Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / A. Sally Davis / William Wilson / Rift Valley fever virus (RVFV) is a zoonotic arbovirus that is a significant threat to livestock and humans. It is listed as #3 for most dangerous animal threats and is in the top 10 pathogens needing urgent research in preventative and control measures. Although RVFV has never been reported in the US or Europe, outbreaks outside the African continent have sparked renewed interest in developing diagnostics and vaccines to protect both agriculture and public health. Having specific and versatile diagnostics is critical for vaccine development and application. For example, diagnostic tools that aid in identifying key immunogens and understanding the virus-host interaction directly contribute to developing protective vaccines. Additionally, vaccines that are used prophylactically or in response to an outbreak require diagnostic tests to differentiate infected from vaccinated animals (DIVA). This is critical for assessing the return to ‘disease free’ status after an outbreak. Unfortunately, there are limited RVFV diagnostic tests that are versatile and DIVA compatible with the newest RVFV vaccines. We describe the development of several diagnostic tools that are DIVA compatible for detecting RVFV nucleic acid, antibodies, and antigens. First, we evaluate a fluorescence microsphere immunoassay (FMIA) for the detection of antibodies against a RVFV surface glycoprotein and the nucleocapsid protein. The targets developed in this assay provide the basis for a DIVA-compatible serological assay with a candidate RVFV Gn/Gc subunit vaccine, as well as, offer a multiplexing platform that can simultaneously screen for several ruminant diseases. Second, we describe a novel chromogenic in situ hybridization (ISH) assay to detect RVFV in formalin-fixed, paraffin-embedded (FFPE) tissues. This molecular assay offers a highly sensitive, multiplexing platform that detects RVFV RNA on the cellular level of diagnostic tissue samples. Moreover, we demonstrate the first application of ISH as a DIVA-compatible assay for candidate RVFV gene-deletion vaccines. Third, we provide working protocols for western blot (WB), immunohistochemistry (IHC), and immunofluorescence (IF) that use monoclonal or polyclonal antibodies against key RVFV antigens. These tools can be applied to pathogenesis research and used in the development of vaccine and therapeutic countermeasures against RVFV. The RVFV diagnostic methods developed and evaluated in this dissertation can serve as a model for developing diagnostic strategies for other transboundary animal diseases.

Carrion’s disease is a neglected tropical disease caused by Bartonella bacilliformis, a vector-borne pathogen restricted to the Andean valleys of Peru, Ecuador and Colombia. Carrion’s disease is a biphasic illness; in the acute phase the case-fatality rate can be as high as 88 %, related to high parasitemia, arriving to almost all erythrocytes, and secondary bacterial infections close related with the development of transient immunosuppression in the earlier illness phases. In addition, there are an undefined number of asymptomatic carriers that are reservoirs of the etiological agent of Carrion’s disease in endemic areas, they make take into account due to they are the perpetuators of this disease. The actual scenario of Carrion’s disease, in which the illness is arriving to new areas, due to the expansion of the vector’s distribution, suggests that now may be a crucial time to design a strategy focusing on its elimination.

