This thesis is about the process and practice of diagnosis and the implications of new diagnostic technologies in low resource settings. As a setting and a disease which has seen significant investment in diagnostics, Lassa fever in Sierra Leone has been selected as a case study to examine these themes. In this thesis, 'new diagnostic technologies' refers to laboratory-based diagnostics which are fast, reliable, accurate and can be used in low income settings. The starting point of this thesis is a narrative surrounding such technologies which suggests that they will revolutionise low income healthcare settings by allowing accurate scientific diagnosis in places where it was not possible before. Various perspectives on diagnosis are examined and some limitations are identified in relation to their accounts of diagnostic process, context, practice and technology. To explore the case, aspects of science and technology studies, the sociology of scientific knowledge and medical anthropology are combined. A multi-sited ethnography of Lassa fever diagnosis was conducted in three settings: a rural village, a laboratory and the wards of a hospital. Documents were reviewed and interviews conducted with key actors and ex-Lassa fever patients. Analysis focused on framings (partial and subjective interpretations), narratives (persuasive storylines which make use of particular framings) and practice in relation to Lassa fever and the development of technology for its diagnosis. Assumptions about the disease, diagnostics and the process of diagnosis are identified and the conclusion considers how they compare with practice in each setting. This thesis argues that diagnosis is a complex negotiated process and that new diagnostics represent only one aspect of that process. Thus, they are not a ‘silver bullet' to transform low resource healthcare contexts. In particular, ‘improved' diagnostics do not always have the expected impacts, sometimes even introducing complexity and uncertainty. In challenging narratives about diagnostics, this thesis provides an alternative, practice-based, approach to thinking about diagnostics and innovations in health systems; this approach acknowledges the importance, and complexity, of the diverse contexts which shape innovations and technology use.

Hemorrhagic fever viruses are a major public health threat in Sub-Saharan Africa. These kinds of viruses cause symptoms ranging from non-specific fevers and body aches to severe, life-threatening bleeding, shock, and multi-organ failure. Previously discovered hemorrhagic fever viruses can cause recurrent or seasonal outbreaks, but new ones continue to emerge. In order to combat these viruses, we need to better understand the aspects of pathogenesis that lead to mortality or survival. I will present analysis of the host immune response to two hemorrhagic fever viruses, Lassa virus and Bundibugyo virus, and how the host response can be used to predict mortality in these diseases. Lassa virus (LASV) was identified over 50 years ago, but it remains understudied and has hence been denoted a “Neglected Tropical Disease”. Clinical studies and experiments were run by our collaborators in Nigeria and Germany. In all, longitudinal blood samples were collected for over two hundred Nigerian Lassa Fever patients and concentrations of over 60 proteins analyzed. I processed the datasets, performed statistical testing, and created logistic regression models for each protein. This modeling allowed me to determine which proteins could be used as a predictive biomarker of mortality and the level of that protein that could best stratify patients who died and survived. I then compared protein levels for the best biomarkers and other markers in the same biological pathways with those of healthy and other febrile illness (non-Lassa Fever) controls. I examined the best biomarkers over time for their utility as biomarkers at later timepoints in hospitalization. Finally, I produced an application using RShiny that incorporated these and other exploratory analyses of the data, which allows users to visualize all the data we had in addition to the plots that were published. The filovirus Bundibugyo ebolavirus (BDBV), a relative of the more well-known Ebola virus (EBOV), first caused an outbreak in people fifteen years ago. Animal models are still being developed and characterized for this virus. Our collaborators in Texas experimentally infected cynomolgus macaques with BDBV and gave them post-exposure treatment with a VSV-based vaccine. These collaborators performed RNA-Seq on longitudinal samples from the infected macaques and sent me these data for analysis. I wrote pipelines to perform RNA-Seq and differential expression analyses on over 600 samples, of which I will focus on a subset here. I found differentially expressed genes for different subsets of the data, and I examined these gene lists using gene set enrichment analysis. I then generated logistic regression models to find differentially expressed genes that could predict mortality or survival. Many of these genes could accurately predict outcome at either late or early timepoints. I then used the top genes found by logistic regression to generate random forest models that could predict mortality over the entire course of disease. / 2025-03-20T00:00:00Z

