Philosophiae Doctor - PhD / Background: Tuberculosis (TB) is a major cause of morbidity and mortality among children (<15 years) in resource-limited countries. The World Health Organization (WHO) identified active contact screening and isoniazid preventive therapy (IPT) as essential actions for detecting and preventing childhood TB. Despite their benefits and inclusion in the policy of most National TB Programme (NTP) guidelines of the resource-limited countries, there is still a wide gap between policy and implementation. The implementation of contact screening for active case finding might be improved by the decentralised use of the Xpert MTB/RIF test in gastric lavage (GL) specimens, but this has not been previously assessed. Furthermore, although the provision of IPT to eligible child contacts has been a focus for implementation by the NTP of Rwanda since 2005, implementation has not previously been evaluated. The assessment of IPT uptake and adherence as well as associated factors could be informative for the programme. Therefore, we aimed to assess the diagnostic yield of Xpert MTB/RIF in GL among child contacts with suspected pulmonary tuberculosis (PTB) and the uptake of and adherence to IPT by eligible child contacts to make recommendations towards strengthening TB diagnostic and prevention in children in Kigali, Rwanda. Methods: The proposed study setting Kigali, the capital city of Rwanda, was the location for 30% of the national PTB case notifications in 2013-14.A conceptual framework based on ecological theory was used in this study. Quantitative, qualitative and mixed (using both quantitative and qualitative research methods in one study) research methods were applied, and various research designs were used depending on the research questions. The study involved a cross-sectional analysis of the diagnostic yield of Xpert MTB/RIF in GL among all child contacts with suspected TB. Across-sectional and prospective cohort study design was used to assess the uptake and adherence of IPT among eligible child contacts.

This study compare immunological and virological outcomes in antiretroviral therapy naïve patients exposed to Isoniazid prevention treatment.Medical records of antiretroviral naïve patients managed in the public sector from 1st January 2006 to 31st December 2006 were analysed.Multivariate analysis of variance showed that each treatment group achieved statistically significant increases in CD4+ cell count and viral load decay at each follow-up time point. Pairwise post hoc contrast tests showed patients in NVPipt-past group and EFVipt-past group to have superior immunological and virological outcomes respectively. / Health Studies / M.A. (Public health)

This study compare immunological and virological outcomes in antiretroviral therapy naïve patients exposed to Isoniazid prevention treatment.Medical records of antiretroviral naïve patients managed in the public sector from 1st January 2006 to 31st December 2006 were analysed.Multivariate analysis of variance showed that each treatment group achieved statistically significant increases in CD4+ cell count and viral load decay at each follow-up time point. Pairwise post hoc contrast tests showed patients in NVPipt-past group and EFVipt-past group to have superior immunological and virological outcomes respectively. / Health Studies / M.A. (Public health)

Thesis (MBA)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Childhood tuberculosis is an infectious disease that can cause serious illness and mortality in especially young children. Following contact with an infectious adult tuberculosis case, the disease is easily preventable through preventive isoniazid treatment, yet very few exposed and at-risk children currently access this healthcare service in most high-burden settings. Previous research pointed out the multifactorial and complex nature of the barriers to accessing preventive care. Specifically, the lack of a formalised recording and reporting tool, such as the universally used tuberculosis treatment registers, possibly contribute to the operational barriers of preventive care delivery to these children. The purpose of this research was to evaluate the cost-effectiveness of an isoniazid preventive treatment register tool used at community level. The study utilised previously reported data from the study population and other high-burden settings to construct a decision analysis model that included varying probabilities of isoniazid preventive treatment across three high risk age groups (<1 year of age, 1 – 2 years of age, 3 – 5 years of age), coupled with disease probabilities and associated treatment costs. The scenarios simulated included 1) the routine isoniazid preventive treatment service (3% started on treatment, 17% identified as eligible); and 2) an isoniazid preventive treatment service supported by a recording register (15% (adherent to six months of treatment) and 38% (started on IPT treatment)). In addition, two hypothetical simulations were included for 76% and 100% isoniazid preventive treatment uptake; these hypothetical simulations required additional community based healthcare worker resources in addition to the register tool. The observations from the literature indicated that more children were identified (24(17%) vs. 54(38%)) and started (4(3%, base case) vs. 54) on isoniazid preventive treatment following the implementation of the register. As expected, the mean number of tuberculosis cases prevented, increased as the proportion of eligible children that received isoniazid preventive treatment, improved; the change in the number of cases prevented per simulation showed incremental improvements which were all significantly better (p<0.01) than the base case.. The incremental cost-effectiveness ratios incurred savings for each of the scenarios simulated since the mean costs for each of the simulations were significantly less (p<0.01) than the costs associated with the base case. The current evidence suggests that the proposed isoniazid preventive treatment register tool is a cost-effective alternative to the current standard of care in place at community level for at-risk children exposed to tuberculosis. It is therefore recommended that the tool be used incrementally on a bigger scale, until such time that sufficient evidence has been generated to support widespread implementation.