La dengue est une préoccupation mondiale majeure en santé publique, touchant principalement les jeunes enfants en Asie du sud est. Les vaccins contre la dengue sont actuellement en développement. Des études coût-efficacité seront nécessaires en cas d'introduction d'un nouveau vaccin. Pour cela, il est indispensable d’obtenir des informations sur le coût économique lié à la dengue (qui sont relativement rares) et des données précises sur l'incidence de la maladie, ceci afin de fournir des estimations solides quant à l'importance de la dengue dans les régions endémiques.Ce travail de thèse propose une approche méthodologique par laquelle le Ministère de la Santé devrait envisager lors de l'évaluation de la pertinence et de la faisabilité de l'adoption d’un vaccin contre la dengue par les programmes de nationaux de vaccination, particulièrement dans les pays en voie de développement ; le Cambodge par exemple. Nous avons mis en place des études prospectives d'incidence de la dengue sur plusieurs années, réalisées dans la population générale, incluant un large éventail de groupes d'âge. Les résultats de ces études ont été comparés avec les données de surveillance nationale réunis dans la même région en calculant des facteurs de multiplication afin d’estimer la sous-déclaration. D'autres études de coûts de la maladie ont été menées pour estimer le poids financier la dengue sur plusieurs années et d'identifier les groupes vulnérables. Cette approche a également permis de mieux comprendre l'épidémiologie de la dengue dans le pays, dont certaines caractéristiques sont nécessaires pour évaluer la faisabilité de l'introduction d’un nouveau vaccin contre la dengue. Les résultats rapportés dans cette thèse vont permettre (1) d’initier à partir des données cambodgiennes des simulations de coût-efficacité à partir des caractéristiques attendues du vaccin et (2) de développer un modèle dynamique afin de permettre aux autorités nationales d'analyser les études de coût-efficacité en tenant compte des spécificités immunologiques et épidémiologiques des pays concernés. Si un vaccin efficace et fiable est disponible à un prix abordable, son adoption par le programme national de vaccination serait probablement rentable pour le Cambodge mais répondrait surtout à un problème d'inéquité sociale. / Dengue is a major public health concern worldwide, particularly in young children in Southeast Asia. Dengue vaccines are currently in development and policymakers need appropriate economic studies to determine their potential financial and public health impact. Alongside economic assessment, accurate disease incidence data are required to provide robust estimates of disease burden across the regions where dengue is endemic. This thesis proposes a methodological approach by which the Ministry of Health should consider when assessing feasibility of adoption dengue vaccines, with specific reference to developing country settings, e.g. Cambodia. We implemented prospective dengue incidence studies over several years, performed in a general population and include a wide range of ages. The results of these studies were compared with national surveillance data gathered in the same region by calculating multiplication factors to estimate underreporting on several years. Additional prospective cost of illness study was conducted to generate accurate economic burden of disease estimates over several years and identify vulnerable groups. This strategy also allowed better understanding of the epidemiology of dengue in Cambodia, of which some characteristics are needed to evaluate the feasibility of introducing dengue vaccine. Designing cost-effectiveness studies before a vaccine has been fully evaluated requires assumptions about variables such as efficacy and effectiveness, dosage and costs. Our next step will be to determine the threshold price – sensitivity analyses - for a vaccine to be cost-effective rather than assigning a specific cost, because it is difficult to predict cost for a vaccine that has not been marketed. Should this vaccine be safe and affordable enough, the adoption of the new vaccine into a National Immunization Programme would probably be cost-effective but, above all, a matter of social equity.

No description available.