M.A. / Two species of multimammate mouse, Mastomys coucha and M. natalensis are common, and widely distributed in southern Africa, occurring sympatrically in some areas, and allopatrically in others. The limits of their distribution are only provisional so far. As they share a high degree of morphological similarity, they are, as yet, impossible to identify with certainty in the field. Each species of multimammate mouse carries important diseases: with M. coucha being a carrier for the bacterium causing plague, and M. natalensis carrying the virus causing Lassa fever. In many areas, multimammate mice, being highly adaptable and ecological generalists, have become co-habitants with humans. This fact, coupled to the medical significance of both species, lends importance to being able to identify each species where it occurs, especially in areas where they occur sympatrically. Thus, a total of 40 specimens of M. natalensis were trapped from Richards Bay and La Lucia ridge in KwaZulu-Natal, and 43 specimens of M. coucha from Montgomery Park in Johannesburg and from the shores of the Vaal Dam in the Free State with the aim of comparing these two species via gel electrophoresis. These specimens were from allopatric populations from the centres of their provisional distributions. It was expected that there would be genetic differences between the two sibling species. Blood, liver, and muscle samples were taken, either in the field from dead specimens caught in snap-traps, or back in the laboratory from live-trapped specimens. Fifteen proteins or enzymes provided interpretable results at a total of 39 loci. Nineteen of these were polymorphic

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.

<p>Abstract</p><p>Crimean-Congo Hemorrhagic Fever virus (CCHF) and Lassa virus are giving sources illness to humans. In addition to zoonotic transmission, CCHF and Lassa virus can spread from person to person. After a short incubation period, CCHF and Lassa virus infections are characterized by a sudden onset of high fever, chills, headache and cough just like flu. Even some people are vomiting and have diarrhoea. After a few days of illness hemorrhagic manifestations occur. Treatment options for CCHF and Lassa viruses are limited, and there is no vaccine available for use in humans. The purpose of the present study was to produce recombinant nucleocapsid protein of Lassavirus and CCHF virus including an aminoterminal His-tag by a Semliki Forest Virus Replicon (pSFV 4.2). The recombinant proteins are planned to be used in future development of diagnostic methods.</p>

Abstract Crimean-Congo Hemorrhagic Fever virus (CCHF) and Lassa virus are giving sources illness to humans. In addition to zoonotic transmission, CCHF and Lassa virus can spread from person to person. After a short incubation period, CCHF and Lassa virus infections are characterized by a sudden onset of high fever, chills, headache and cough just like flu. Even some people are vomiting and have diarrhoea. After a few days of illness hemorrhagic manifestations occur. Treatment options for CCHF and Lassa viruses are limited, and there is no vaccine available for use in humans. The purpose of the present study was to produce recombinant nucleocapsid protein of Lassavirus and CCHF virus including an aminoterminal His-tag by a Semliki Forest Virus Replicon (pSFV 4.2). The recombinant proteins are planned to be used in future development of diagnostic methods.

