open-ended questions aild focussing on their illness explanations, experiences of treatment, and their understanding and/or experience of "nerves", was conducted within ten days, in their own homes. The analysis of the interview material drew on both quantitative and qualitative methodologies. The quantitative analysis provides indications of the frequency of types of illness and patient responses to treatment. The qualitative analysis draws on Kleinman's (1980) explanatory model/ framework with the aim of understanding the illness experience of each participant. The participants' understanding and/or experience of nerves is also described. This study provides additional insights in understanding the process of illness identification r and help-seeking and the contribution of psychological issues in the presentation for , primary health care.

Two separate bioanalytical methods were developed, validated and applied to determine agricultural exposure to organochlorine and organophosphorus pesticides using different biological matrices as reference sources. The method that was validated for the quantification of the organochlorine compounds was used to simultaneously determine β-endosulfan [6, 7, 8, 9, 10, 10- hexacloro-1, 5, 5a, 6, 9, 9a- hexahydro- 6, 9-methano-2, 4, 3-benzodioxathiepin-3- oxide] and one of its main metabolites, endosulfan sulfate, in human serum. In a second bioanalytical method, urinary dialkylphosphate metabolites have been assessed as markers to estimate the exposure to organophosphorus pesticides, focusing on three of the six organophosphorus urinary metabolites, namely dimethyl phosphate, dimethyl thiophosphate and diethyl phosphate. For both the bioanalytical methods, liquid-liquid extraction was used for sample preparation and high performance liquid chromatography with tandem mass spectrometry as detection method due to its high sensitivity and selectivity. Chromatographic separation for both bioanalytical methods was achieved by performing reverse phase chromatography on C18 analytical columns. Isocratic elution with a mobile phase composed of acetonitrile, methanol and water was employed for the analysis of the organochlorine compounds while the organophosphorus compounds were eluted using gradient elution with a mobile phase consisting of acetonitrile and 20 mM ammonium acetate. A triple quadrupole mass spectrometer equipped with an electrospray ionization source operating in the negative ionization mode was used for mass detection of all the analytes, employing multiple reaction monitoring as scan mode. Calibration standards and quality control samples for both analyses were prepared in the biological matrix in which the samples for each determination were collected, i.e. serum for the determination of the organochlorine compounds and stripped urine for the organophosphorus compounds. Deuterated internal standards were used in the bioanalytical method for the determination of the organophosphorus compounds whereas the organochlorine compounds were determined without the use of an internal standard due to unavailability of suitable internal standards. The calibration ranges for the determination of β-endosulfan and endosulfan sulfate were 0.8 ng/ml to 200 ng/ml and 0.117 ng/ml to 30 ng/ml, respectively, and 1.0 ng/ml to 30 ng/ml for the dialkylphosphate metabolites of the organophosphorus compounds. These sensitive and robust quantitation methods were successfully applied to quantify 219 serum and 187 urine samples that were collected from agricultural workers with the purpose to determine whether they were exposed to any of the investigated organochlorine or organophosphorus compounds. No traces of β-endosulfan and endosulfan sulfate were found in any of the serum samples that were analyzed, however, significant amounts of the three organophosphorus compounds dimethyl phosphate, dimethyl thiophosphate and diethyl phosphate were present in the urine samples.

Includes bibliographical references. / 'Sexting' is defined as the act of sending sexually suggestive text messages (SSTMs), or the self-generation of nude or semi-nude images (NSNIs) and sending them to others via digital devices. It has recently emerged as a risk behaviour, particularly as it relates to adolescent sexuality. The consequences of sexting may include humiliation, ostracism, depression, anxiety, suicide and criminal or legal action. As such, research into the practice is vital in order to mitigate the risk to adolescents. The current study aimed to investigate the prevalence and characteristics of sexting among a sample of adolescents and adults in Cape Town, South Africa.

Bibliography: leaves 49-57. / Since the 1970's there has been prevalence of eating disorders in especially among adolescent and young women, notably university students. While eating disorders are multidetermined, sociocultural factors have been particularly implicated, among them, pressures to be thin and successful. In developing countries and among ethnic minorities in Western societies, increasing numbers of women, especially achievement-orientated, Westernised young women from upwardly mobile families, have also been found to be at risk for developing eating disorders. This study seeks to establish the prevalence of eating disorders in a sample of South African university students, to investigate the presentation of eating disturbance in African and coloured students, and to establish the extent of body shape dissatisfaction.

Bibliography: leaves 51-55. / The aim of this study is to answer the question: is celibacy psychologically heal thy with specific reference to Christianity? Jungian theory is utilized to develop a theoretical framework in which celibacy may be viewed. The meaning of celibacy is initially examined from a Christian perspective as a form of love and contrasted with celibacy's meaning and implications from a Jungian perspective. This is done by means of a comprehensive exposition of Jung's concept of individuation, what is understood by "psychological health" and how this is linked with religious experience. The integration of instinctuality and spirituality is then centrally addressed in an elaboration of the concepts of sexuality, religion and mysticism and Jung's critique of Christianity. Further levels of the meaning of celibacy, as possible specific and unique constellations of an individual's psychic development are examined in both negative and positive forms. It is concluded that from a Jungian perspective celibacy in the main is not psychologically healthy, and specific points of departure between Jung and Catholicism are highlighted.

Bibliography : pages 48-50. / The study attempted an exploration of the relation between aspects of autonomy and cognitive development, at the concrete operational stage. This was intended as a contribution to extending Piagetian theory in order to encompass an understanding of variations in cognitive development. Thirty Sub A boys and girls were individually tested on a modified Piagetian Inventory, and a modified sentence completion test of autonomy. The Vineland Scale was administered to their parents, and teachers completed a teacher rating scale of autonomy, developed for this study. Through regression analyses, it was found, in line with previous research, that autonomy was situationally specific. Linkages between some aspects of autonomy and specific areas of concrete operational thinking were also evident. A number of preliminary interpretations were offered for the findings. It was concluded that the linkage between aspects of autonomy and areas of concrete operational thinking could be understood within a Piagetian framework, with implications for a revision of Piagetian theory.