Magister Public Health - MPH / In Zambia, there had been a large scaling up of new interventions to control malaria since 2003, which included the distribution of rapid diagnostic tests (RDTs), used to immediately determine if someone with symptoms suggestive of malaria actually has malaria; training of health workers in the use of the RDTs; and the prescription of artemisinin-based combination therapy (ACT) to which the malaria parasite is sensitive, rather than the old treatment regime of chloroquine to which the malaria parasite had become resistant. The use of RDTs to confirm the presence of malaria before treating for it with ACT became known as the „test and treat‟ policy. Previously, since the 1960s, in malaria endemic areas such as Zambia, children presenting with fever (the commonest symptom of malaria) without any obvious other cause for the fever, were assumed to have malaria and were hence treated for it with chloroquine. This was known as "presumptive treatment" of malaria. The combination of "presumptive treatment" and the use of a single medication led to the development of high levels of resistance to chloroquine, to the extent that it is now no longer an effective treatment for malaria. Years after the introduction of the "test and treat" policy, it was still unclear to what extent it was being implemented, as there was initial reluctance by health workers to test all children presenting with fever for malaria and if they did test they may not have followed the management guidelines of treating those who test positive with ACT and further investigating those who test negative for the cause of the fever. It seemed that staff had gotten used to the "presumptive treatment" approach to malaria over almost 4 decades and hence were quite reluctant to abandon it. The conflicting guidelines for malaria treatment in children between IMCI and "test and treat‟ has promoted a paradox between presumptive treatment for malaria and "test and treat" approach as IMCI teaches health workers to treat febrile children presumptively for malaria whereas the "test and treat" approach requires them to first make a definitive diagnosis before treating. Hence although the "test and treat" approach was instituted to overcome the problems with presumptive treatment approach it now had to contend with the competing and contradictory influence of the IMCI approach. This study therefore aimed to assess what proportion of children aged five years and younger who presented with fever were managed via the "test and treat" guidelines and which factors were associated with this, in Lusaka District, Zambia. Methodology: A cross sectional analytical study design was used based on a review of medical records. A sample size of 800 medical records of children presenting with fever was selected from 10 out of the 23 health care facilities in Lusaka, using a multistage stratified random sampling technique. Four hundred records were sampled from 2008 records (five years after commencement of the "test and treat" policy) and 400 from 2011 records (eight years after commencement of the "test and treat" policy). Trained data collectors used a data extraction tool to transcribe demographic and clinical data from the medical records in a standardized manner. Data Analysis: Univariate descriptive statistics analysis was performed using measures of central tendency and measures of dispersion to analyze numerical (continuous) variables such as age, weight and body temperature; and using frequencies for categorical variables such as gender, area of residence, RDTs/microscopy malaria tests conducted, received ACT if RDT positive, presence of an ACT treatment chart on the health centre wall and availability of a weighing scale. To determine the relationship between variables, bivariate analysis via the prevalence ratio was conducted. Results: Just over half (55%) of all children with fever were tested for malaria in 2008 and this gratifyingly increased to (73%) in 2011. Overall, the proportion of children correctly and appropriately treated with ACT, which means that those who tested positive for malaria were given ACT, was 85% in 2008 but regrettably dropped to 72% in 2011. Although "presumptive treatment" decreased from 24% in 2008 to 11% in 2011, the proportion of children with fever not tested for malaria, and although not treated for malaria, but left without a definitive diagnosis of their fever being made, remained high but dropping (22% in 2008 and 16% in 2011). Similarly the proportion of children who tested negative for malaria but then did not undergo any further investigation also unfortunately remained very high and rising (57% in 2008 and 89% in 2011). A combination of the above poor clinical management practises resulted in only 38% of children with fever in 2008 and unfortunately dropping to only 33% in 2011 being correctly managed (tested for malaria via RDT or microscopy and treated with ACT if positive, while further investigated for the cause of fever if negative). On preparedness of the health facility to implement the "test and treat" policy, it was noted that only 4 out of 10 health facilities were at least minimally prepared to do so, but paradoxically on bivariate analysis those minimally prepared were less likely (PR 0.62; 95% CI 0.41-0.94) to correctly manage the patients in 2011 than those who were unprepared. A similar paradox occurred for those correctly treated with ACT after testing positive, with facilities which were minimally prepared being less likely to do so (PR 0.28; 95% CI 0.14-0.58) in 2011 than those facilities which were unprepared to implement the "test and treat" policy. However these associations were inconsistent over time, as the associations were not present in 2008. Similarly all other factors such as staff category (doctor, nurse, clinical officer) and type of presenting symptoms besides fever (anorexia, lethargy, pallor) assessed, were not consistently associated with testing for malaria in both 2008 and 2011. The same applied for the other two main outcome variables of 'treated with ACT after test positive for malaria' and 'correctly managed child with fever', in that there were no factors that showed a consistent association with them in both 2008 and 2011. Conclusion: Testing of children with fever for malaria is at a low level but rose between 2008 and 2011. Paradoxically the proportion of those diagnosed with malaria who were correctly treated with ACT dropped between 2008 and 2011, as did the proportion of children with fever who were correctly managed. No factors assessed in this study were found to be consistently associated in both 2008 and 2011 with either testing for malaria, or treating confirmed malaria cases with ACT, or managing patients with fever correctly. Recommendations: In order for health workers to correctly implement the "test and treat" policy, which involves a series of complex steps, they ought to be formally trained to do so, mentored and constructively supervised. Additionally health facilities should be adequately equipped to enable health workers to fully implement the policy. Further studies to assess factors associated with the correct management of malaria via the "test and treat" policy are warranted.

The major outer membrane protein, MOMP, is the main protein in the outer membrane of pathogenic Campylobacter bacteria. Infection with Campylobacter is the principle cause of severe enteritis and untreated may result in non-trauma related paralysis. Studies have shown, that MOMP can act as antigen and thus has the potential to provide protection by induced humoral immunity. In our study, we expressed recombinant MOMP in Escherichia1coli, developed an alternative method to extract the outer membrane protein from its lipid environment and solved and characterised its crystal structure. The information acquired through these structural studies sheds new light on the structural characteristics of this important membrane protein. The West-African Lassa virus can cause deadly haemorrhagic fever. Lassa virus only possesses five proteins, which are synergistically responsible for the virus' life cycle, and virulence. The way in which the individual proteins act with one another and with host cell proteins is not fully understood. The polymerase L is the largest of the five proteins and has multiple functions. In this study, we first divided the L protein into different domains and tested their recombinant expression in Escherichia1coli. For first time, we solved the crystal structure of the putative endonuclease domain of Lassa virus and validated its endonucleolytic function by means of RNA digestion assays and alanine point mutations.