The introduction of artemisinin-based combination therapy (ACT) substantially reduced malaria-related mortality and morbidity during the past decade. Despite the widespread use of ACT, there is still a considerable knowledge gap with regards to safety, efficacy and pharmacokinetic properties of these drugs, particularly in vulnerable populations like children and pregnant women. In addition, there is growing evidence of widespread artemisinin-resistance across the Greater Mekong Subregion. Expedited delivery of novel antimalarial drugs with different mechanisms of action to the clinical setting is still far off; therefore, it is crucial to improve the use of existing antimalarial drugs for optimal outcome in order to prolong their therapeutic life span. This thesis focuses on utilizing pharmacometric tools to support this effort for malaria prevention and treatment. An extensive simulation framework was used to explore alternative malaria chemopreventive dosing regimens of a commonly used ACT, dihydroartemisinin-piperaquine. Different monthly and weekly dosing regimens were evaluated and this allowed an understanding of the interplay between adherence, loading dose and malaria incidence. A weekly dosing regimen substantially improved the prevention effect and was less impacted by poor adherence. This is also expected to reduce selection pressure for development of resistance to piperaquine. Population pharmacokinetics-pharmacodynamic models were developed for artesunate and the active metabolite dihydroartemisinin, effect on parasite clearance, in patients with artemisinin-resistant and -sensitive malaria infections in Southeast Asia. The modeling identified an association between parasite density and drug bioavailability. It predicted the presence of high levels of artemisinin resistant infection among patients in Cambodia and its spread into Myanmar. A nomogram to identify patients with artemisinin resistant infections was developed. Furthermore, the model was used to demonstrate the need for extended treatment duration to treat patients with artemisinin resistant infections. A population pharmacokinetic model developed from data on pregnant women in East Africa allowed further understanding of artemether-lumefantrine exposure in pregnant populations. It also suggested that the lumefantrine exposure in this population is not compromised. In summary, the results presented in this thesis demonstrate the value of pharmacometric approaches for improving antimalarial drug treatment and prevention. This ultimately contributes to overcoming the prevailing challenges to malaria control.