Introduction/background: Epidemics of infectious diseases (ID) are re-occurring now more often and spreads faster into many different parts of the world due to globalization. The increasing evidence of climate change and man-made events have shown impacts to increase the emergency and re-emerging of animal- borne IDs. Studies claims that background factors of these IDs are biological, environmental and human-lifestyle related changes. The pathogen Lassa fever virus (LASV) is a zoonotic organismthat circulates in rodent reservoirs, and the animal´s hosts are rodent species (rats) of the genus Mastomys natalensis. Mastomys natalensis is primarily the reservoir species of the animal-borne disease of Lassa fever (LF) which is most prevalent in west Africa, particularly in Nigeria. Lassa fever (LF) has limited information with under-documented cases, its health effect on pregnant women especially in Nigeria is within the rural areas of Edo, Ondo, Delta, Ebony, Bauchi, Taraba and Plateau states where maternal mortalities are higher.  Aim: The overarching aim of this thesis is to analyse and discuss the health effects of Lassa fever occurrence and outcomes on pregnant women in Nigeria, with emphasis on the maternal mortality and fatality during pregnancy. Method: A statistical and systematic review was performed from retrospective studies of case series, case-control, observational and cohort studies of patients in Nigeria (pregnant women with gestation ages of pregnancy from 2 weeks –32 weeks) that tested positive to LASV. Publication status and publication date was applied for the inclusion of respective studies by electronic searches via Web of Science, Google scholar, MEDLINE and PubMed. Preferred Reporting Items for Systematic reviews and Meta-Analysis guidelines (PRISMA) was used to illustrate the flow of relevant articles in my study. R-commander and R-Studio software was used to analyze the data and to find the causal significant relationship between LF and maternal mortality using “Linear regression and linear model plot”. Result: The total number of full-text and Peer-view publications on Lassa fever virus cases was 1 609 articles. 94 articles out of the 1 609 articles were eligible for full text revision. Exclusion criteria finally yielded 6 studies that were relatively relevant to my study. However, 3 out of the 6 articles were statistically reviewed to know the influence of Lassa fever and the risk of maternal mortality during pregnancy.  Conclusion: Lassa fever occurrence have significantly shown potential increase in the severity of maternal mortality, and is predominant among pregnant women from 39 - 45 years old in Nigeria especially within the risk endemic areas of Ondo, Edo, Ebony and Bauchi states showing significant long-term diseases on LF affected pregnant women, such as encephalopathy, acute kidney dysfunction and acute kidney failure that leads to further health problems or complications like coma and sensorineural deafness.

Le virus Lassa entraine une fièvre hémorragique endémique en Afrique de l’Ouest et représente un problème de santé publique. Les connaissances sur la pathogénèse et les réponses immunitaires associées à la maladie sont partielles. Nous avons suivi les paramètres pathologiques, virologiques et immunologiques associés aux infections létales et non létales du LASV chez le singe cynomolgus. Le tableau clinique a été caractérisé par une dépression, une anorexie, une perte de poids et une asthénie chez les animaux survivants, tandis que ces mêmes symptômes ont été accompagnés de fièvre, de difficultés respiratoires et d’épistaxis chez les animaux infectés par une dose létale. Seuls ces derniers ont montré une perturbation des paramètres de coagulation, une rhabdomyolyse et une hausse des marqueurs de lésions rénales. Nous avons observé un tropisme radicalement différent en fonction de la sévérité de la maladie, avec une dissémination virale dans les organes plus importante et plus rapide chez les animaux décédés, la présence de particules infectieuses plus nombreuses et des modifications anatomopathologiques plus sévères. Une réponse immunitaire innée et adaptative précoce et puissante a été associée avec le contrôle de l’infection et la survie tandis que les infections fatales ont été caractérisées par une réponse inflammatoire ressemblant au choc septique, une défaillance de la réponse immunitaire ainsi qu’une réplication virale incontrôlée. Cette étude permet d’améliorer nos connaissances de la pathogénèse de la fièvre de Lassa et d’apporter des marqueurs d’infection prédictifs de la maladie / Lassa virus causes a hemorrhagic fever endemic in West Africa and represents a threat for civilians. The pathogenesis and the immune responses associated with the disease are poorly understood. We followed pathological, virological and immunological parameters associated with fatal and non-fatal Lassa virus infection in the cynomolgus monkey. The clinical picture was characterized by depression, anorexia, weight loss and asthenia in survivors whereas the same symptoms were supported by fever, respiratory difficulties and epistaxis in animals infected with the lethal dose. Only fatalities have shown coagulation parameters dysfunction, rhabdomyolysis and an increase of renal function markers. We observed a different viral tropism in a function of the disease severity, with viral dissemination in organs that was more important and faster in fatalities, the appearance of numerous infectious particles number and more severe pathologic changes. Early and robust innate and adaptive immune response has been associated with the control of infection and recovery whereas fatal infections were characterized by a sepsis like inflammatory response, defective immune response as well as uncontrolled viral replication. This study sheds light on the pathogenesis of Lassa fever and reveals infection markers predictive of the disease outcome