La fièvre de la Vallée du Rift (FVR) est une zoonose émergente provoquée par un virus. La FVR affecte principalement le bétail. Chez l'Homme, la maladie peut évoluer sous deux formes mortelles: une fièvre hémorragique et une encéphalite. Malgré l'importance du fonds génétique dans l'issue de la FVR, l'identité des gènes responsables reste inconnue. Nous avons étudié les facteurs génétiques qui déterminent la sensibilité de la lignée de souris MBT/Pas et la résistance de la lignée BALB/c à la FVR. Nous avons identifié trois QTLs sur les chromosomes 2, 5 et 11, nommés, respectivement, Rvfs1, Rvfs3 et Rvfs2. Une infection des lignées congéniques correspondantes, C.MBT Rvfs1, -2 et -3, a confirmé le rôle de la région Rvfs2 dans la sensibilité à la FVR. Une analyse pathophysiologique a montré que les souris C.MBT Rvfs2 et BALB/c développent précocement une hépatite. Les souris C.MBT Rvfs2 meurent de cette hépatite aiguë. En revanche, les souris BALB/c régénèrent leur foie, ce qui leur permet de mieux tolérer l'atteinte du foie. La majorité des souris BALB/c sont décédées plus tard d'une encéphalite. Ces observations montrent que les modèles étudiés reproduisent chacune des deux formes de la maladie observées chez l'Homme. Nous avons produit des lignées sous-congéniques pour la région Rvfs2 et avons testé leur sensibilité à la FVR. Les résultats ont été croisés avec une analyse des variants de structure et de régulation présents dans l'intervalle. Cette stratégie a permis d'identifier 3 gènes candidats : Rnf213, Cd7 et Fasn. La fonction du gène Fasn suggère qu'il puisse jouer un rôle lors de la régénération du foie et ainsi être le facteur génétique que nous recherchons. / Rift Valley fever (RVF) is an emerging zoonosis caused by an arbovirus. The disease affects mainly livestock, but it can have a severe impact on human health. In humans, RVF may progress into fatal outcomes due to acute hepatitis or encephalitis. Despite the influence of the host genetic background on the outcome of the disease, the identity of causative genes remains unknown. We studied the genetic factors determining the susceptibility of MBT/Pas and the resistance of BALB/c mouse strains to RVF. We identified 3 QTLs linked to survival on chromosomes 2, 5 and 11 and named them, respectively, Rvfs1, Rvfs3 and Rvfs2. The infection of the corresponding congenic strains, C.MBT Rvfs1, 2 and 3, confirmed the role of Rvfs2 on the susceptibility to RVF. A pathophysiological investigation showed that both C.MBT Rvfs2 and BALB/c mice exhibit early onset severe hepatitis. However, while C.MBT Rvfs2 died rapidly from liver failure, BALB/c mice tolerated the liver disease and regenerated the hepatic tissue. These mice eventually died at a later stage from encephalitis. These observations indicate that each of the studied mouse models recapitulates one form of the human disease. We generated subcongenic strains harboring the Rvfs2 region and tested their susceptibility to RVF. The results were combined with high-throughput analyses of the structural and regulatory variants found in the region. Our combined approach allowed the identification of three candidate genes: Rnf213, Cd7 and Fasn. The function of the Fasn gene suggests that it could play a role in the mechanism of liver regeneration and, thus, Fasn is our best candidate gene to account for the susceptibility phenotype.