Le virus Epstein-Barr (VEB) est fortement associé au développement de syndromes lymphoprolifératifs (SLP) en greffe pédiatrique. Ce virus a la capacité d’immortaliser les lymphocytes B et de provoquer leur prolifération incontrôlée chez l’hôte immunodéprimé. Plusieurs études démontrent que le cycle lytique du virus jouerait un rôle primordial dans la genèse des SLP en produisant des particules virales pouvant infecter les cellules B adjacentes. Chez un individu immunodéprimé, ces cellules B nouvellement infectées peuvent donner naissance à une expansion lymphocytaire. Le projet présenté dans ce mémoire fait partie d’un programme de recherche visant à élucider le rôle de l’infection productive par le VEB dans le développement des SLP. L’objectif précis de ce projet est de développer un anticorps monoclonal chimère contre la glycoprotéine gp350 du VEB dans le but de neutraliser le virus et d’ainsi prévenir son entrée dans les cellules B. Notre laboratoire a construit une version chimère de l’anticorps monoclonal murin 72A1, lequel se lie à la gp350 et bloque l’infection. Les premiers essais ont révélé la présence de chaînes non fonctionnelles (aberrantes) dans l’hybridome produisant l’anticorps 72A1. La construction de la chaîne légère authentique est maintenant complète alors que celle de la chaîne lourde est toujours en cours. Le processus de caractérisation de l’anticorps chimère inclura des essais de cytotoxicité à médiation cellulaire dépendante des anticorps (ADCC). Dans cette optique, une lignée cellulaire exprimant de façon stable la gp350 a été établie. Notre anticorps chimère anti-gp350 pourrait éventuellement être utilisé comme thérapie préventive chez les greffés présentant un risque élevé de SLP en empêchant l’infection des cellules B adjacentes. / The Epstein-Barr virus (EBV) is associated with B-cell post-transplant lymphoproliferative disease (PTLD). EBV has the unique property of immortalizing B lymphocytes, thereby causing their uncontrolled proliferation in an immunocompromised host. Certain evidence suggests that EBV productive infection may play a primary role in the genesis of PTLD by generating virus particles which can infect bystander B cells. In an immunocompromised individual, these infected B cells may then give rise to expanding B-cell clones. The project presented in this thesis is part of a research program seeking to elucidate the role of EBV productive infection in the genesis of PTLD. The specific aim of this work was to design a chimeric monoclonal antibody against the EBV envelope glycoprotein gp350 in order to neutralize the virus, thereby preventing entry into B cells. Our laboratory constructed a chimeric version of the murine monoclonal antibody, 72A1, which binds to gp350 and blocks infection. The initial cloning attempts revealed the presence of nonfunctional (aberrant) transcripts in the hybridoma line producing the 72A1 antibody. The chimeric version of the authentic light chain is now completed while the chimeric heavy chain construction is ongoing. As part of the characterisation process for the chimeric antibody, a cell line stably expressing surface gp350 was generated. This gp350-expressing cell line will be used for antibody dependent cellular cytotoxicity assays (ADCC). This anti-gp350 chimeric antibody could be useful as a preventive therapy in transplant patients at high risk for PTLD by blocking the infection of bystander B cells.

Le virus Epstein-Barr (VEB) est un pathogène opportuniste qui a la capacité d’immortaliser les lymphocytes B et de provoquer une prolifération maligne, appelée syndrome lymphoprolifératif post-transplantation (SLP), chez les individus immunodéprimés. A l’intérieur de ce groupe, les personnes à plus haut risque sont les enfants, puisqu’ils sont à risque de développer une infection primaire par le VEB pendant leur régime d’immunosuppression post-greffe. Dans le but de développer un anticorps préventif, notre laboratoire s’est attardé au rôle du cycle lytique du VEB dans le développement du SLP. À cette fin, le premier objectif du présent projet vise à fournir la preuve expérimentale de l’existence ou non d’une phase réplicative productive pendant l’infection aiguë des lymphocytes B sanguins. Un examen des événements qui se déroulent au tout début de l’infection par le VEB tant au niveau de la réplication virale qu’au niveau de l’expression des gènes lytiques précoces et tardifs a révélé l’existence d’une phase réplicative productive pendant l’infection aiguë. Ceci a permis de justifier l’élaboration, dans notre laboratoire, d’un anticorps chimère (murin-humain) neutralisant, dirigé contre la protéine gp350 située sur l’enveloppe virale. Le deuxième objectif, quant à lui, vise à fournir la preuve expérimentale de la capacité neutralisante de cet anticorps chimère. Des essais de caractérisation in vitro ont démontré une capacité de reconnaissance de la protéine cible, notamment la gp350, et une capacité de neutralisation du virus par l’anticorps chimère. L’anticorps chimère anti-gp350 pourra faire l’objet d’essais précliniques in vivo en vue d’évaluer sa capacité à reconnaître le virus et à prévenir l’apparition de tumeurs de type SLP chez les souris SCID. Il pourrait être éventuellement utilisé, par la suite, comme traitement préemptif contre les tumeurs dans l’espoir de mieux gérer les patients à risque de développer un SLP. / Epstein-Barr virus (EBV) is an opportunistic pathogen in immunocompromised transplant patients. In these patients EBV infection can lead to malignant B-cell lymphoproliferation, called post-transplant lymphoproliferative disease (PTLD). This thesis project aimed to investigate the role of lytic EBV infection in the genesis of PTLD. The first experimental objective was to provide in vitro proof that EBV could induce productive replication upon acute in vitro infection of B cells. Data obtained through study of viral DNA replication and transcription during the first 96 hours post-infection indicate that lytic infection does occur. These results provided justification for proceeding to the second experimental objective which involved the characterization of an anti-gp350 human-mouse chimeric antibody for its capacity to recognize and neutralize EBV. Results showed that this antibody did possess neutralization activity. Further study of this anti-gp350 chimeric antibody in SCID mice is necessary in order to evaluate its in vivo efficacy against PTLD.

Le virus Epstein-Barr (VEB) est fortement associé au développement de syndromes lymphoprolifératifs (SLP) en greffe pédiatrique. Ce virus a la capacité d’immortaliser les lymphocytes B et de provoquer leur prolifération incontrôlée chez l’hôte immunodéprimé. Plusieurs études démontrent que le cycle lytique du virus jouerait un rôle primordial dans la genèse des SLP en produisant des particules virales pouvant infecter les cellules B adjacentes. Chez un individu immunodéprimé, ces cellules B nouvellement infectées peuvent donner naissance à une expansion lymphocytaire. Le projet présenté dans ce mémoire fait partie d’un programme de recherche visant à élucider le rôle de l’infection productive par le VEB dans le développement des SLP. L’objectif précis de ce projet est de développer un anticorps monoclonal chimère contre la glycoprotéine gp350 du VEB dans le but de neutraliser le virus et d’ainsi prévenir son entrée dans les cellules B. Notre laboratoire a construit une version chimère de l’anticorps monoclonal murin 72A1, lequel se lie à la gp350 et bloque l’infection. Les premiers essais ont révélé la présence de chaînes non fonctionnelles (aberrantes) dans l’hybridome produisant l’anticorps 72A1. La construction de la chaîne légère authentique est maintenant complète alors que celle de la chaîne lourde est toujours en cours. Le processus de caractérisation de l’anticorps chimère inclura des essais de cytotoxicité à médiation cellulaire dépendante des anticorps (ADCC). Dans cette optique, une lignée cellulaire exprimant de façon stable la gp350 a été établie. Notre anticorps chimère anti-gp350 pourrait éventuellement être utilisé comme thérapie préventive chez les greffés présentant un risque élevé de SLP en empêchant l’infection des cellules B adjacentes. / The Epstein-Barr virus (EBV) is associated with B-cell post-transplant lymphoproliferative disease (PTLD). EBV has the unique property of immortalizing B lymphocytes, thereby causing their uncontrolled proliferation in an immunocompromised host. Certain evidence suggests that EBV productive infection may play a primary role in the genesis of PTLD by generating virus particles which can infect bystander B cells. In an immunocompromised individual, these infected B cells may then give rise to expanding B-cell clones. The project presented in this thesis is part of a research program seeking to elucidate the role of EBV productive infection in the genesis of PTLD. The specific aim of this work was to design a chimeric monoclonal antibody against the EBV envelope glycoprotein gp350 in order to neutralize the virus, thereby preventing entry into B cells. Our laboratory constructed a chimeric version of the murine monoclonal antibody, 72A1, which binds to gp350 and blocks infection. The initial cloning attempts revealed the presence of nonfunctional (aberrant) transcripts in the hybridoma line producing the 72A1 antibody. The chimeric version of the authentic light chain is now completed while the chimeric heavy chain construction is ongoing. As part of the characterisation process for the chimeric antibody, a cell line stably expressing surface gp350 was generated. This gp350-expressing cell line will be used for antibody dependent cellular cytotoxicity assays (ADCC). This anti-gp350 chimeric antibody could be useful as a preventive therapy in transplant patients at high risk for PTLD by blocking the infection of bystander B cells.