La fièvre est l’une des principales raisons des consultations hospitalières en Afrique Sub-saharienne. Le paludisme est la cause la plus souvent suspectée, posant ainsi un grand problème: la prescription abusive des anti-paludiques aux sujets fébriles dont le test de diagnostic est négatif pour le paludisme.Vue que les principales causes virales de fièvre sont sporadiques et les patients fébriles sont souvent diagnostiqués négatifs au paludisme au Gabon, l’objectif prioritaire de ces travaux a donc été d’évaluer la présence des bactéries dans le sang des sujets fébriles et afébriles via les outils de biologie moléculaire. La flore bactérienne intestinale a aussi été évaluée à partir des échantillons de selles grâce à la méthode de culturomics.Au total, 1.363 échantillons d’ADN dont 1.203 appartenant aux sujets fébriles ont été analysés. Rickettsia felis a été la bactérie la plus détectée chez 50 patients fébriles suivie de Staphylococcus aureus (26), Streptococcus pneumoniae (12), Salmonella spp (11), Tropheryma whipplei (1) et Streptococcus pyogenes (1). Borrelia spp a été détectée chez 2 sujets afébriles. Le Plasmodium, Mansonella perstans, Loa loa et Mansonella sp "DEUX" ont été aussi détectés. L’évaluation de la flore bactérienne intestinale chez les sujets Gabonais a montré qu’elle était riche en Firmicutes suivie des Bacteroidetes, Actinobacteries et de Proteobacteries. On note aussi la découverte de nouvelles bactéries: Gabonia massiliensis, Gabonibacter massiliensis, Kallipyga gabonensis, Intestinimonas gabonensis et Bacillus massiliogabonensis.Ces travaux apportent beaucoup de ressources dans la lutte contre la fièvre au Gabon. / Fever is one of the main reasons of hospital consultations in Sub-Saharan Africa. Malaria is the most frequently suspected cause, posing a big problem: the wrong prescription of anti-malarial drugs to febrile patients without malaria.In a context of the sporadic existence of the major viral causes of fever added to the fact that febrile patients are often diagnosed negative for malaria, this work aimed to assess, especially, the presence of bacteria in the blood samples from febrile and afebrile Gabonese people using molecular tools. The intestinal bacterial flora was also evaluated from stool samples using culturomics method.A total of 1,363 DNA extracts including 1,203 from febrile patients were analyzed. Rickettsia felis was the most frequently detected bacterium in 50 febrile patients followed by Staphylococcus aureus (26), Streptococcus pneumoniae (12), Salmonella spp (11), Tropheryma whipplei (1) and Streptococcus pyogenes (1). Borrelia spp was detected in 2 afebrile cases. Plasmodium, Mansonella perstans, Loa loa and Mansonella sp "DEUX" were also detected. The evaluation of the gut bacterial flora from Gabonese people showed that Firmicutes were abundant followed by Bacteroidetes, Actinobacteria and Proteobacteria. New bacteria were discovered: Gabonia massiliensis, Gabonibacter massiliensis, Kallipyga gabonensis, Intestinimonas gabonensis and Bacillus massiliogabonensis.This work gives data enhancing the fight against the fever in Gabon.

Lumpy skin disease (LSD) and Rift Valley fever (RVF) are transboundary viral diseases occurring in Africa and the Middle East (e.g. Israel, Saudi Arabia and Yemen) with increasing potential for global spread. Although the role of wildlife in the epidemiology of these diseases is still not clearly understood, the African buffalo (Syncerus caffer) is thought to play a role in the epidemiology of these diseases. This study sought to expand our understanding of the role of buffalo in the maintenance of RVF and LSD by determining seroprevalence to these viral diseases in buffalo during the inter-epidemic period. Lumpy skin disease is endemic in Africa, and has spread to the Middle East (e.g. Israel); consequently there is a high risk of lumpy skin disease virus (LSDV) expanding its geographical distribution to other areas and due to its economic importance it is included in the list of Notifiable Diseases of the World Organization of Animal Health (OIE). The African buffalo is also suspected to play a role in the epidemiology of RVF. Like LSD, RVF was, until recently, only endemic in Africa. However, it spread to the Arabian Peninsula (Saudi Arabia and Yemen) in 2000 exacerbating concerns that it will extend to other regions of the world. Studies have already established that competent mosquito vectors for RVFV exist in North America and other parts of the world. A total of 248 buffalo sera was tested for antibodies to capripoxviruses and neutralising antibodies against LSDV and RVFV using an indirect enzyme-linked immunosorbent assay (I-ELISA) as well as the serum neutralisation test (SNT). The samples were obtained from the Kruger National Park (KNP) and Hluhluwe-iMfolozi Park (HiP) in South Africa. The prevalence of antibodies to LSDV and RVFV in the sera tested was 70/248 (28.2%) and 15/248 (6.1%), respectively using an I-ELISA. The LSDV I-ELISA, using a sheeppox virus as antigen, has not been validated for use in African buffalo. The high percentage of LSDV positive antibody results obtained in this study is however a concern. Results obtained is in contrast with other published results as well as results obtained with the SNT for antibodies against LSDV. The SNT is currently the gold standard for LSDV antibody testing. Using this test for LSDV in this study, 5/66 (7.6 %) samples tested positive. The results of the RVF I-ELISA, which had previously been validated for use in the African buffalo, correlated with the SNT results. From 12 SNT RVFV-positive sera, 3 (25%) had very high SNT titres of 1:640. Neutralising antibody titres of more than 1:80 were found in 80% of the positive sera tested. Eleven buffaloes (4.4% of the total samples) also showed evidence of antibodies to both LSDV and RVFV. The results obtained in this study complement other reports indicating the role of African buffalo in the epidemiology of these diseases during inter-epidemic periods. / Dissertation (MSc)--University of Pretoria, 2012. / Veterinary Tropical Diseases / unrestricted

Please read the abstract in the 00front part of this document / Dissertation (MSc (Clinical Epidemiology))--University of Pretoria, 2006. / Clinical Epidemiology / unrestricted