Le virus Epstein-Barr (VEB) est un pathogène opportuniste qui a la capacité d’immortaliser les lymphocytes B et de provoquer une prolifération maligne, appelée syndrome lymphoprolifératif post-transplantation (SLP), chez les individus immunodéprimés. A l’intérieur de ce groupe, les personnes à plus haut risque sont les enfants, puisqu’ils sont à risque de développer une infection primaire par le VEB pendant leur régime d’immunosuppression post-greffe. Dans le but de développer un anticorps préventif, notre laboratoire s’est attardé au rôle du cycle lytique du VEB dans le développement du SLP. À cette fin, le premier objectif du présent projet vise à fournir la preuve expérimentale de l’existence ou non d’une phase réplicative productive pendant l’infection aiguë des lymphocytes B sanguins. Un examen des événements qui se déroulent au tout début de l’infection par le VEB tant au niveau de la réplication virale qu’au niveau de l’expression des gènes lytiques précoces et tardifs a révélé l’existence d’une phase réplicative productive pendant l’infection aiguë. Ceci a permis de justifier l’élaboration, dans notre laboratoire, d’un anticorps chimère (murin-humain) neutralisant, dirigé contre la protéine gp350 située sur l’enveloppe virale. Le deuxième objectif, quant à lui, vise à fournir la preuve expérimentale de la capacité neutralisante de cet anticorps chimère. Des essais de caractérisation in vitro ont démontré une capacité de reconnaissance de la protéine cible, notamment la gp350, et une capacité de neutralisation du virus par l’anticorps chimère. L’anticorps chimère anti-gp350 pourra faire l’objet d’essais précliniques in vivo en vue d’évaluer sa capacité à reconnaître le virus et à prévenir l’apparition de tumeurs de type SLP chez les souris SCID. Il pourrait être éventuellement utilisé, par la suite, comme traitement préemptif contre les tumeurs dans l’espoir de mieux gérer les patients à risque de développer un SLP. / Epstein-Barr virus (EBV) is an opportunistic pathogen in immunocompromised transplant patients. In these patients EBV infection can lead to malignant B-cell lymphoproliferation, called post-transplant lymphoproliferative disease (PTLD). This thesis project aimed to investigate the role of lytic EBV infection in the genesis of PTLD. The first experimental objective was to provide in vitro proof that EBV could induce productive replication upon acute in vitro infection of B cells. Data obtained through study of viral DNA replication and transcription during the first 96 hours post-infection indicate that lytic infection does occur. These results provided justification for proceeding to the second experimental objective which involved the characterization of an anti-gp350 human-mouse chimeric antibody for its capacity to recognize and neutralize EBV. Results showed that this antibody did possess neutralization activity. Further study of this anti-gp350 chimeric antibody in SCID mice is necessary in order to evaluate its in vivo efficacy against PTLD.

This study assessed healthcare providers’ knowledge, attitudes and practices towards IPT provision for people living with HIV (PLHIV) in Addis Ababa, the capital city of Ethiopia. A quantitative, descriptive, cross-sectional study design was used for the study and data was collected using a self-administered questionnaire from 104 clinicians working in ART clinics. The findings show that healthcare providers who participated in this study had a mean value of high knowledge, positive attitude and good practice towards IPT provision for PLHIV. Significant association was found between knowledge and attitude (P=0.000) but no significant associations were found between knowledge and practice, attitude and practice as well as between the type of facility (public/private) and level of practice. This implied that, the low level of IPT implementation in Addis Ababa doesn’t seem due to health care providers’ lack of knowledge and resistance to provide IPT for people living with HIV. As a result, the researcher recommends for further researches of other possible factors like; the reliability of IPT information/data management, drug supply and the leadership and governance of the health system that IPT program is a direct concern. / Health Studies / M. Public Health (with specialisation in Medical Informatics)